Drosophila gene families: Protein targeting, Asymmetric cell division and apical/basal polarity

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of G&alpha:i depends on Partner of Inscuteable (Pins). This study establishes that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Gαi, regulates cortical localization of the subunits G&alpha: and Gβ13F. Ric-8, Gαi and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis (David, 2005).

Analysis of spindle orientation in epithelial cells revealed that division takes place in the plane of the epithelium in both wild-type and pins mutant cells. This demonstrates, first, that the requirement for Pins/Gαi to maintain the planar orientation of the spindle is specific to pI cells and, second, that a pI-specific activity tilts the spindle in the absence of Pins/Gαi signalling. Fz signalling was an obvious candidate for this pI-specific activity for two reasons. First, Fz signalling is still active in the pins and Gαi mutants as the spindle was correctly oriented along the antero-posterior axis in these mutants. Second, Fz accumulates at the posterior apical cortex of pI cells and this accumulation of Fz is maintained in Gαi pI cells. It is therefore envisaged that, although orienting the spindle along the antero-posterior axis, Fz signalling may also be responsible for tilting the spindle along the apico-basal axis in the absence of Pins/Gαi signalling. fz,pins double mutants were analyzed to test this hypothesis. Strikingly, in the absence of both Fz and Pins, the spindle was parallel to the plane of the epithelium. Therefore, in the absence of Pins/Gαi signalling, the activity tilting the spindle along the apico-basal axis is Fz-dependent. Intriguingly, in fz,pins pI cells, the spindle was even less tilted than in wild-type cells, indicating that Fz may also tilt the spindle in wild-type cells along their apico-basal axis. To test this, spindle orientation was analyzed in the fz mutant. In the absence of Fz, division takes place within the plane of the epithelium, the spindle being less tilted than in wild-type cells. Together, these results demonstrate that in pI cells, a Fz-dependent activity tends to tilt the spindle along the apico-basal axis. This activity is counterbalanced by a Ric-8a/Pins/Gαi-dependent one that maintains the spindle in the plane of the epithelium. Orientation of the spindle in wild-type cells arises from this balance. Finally, the analysis of spindle orientation in baz mutant pI cells revealed that Fz exerts its activity on the spindle independently of Baz, and hence probably independently of the Par complex. The tight control of the spindle apico-basal orientation probably regulates the morphogenesis of the pIIb cell and of the differentiated sensory organs (David, 2005).

In C. elegans, ric-8 regulates spindle positioning in anaphase, downstream of the par genes and upstream or downstream of the GPR-Gαi complex, which is the homologue of the Pins-Gαi complex. These data demonstrate that, in the dividing pI cell, Ric-8a is required for asymmetric localization of Pins, Baz and Numb and for mitotic-spindle positioning. It is proposed that these activities of Ric-8a depend on an unexpected function of Ric-8a: localizing Gαi and G<β13F at the plasma membrane. This study of ric-8a also revealed that, in the pI cell, ric-8a, pins, Gαi and Gγ1 are all required for orientation of the spindle within the plane of the epithelium. The milder apico-basal phenotype that was observed in ric-8a pI cells could be accounted for by some persistence of the Ric-8a protein in somatic clones. Alternatively, an intriguing possibility is that ric-8a may also affect Gαo activity, which has recently been proposed to act downstream of Fz signalling. ric-8a loss of function would thereby affect both the Fz- and Gαi-dependent activities exerted on the spindle, resulting in a milder apico-basal tilt (David, 2005).

Importantly, developmental processes ranging from gastrulation, neural-tube closure, neurogenesis and retina formation to asymmetric segregation of cell-fate determinants require that spindle orientation is controlled in two directions: along the polarity axis of the tissue (antero-posterior, animal-vegetal, central-peripheral, etc) and parallel to the plane of the epithelium. This study shows that, in dividing pI cells, these two orientations are controlled by different and opposing activities. A Fz-dependent activity orients the spindle along the antero-posterior axis but tends to tilt it along the apico-basal axis, and a Gαi-dependent activity maintains the spindle parallel to the plane of the epithelium. The Fz- and Gαi-dependent activities are likely to act through forces pulling on astral microtubules. Fz and heterotrimeric G signalling are implicated in mitotic-spindle positioning during both symmetric and asymmetric cell division. The elucidation of the molecular mechanisms underlying these forces in the pI cell might therefore generally contribute to understanding of the mechanisms that control mitotic-spindle positioning (David, 2005).

Inscuteable regulates the Pins-Mud spindle orientation pathway

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). This study investigated the mechanism of spindle orientation by the Insc-Pins complex. Previously, two Pins spindle orientation pathways were defined: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. The current study examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. It was found that the Insc-Pins complex requires Galphai for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. These results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud) (Mauser, 2012).

Spindle positioning is important in many physiological contexts. At a fundamental level, spindle orientation determines the placement of the resulting daughter cells in the developing tissue, which is important for correct morphogenesis and tissue organization. In other contexts, such as asymmetric cell division, spindle position ensures proper segregation of fate determinants and subsequent differentiation of daughter cells. This study examined the function of a protein thought to provide a 'passive' mark on the cortex for subsequent recruitment of the spindle orientation machinery. During neuroblast asymmetric cell division, Insc has been thought to mark the cortex based on the location of the Par polarity complex (Mauser, 2012).

Ectopic expression of Insc in cells that normally do not express the protein has revealed that it is sufficient to induce cell divisions oriented perpendicular to the tissue layer, reminiscent of neuroblast divisions. Expression of the mammalian ortholog of Inscuteable, mInsc, in epidermal progenitors has shown that this phenotype is not completely penetrant over time. Expression of mInsc leads to a transient re-orientation of mitotic spindles, in which mInsc and NuMA initially co-localize at the apical cortex. After prolonged expression, however, the epidermal progenitors return to dividing along the tissue polarity axis, a scheme in which mInsc and NuMA no longer co-localize. These results indicate that Insc and Mud can be decoupled from one another (Mauser, 2012).

This study examined the effect of Insc-Pins complex formation both in an induced polarity spindle orientation assay and in in vitro binding assays. The results indicate that Insc plays a more active role in spindle positioning than previously appreciated. Rather than passively coupling polarity and spindle positioning systems, Insc acts to regulate the activity of downstream Pins pathways. The Dlg pathway is unaffected by Inscuteable expression while the Mud pathway is inhibited by Insc binding (Mauser, 2012).

Recent work on the mammalian versions of these proteins explains the structural mechanism for competition between the Insc-Pins and Pins-Mud complexes. The binding sites on Pins for these two proteins overlap making binding mutually exclusive because of steric considerations. The observation of Insc dissociation of the Pins-Mud complex in Drosophila (this work) and mammalian proteins (LGN-NuMA) suggests that Insc regulation of Mud-binding is a highly conserved behavior (Mauser, 2012).

This competition between Mud and Insc for Pins binding is consistent with previous work done with a chimeric version of Inscuteable/Pins (Yu, 2000). This protein, in which the Pins TPR domain was replaced with the Inscuteable Ankyrin-repeat domain, bypasses the Insc-Pins recruitment step of apical complex formation. In these cells, the chimeric Insc-Pins protein was able to rescue apical/basal polarity and spindle orientation in metaphase pins mutant neuroblasts. As this protein lacks the Mud-binding TPR domain, Mud binding to Pins is not absolutely necessary for spindle alignment. Importantly, the PinsLINKER domain is still intact in the Insc-Pins fusion, implying that Dlg, not Mud, function is sufficient for partial activity, as observed in the S2 system (Mauser, 2012).

The Mud and Dlg pathways may play distinct roles in spindle positioning. The Dlg pathway, through the activity of the plus-end directed motor Khc73, may function to attach the cortex to the spindle through contacts with astral microtubules. In contrast, the Mud pathway, through the minus-end directed Dynein/Dynactin generates force to draw the centrosome towards the center of the cortical crescent. Fusion of the Pins TPR motifs, which recruit Mud, to Echinoid does not lead to spindle alignment, indicating that the Mud pathway is not sufficient for spindle alignment. The PinsLINKER domain does have partial activity on its own, however, and when placed in cis with the TPRs leads to full alignment. In this framework, the function of Insc may be temporal control, ensuring that microtubule attachment by the Dlg pathway occurs before the force generation pathway is activated (Mauser, 2012).

In the temporal model of Insc function, what might cause the transition from the Insc-Pins-Dlg complex, which mediates astral microtubule attachment, to the Mud-Pins-Dlg complex, which generates spindle pulling forces? By early prophase, Inscuteable recruits Pins and Gαi to the apical cortex. During this phase of the cell cycle, Mud is localized to the nucleus in high concentration. Apically-localized Pins binds Dlg, creating an apical target for astral microtubules. During early phases of mitosis, Inscuteable would serve to inhibit binding of low concentrations of cytoplasmic Mud to the Pins TPRs to prevent spurious activation of microtubule shortening pathways. After nuclear envelope breakdown, Mud enters the cytoplasm in greater concentrations and could then act to compete with Insc for binding to Pins, allowing Pins output to be directed into microtubule-shortening pathways (see Proposed model for Inscuteable regulation of spindle orientation). Future work will be directed towards testing additional aspects of this model (Mauser, 2012).

A role for a novel centrosome cycle in asymmetric cell division

Tissue stem cells play a key role in tissue maintenance. Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asymmetric division. It has been shown that their mitotic spindle orientation is established before spindle formation. This study investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the two centrioles separate, but only one is active, retaining pericentriolar material and forming a 'dominant centrosome.' This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming one pole of the future spindle. The second centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asymmetric localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asymmetric division. A two-step mechanism is proposed to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment (Rusan, 2007).

By prophase, NBs contain two MTOCs that are almost fully separated and aligned along the NB/GMC axis, but analysis of fixed NBs revealed a single MTOC positioned opposite the GMCs before mitotic entry. Thus MTOC behavior was examined throughout the cell cycle as an initial approach to test the hypothesis that fixing the position of one MTOC through successive divisions helps ensure persistent spindle orientation. Live NBs expressing GFP-G147, an MT-associated protein were examined, revealing a striking temporal difference in MTOC activity. During interphase, a single detectable MTOC persists opposite the previous division site; this is referred to as the dominant MTOC. As NBs approach mitosis, this MTOC increases activity (matures; empirically judged by size and MT fluorescence intensity), forming an MT basket around the nucleus. This is referred to as preprophase; this stage is also seen in fixed samples stained for tubulin. Soon after, sometime before the dominant MTOC fully matures, something striking happens: a second MTOC appears distant from the first. This is the second MTOC and this stage is referred to as prophase onset. The second MTOC increases activity, maturing ~10 min before nuclear envelope breakdown (NEB). Using 4D imaging, the possibility was excluded that the second MTOC was present earlier in another focal plane. To further assess this, forming spindles were imaged end on. It is clear that the second MTOC did not emerge from the dominant MTOC or travel around the nucleus. Instead, the second MTOC appeared roughly opposite the dominant MTOC. MTOC separation began immediately, and by NEB, they were 146 ± 20° apart. Thus, NBs form two distinct MTOCs: an MTOC persisting from the previous division and another only activated at mitotic entry (Rusan, 2007).

This distant activation of the second MTOC raised questions about the centrosome cycle. One possibility is that NBs have two MT nucleating centrosomes, but only one can retain MTs and act as an MTOC during interphase, whereas the second acquires MT retention ability during mitotic entry, explaining the second MTOC's sudden appearance. There is precedent for this: mouse L929 cells have two γtub-bearing centrosomes that can nucleate MTs, but only one contains Ninein and can retain MTs to form an MTOC (Rusan, 2007).

To test this hypothesis in NBs, EB1-GFP was used. This binds growing MT plus ends and reliably identifies MT nucleation sites. Only one nucleation site was present in interphase and preprophase, and a new nucleation site appeared distant from the first, consistent with spatially and temporally distinct second MTOC activation. Thus, NBs regulate MT nucleation and not just MT retention (Rusan, 2007).

To examine how the new nucleation center forms, centrosomes were imaged using a PCM protein, GFP-Cnn. NBs contain a single detectable centrosome during interphase. When NBs reenter mitosis, a second Cnn-positive centrosome appears distant from the first, mimicking activation of the second MTOC. To verify that these occur simultaneously, NBs expressing mCherry-Tubulin (chTub) and GFP-Cnn were imaged. This revealed perfect temporal and spatial correlation between the appearance of the second centrosome and activation of the second MTOC. Physical separation of two centrosomes/MTOCs was never seen. This is the first example of asynchronous and physically distant centrosome maturation, suggesting that NBs use a novel centrosome cycle (Rusan, 2007).

Higher temporal/spatial resolution imaging revealed that two GFP-Cnn spots separate during mitotic exit. One GFP-Cnn spot persists as the NB interphase centrosome, forming the dominant MTOC, whereas the other spot disappears. The persistent Cnn spot (centrosome) remains relatively stationary in interphase, consistent with the hypothesis that coarse spindle alignment begins in interphase by anchoring the dominant centrosome (Rusan, 2007).

Centrosome fate was examined in the two daughters (new NB and GMC). They differ dramatically in PCM retention, in contrast to mammalian cells, where both daughters' centrosomes retain PCM. The GMC centrosome sheds all PCM. The new NB centrosome (that becomes the dominant MTOC) retains PCM throughout interphase and further accumulates PCM during the next mitosis. The complete shedding of PCM in GMCs appears to be the normal behavior of interphase centrosomes in most fly cells, whereas in syncytial early embryos, both daughters retain PCM foci through the cell cycle. In contrast to both cell types, the NB daughters exhibit differential PCM retention (Rusan, 2007).

These data suggest that NBs have a novel centrosome cycle in which the second centrosome matures distant from the dominant centrosome/MTOC. One hypothesis to explain this would be the distal positioning of a differentially regulated centriole that is blocked from recruiting PCM in interphase and thus cannot form an MTOC until 'activated' during mitosis. If this centriole is always inherited by the GMC, it might also explain complete PCM loss as GMCs exit mitosis (Rusan, 2007).

Centrioles were examined live to test this hypothesis, using the centriole marker GFP-PACT and Histone-GFP Mother/daughter centrioles disengaged in late telophase, as in mammalian cells and fly embryos. Thus, two NB centrioles are present throughout interphase despite the presence of only one MTOC (Rusan, 2007).

The two centrioles then exhibit different behaviors. One remains fairly stationary, whereas the second moves to roughly the other side of the nucleus. Disengagement perfectly correlates with separation of Cnn spots, suggesting that the stationary centriole retains PCM to form the dominant MTOC and the mobile centriole completely sheds PCM. To test this, NBs were imaged expressing chTub and GFP-PACT. The stationary centriole retained MTs, whereas the mobile centriole did not. Upon reentering mitosis, the mobile centriole regained nucleation activity, forming the second MTOC (Rusan, 2007).

This suggests that full separation of the MTOCs that organize the spindle is biphasic. It begins in interphase, when one centriole retains PCM, remains stationary, and forms the dominant MTOC, whereas the second centriole sheds PCM and becomes mobile. Movement of the second centriole away from the dominant MTOC in interphase accounts for ~70% of the separation needed to form a spindle. Mechanisms of transporting the mobile centriole remain to be identified, but it is nonrandom, as in 26/30 NBs, the second MTOC emerged more than 90° from the dominant MTOC. After the second MTOC is activated, the two separate the last 30%, most likely via MT sliding forces. This might explain defects in lissencephaly1 mutants, where MTOCs are only separated by 124° at NEB. Perhaps interphase centriole movement is normal, but MT-based MTOC separation is defective (Rusan, 2007).

These data suggest that NBs differentially regulate the activity of their two centrioles within the same cytoplasm. Interestingly, a similar observation was made in clam eggs, which have three centrosomes just after fertilization. The sperm centrosome is functionally inactivated, whereas female centrosomes organize the meiotic spindle (Rusan, 2007).

Next NB centrosome regulation was examined. In preprophase, one centriole (marked by anti-DPLP) formed the dominant MTOC, whereas the second centriole had no associated MTs and was randomly positioned, confirming the live-cell data. Thus whether γtub is recruited asymmetrically was examined. Fixed preprophase NBs had two centrioles; only that opposite the GMCs accumulated γtub. Further, both γtub and Cnn are absent from the NB centriole nearest the GMCs in interphase/preprophase (Rusan, 2007).

Polo kinase promotes centrosome maturation by promoting γtub recruitment during mitotic entry. Differences in Polo localization/activity might underlie differences in timing of NB centrosome maturation. NBs expressing Polo-GFP and the centriole marker mCherry-DSAS-6 were examined. Only the centriole pair that forms the dominant centrosome was Polo-GFP positive during preprophase. Polo-GFP accumulated on the mobile centriole pair as the NB entered mitosis, increased on both centriole pairs through prophase, and moved on to kinetochores. When Polo-GFP was examined in cells exiting mitosis, it was retained at low levels on the dominant centrosome. In the future, it will be interesting to examine the localization of Aurora A, another centrosome regulator (Rusan, 2007).

Unlike the distal appendages of mammalian mother centrioles, fly mother and daughter centrioles have been thought to have no known ultrastructural or molecular differences. These data suggest that differences exist. It is unlikely that this differential regulation is a result of location, as both centrioles are initially adjacent after disengagement. The differences may be due to centriole age or procentriole maturation state (Rusan, 2007).

The NB spindle is largely aligned by NEB. Based on the current data, the hypothesis was tested that the dominant centrosome helps define one spindle pole before prophase. The angle between the dominant centrosome/MTOC axis and the anaphase axis was calculated, using the nuclear centroid as a fixed reference. This revealed two phases in defining the future spindle axis. Through prophase onset, the dominant centrosome remains fairly stationary roughly opposite the GMCs, agreeing with fixed images, whereas the second centriole moves to a distal position (to within 46 ± 33° of the anaphase axis). This is consistent with the hypothesis. The dominant centrosome may be immobilized by aster–cortex interactions or by absence of an active displacement mechanism. In the second phase, alignment is refined in prophase and prometaphase (the angle between the NB centrosome and anaphase axes decreases from 31 ± 29° to 15 ± 12° (Rusan, 2007).

To further test whether anchoring the dominant centrosome helps roughly align the spindle, asl mutant NBs were imaged live. They lack functional centrosomes and astral MTs. Mutant NBs lack a dominant interphase centrosome, allowing assessment of its role in spindle orientation and asymmetric cell division. Live imaging revealed robust chromatin-mediated MT nucleation and spindle assembly producing fairly normal spindles. Spindle poles emerge from a disorganized MT array near the chromosomes that focuses as the spindle lengthened. Spindles do not rotate during formation, always forming along the initial pole separation axis, but do rotate during metaphase, suggesting that rotation can occur without astral MTs or that asl mutants have a reduced astral array sufficient for rotation. Surprisingly, consecutive divisions in asl mutants usually produce adjacent or near-adjacent daughters, as in wild type. In a few cases, however, spindles form parallel to the GMC cap and, presumably, the polarity axis (2/13; ~15%); these NBs divide symmetrically. This suggests that the second phase of spindle alignment can occur without a dominant centrosome and can rescue misalignment, as long as it is not too extreme, but occasional atypical symmetric divisions occur. This results in defective brain anatomy, with ectopic paired, smaller NBs, presumably progeny of symmetric divisions (Rusan, 2007).

These data reveal two new aspects of asymmetric division in this stem cell model. First, cells can adjust the canonical centrosome cycle to allow novel cell behaviors, as was observed during clam meiosis. Central brain NBs also alter this cycle: rather than both centrosomes maturing in synchrony and proximity, the two centriole pairs are differentially regulated, maturing asynchronously and distant from one another. One retains MT nucleating activity throughout the cell cycle, forming the dominant MTOC during interphase, whereas the second is initially inactive, only forming a functional centrosome and nucleating MTs at mitotic entry. One speculative possibility is that these are mother and daughter centrioles and that one is preferentially retained in the stem cell, a hypothesis that will now be tested. It is also of interest to ask whether other stem cells use this mechanism (Rusan, 2007).

Second, the data suggest that this novel centrosome cycle helps ensure high-fidelity spindle positioning and thus asymmetric division. A model is proposed in which NB mitotic spindles are aligned in two phases to ensure that GMC daughters are born next to the previous GMC. Rough alignment is achieved by confining the dominant MTOC to a relatively fixed position from the previous division and moving the second centriole to the other side of the cell. As spindles form, a second process refines this initial alignment. In asl mutants, without centrosomes, the first mechanism is inactive, but the second mechanism can align the spindle unless initial alignment is wildly off axis. In mud mutants, centriole separation must occur normally, as prophase MTOCs are nearly fully separated, but alignment of spindle poles to cortical polarity cues is defective. The normal two-step process is a robust mechanism ensuring successful asymmetric divisions and reproducible brain anatomy (Rusan, 2007).

Asymmetric centrosome inheritance maintains neural progenitors in the mammalian neocortex

Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. This study shows that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex (Wang, 2009).

Radial glia cells constitute a major population of neural progenitor cells that occupy the proliferative VZ in the developing mammalian neocortex. In addition to their well-characterized function as a scaffold in supporting neuronal migration, radial glia cells display interkinetic nuclear oscillation and proliferate extensively at the luminal surface of the VZ (that is, the VZ surface). During the peak phase of neurogenesis [around embryonic day 13-18 (E13-E18) in mice] they predominantly undergo asymmetric division to self-renew while simultaneously giving rise either directly to a neuron, or to an intermediate progenitor cell which subsequently divides symmetrically to produce neurons. Whereas differentiating progeny progressively migrate away from the VZ to form the cortical plate (CP) (the future neocortex) renewing radial glia progenitors remain in the VZ for subsequent divisions. The distinct migratory behaviour of radial glia progenitors and their differentiating progeny is fundamental to the proper development of the mammalian neocortex; however, little is known about the basis of these behavioural differences (Wang, 2009).

Centrosomes, the main microtubule-organizing centres in animal cells, have an important role in many cell processes, particularly during cell division. All normal animal cells initially inherit one centrosome, consisting of a pair of centrioles surrounded by an amorphous pericentriolar material. The two centrioles differ in their structure and function. The older 'mother' centriole, which is formed at least one-and-a-half generations earlier, possesses appendages/satellites that bear specific proteins, such as cenexin (also known as Odf2), and anchor microtubules and support ciliogenesis. In contrast, the younger 'daughter' centriole, which is formed during the preceding S phase, lacks these structures. Full acquisition of appendages/satellites by the daughter centriole is not achieved until at least one-and-a-half cell cycles later. During each cell cycle, the centrosome replicates once in a semi-conservative manner, resulting in the formation of two centrosomes: one of which retains the original old mother centriole (that is, the mother centrosome) while the other receives the new mother centriole (that is, the daughter centrosome). This intrinsic asymmetry in the centrosome has recently been demonstrated to be important for proper spindle orientation during the division of male germline stem cells and neuroblasts in Drosophila, although female germline stem cells appear to divide normally in the absence of centrioles/centrosomes. These studies indicate a critical role for the differential behaviour of centrosomes with differently aged mother centrioles in asymmetric division of the progenitor/stem cells, although it remains unclear whether proper behaviour and development of the progenitor/stem cells and their differentiating daughter cells depend on centrosome asymmetry. Asymmetric division of radial glia progenitors accounts for nearly all neurogenesis in the developing mammalian neocortex. Three out of four autosomal recessive primary microcephaly (MCPH) genes identified so far encode centrosomal components, suggesting that proper neocortical neurogenesis and development entail a tight regulation of the centrosome, which is so far poorly understood. To address these issues, centrosome regulation during the peak phase of mammalian neocortical neurogenesis was investigated (Wang, 2009).

To examine centrosome behaviour, a plasmid encoding centrin 1, a central component of the centriole, fused with enhanced green fluorescent protein (EGFP-CETN1) was introduced into the developing neocortex of E13.5 mouse embryos by in utero electroporation. As expected, EGFP-CETN1 formed pairs of dots that co-localized with γ-tubulin, a centrosomal marker, suggesting that transient expression of EGFP-CETN1 reliably labels the two centrioles of individual centrosomes in the developing neocortex in vivo. Moreover, it was observed that at the onset of peak neurogenesis (E13-E14), EGFP-CETN1-labelled centrosomes were predominantly located at the VZ surface with a small subset located in the subventricular zone (SVZ) and the intermediate zone (IZ) (Wang, 2009).

To identify the cell types harbouring EGFP-CETN1-labelled centrosomes, a plasmid encoding DsRedexpress (DsRedex), a red fluorescent protein that diffuses throughout cells and thereby reveals their morphology, was co-electroporated . In bipolar radial glia progenitors in the VZ the centrosome was located in their ventricular endfeet at the VZ surface, whereas in multipolar cells in the IZ and the SVZ the centrosome was harboured in their cell bodies. Moreover, dividing radial glia progenitors were observed that possess a pair of centrosomes together with condensed chromosomes at the VZ surface. Consistent with this differential centrosome localization between radial glia progenitors and their differentiating daughter cells, a progressive increase was observed in the appearance of EGFP-CETN1-labelled centrosomes in the IZ and the CP as development proceeded, in addition to some that remained at the VZ surface. This gradual increase in centrosome localization in the IZ and the CP coincided with the production and migration of differentiating cells such as neurons to these regions during this period (Wang, 2009).

The distinct positioning of the centrosome in radial glia progenitors versus their differentiating progeny prompted asking whether the centrosomes in these two cell populations/types are different. To explore this, a plasmid encoding ninein, a mature centriole-specific protein that localizes to appendages/satellites, fused with EGFP (EGFP-Nin) together with a plasmid encoding CETN1 fused to DsRedex (DsRedex-CETN1) were electroporated into the developing mouse neocortex at E13.5. As expected, both EGFP-Nin and DsRedex-CETN1 formed dot-like structures and co-localized to the centrosomes, especially those at the VZ surface, as identified by an antibody to the integral centrosomal protein pericentrin 1. Notably, EGFP-Nin was preferentially concentrated at one of the two centrioles marked by DsRedex-CETN1 in individual centrosomes, suggesting that the two centrioles in interphase radial glia progenitors are not identical. Given that Nin specifically associates with mature centrioles, these results indicate that the centriole with abundant EGFP-Nin is the more mature mother centriole, whereas the one with little EGFP-Nin is the less mature daughter centriole. A similar inequity in the recruitment of EGFP-Nin by the duplicated centrosomes was also observed in dividing radial glia progenitors at the VZ surface, indicating that the duplicated centrosomes are not identical during late mitosis. The centrosome with abundant EGFP-Nin is probably the centrosome that retains the mature old mother centriole and the centrosome with little EGFP-Nin is probably the centrosome that bears the relatively immature new mother centriole (Wang, 2009).

Having found that the centrosomes in dividing radial glia progenitors exhibit asymmetry in their maturity, it was next asked whether this centrosome asymmetry is related to the distinct behaviour of radial glia progenitors and their differentiating progeny in the developing neocortex during neurogenesis. To address this, the relative distribution of centrosomes labelled by DsRedex-CETN1 versus those labelled by EGFP-Nin in the developing neocortex were compared as development proceeded. Interestingly, whereas DsRedex-CETN1-labelled centrosomes progressively occupied the IZ and the CP, where differentiating cells are situated, EGFP-Nin-labelled centrosomes were mostly found in the VZ, where radial glia progenitors are located. Given that DsRedex-CETN1 labels all centrosomes whereas EGFP-Nin selectively labels mature centrosomes, these results point to an intriguing possibility that the duplicated centrosomes in dividing radial glia cells are differentially inherited depending on their age and maturity during neocortical neurogenesis. It is known that during each cell division one centrosome retains the old mature mother centriole and the other bears the new less mature mother centriole. Thus, these results suggest that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors (Wang, 2009).

To test this, an assay was developed to distinguish explicitly between the centrosome containing the old mother centriole and the centrosome containing the new mother centriole in the developing neocortex in vivo. The assay takes advantage of the photoconvertible fluorescent protein Kaede, which changes from green to red fluorescence on exposure to violet light. Centrioles in the developing neocortex were labelled by transient expression of CETN1 fused with Kaede (Kaede-CETN1). Photoconversion was then performed to switch labelled centrioles from green to red fluorescence. This green-to-red fluorescence conversion of Kaede proteins is irreversible and the red protein is very stable, thus allowing long-term tracking of the existing photoconverted proteins and the structures with which they are associated. Moreover, all newly synthesized Kaede proteins are green fluorescent. It is known that centriole duplication requires new protein synthesis of centrin. As a result, newly duplicated centrioles that contain newly synthesized Kaede-CETN1 are green fluorescent, whereas previously existing centrioles are red fluorescent. Hence, in the first cell cycle after photoconversion, both centrosomes contain a red fluorescent mother centriole and a green fluorescent daughter centriole. However, in the second and subsequent cell cycles, centrosomes with the new mother centriole contain only green fluorescent centrioles, whereas centrosomes retaining the original old mother centriole harbour both red and green fluorescent centrioles, thus distinguishing between centrosomes with differently aged mother centrioles (Wang, 2009).

To carry out this assay in the developing neocortex in vivo, an in utero photoconversion procedure was developed and and combined with in utero electroporation. Kaede-CETN1, which localized specifically to the centrosomes, was introduced into the developing mouse neocortex at E13.5. One day later, that is, E14.5, the forebrain of electroporated embryos was treated with a short exposure of violet light while still in the uterus, which effectively converted nearly all Kaede-CETN1 proteins and their labelled centrosomes from green to red fluorescence [E13.5-E14.5(PC)]. The uterus was replaced and the embryos continued to develop in vivo. The localization and inheritance of centrosomes were analysed at different developmental stages thereafter (Wang, 2009).

It was found that one day after photoconversion [E13.5-E14.5(PC)-E15.5], around 95% of centrosomes contained both red and green fluorescent centrioles (indicated by yellow colour in the merged image), consistent with the notion that the labelled cells have undergone one round of division and have duplicated their centrioles during the 24-h period after photoconversion. This was shown directly by imaging centrosomes at high magnification, revealing that each centriole was mostly only red or green fluorescent. This also demonstrates that there is little diffusion of centrin proteins between duplicated centrioles, or between the centrioles and a cytoplasmic pool which was confirmed by fluorescence recovery after photobleaching (FRAP) experiments. Moreover, it was found that more than 30% of centrosomes possessed only green fluorescent centrioles 2 days after photoconversion [E13.5-E14.5(PC)-E16.5]. The appearance of the solely green fluorescent centrosomes 48 h after photoconversion indicates that the initially labelled radial glia progenitors have undergone two rounds of division during this period; this is consistent with the previous observation that the duration of the neocortical progenitor cell cycle is about 12 to 20 h around this developmental stage. The ongoing division of labelled radial glia cells at a normal rate suggests that expression of Kaede-CETN1 and the photoconversion procedure had no effect on their cell cycle. In addition, no obvious DNA damage or cell death was induced by the photoconversion treatment. Besides the green and yellow fluorescent centrosomes, about 4% of solely red fluorescent centrosomes were observed, indicating that a few labelled cells do not undergo cell division during this period (Wang, 2009).

Having successfully distinguished the centrosomes with differently aged mother centrioles in the developing neocortex in vivo, their distribution was examined to determine whether they are asymmetrically segregated. Remarkably, it was found that more than 76% of centrosomes with the new mother centriole (that is, only green fluorescent) were located in the IZ and the CP, whereas around 78% of centrosomes with the old mother centriole (that is, both green and red fluorescent) were located in the VZ in addition to the SVZ. These results demonstrate that the centrosomes with differently aged mother centrioles are asymmetrically segregated in the developing neocortex during the peak phase of neurogenesis. It is worth noting that a small fraction of both green and red (that is, yellow) fluorescent centrosomes was found in the IZ and the CP and that these centrosomes probably originated from the first cell cycle after photoconversion (Wang, 2009).

The asymmetric segregation of centrosomes suggests differential regulation of the duplicated centrosomes in dividing radial glia progenitors. To gather further evidence for this, time-lapse imaging experiments were carried out to monitor the behaviour of centrosomes with differently aged mother centrioles in dividing radial glia progenitors at the VZ surface in situ. Kaede-CETN1 was introduced into radial glia cells together with mPlum, a far-red fluorescent protein, to label cell morphology. Around 24 h later, cortical slices were prepared. Photoconversion of the existing Kaede-CETN1 proteins was then performed in individual slices, which were then cultured for another 24 h before being subjected to time-lapse imaging. Labelled dividing radial glia cells with enlarged and rounded cell bodies possessing a pair of centrosomes at the VZ surface were monitored at 10-min intervals over a period of 5 to 8 h. In six out of seven dividing radial glia cells that proceeded through mitosis at the VZ surface and reached the two-cell stage, the centrosome retaining the old mother centriole in both red and green fluorescence stayed at the VZ surface, whereas the centrosome containing the new mother centriole in solely green fluorescence migrated away from the VZ surface. These results demonstrate that the centrosomes with differently aged mother centrioles in dividing radial glia progenitors exhibit distinct behaviour during the peak phase of neurogenesis (Wang, 2009).

The distinct behaviour of the centrosomes suggests that they are differentially inherited by the two daughter cells embarking on different routes of fate specification and development. On the basis of their behaviour, it is postulated that the centrosome with the new mother centriole is largely inherited by differentiating cells, such as neurons, whereas the centrosome with the old mother centriole that remains located at the VZ is mostly inherited by radial glia progenitors. Indeed, it was found that 2 days after photoconversion [E13.5-E14.5(PC)-E16.5] the centrosomes with the new mother centriole, marked by green fluorescence alone, were mostly associated with cells expressing TUJ1, a differentiating neuronal marker, in the CP and the IZ. In contrast, the centrosomes that retained the old mother centriole in yellow (that is, both green and red) fluorescence at the VZ were largely associated with cells expressing Pax6, a radial glia progenitor marker. These results show that the centrosomes with differently aged mother centrioles in dividing radial glia cells are asymmetrically inherited by the two daughter cells: whereas the renewing radial glia progenitor inherits the centrosome with the old mother centriole, the differentiating daughter cell inherits the centrosome with the new mother centriole (Wang, 2009).

These data thus far show that centrosomes with differently aged mother centrioles are differentially inherited by the two daughter cells of asymmetrically dividing radial glia progenitors in the developing neocortex. Next, whether the selective inheritance of the centrosome with the old mature mother centriole by radial glia progenitors is necessary for their maintenance at the VZ was tested. Should this be the case, given that Nin is an essential component of the appendage/satellite structures specific to the mature centriole, it was predicted that removal of Nin, which prevents centriole maturation, would disrupt asymmetric segregation of centrosomes with differently aged mother centrioles and impair the maintenance of radial glia progenitors in the developing neocortex (Wang, 2009).

To test this, short hairpin RNA (shRNA) sequences were developed against Nin, that effectively suppressed its expression (Nin shRNAs). Consistent with the prediction, expression of Nin shRNA, but not control shRNA, disrupted asymmetric segregation of centrosomes with differently aged mother centrioles labelled with Kaede-CETN1 in the developing neocortex, suggesting that Nin is necessary for centriole maturation, thereby generating asymmetry between duplicated centrosomes. The presence of solely green fluorescent centrosomes in Nin shRNA-expressing cortices indicates that centrosome duplication and segregation and cell division are not severely affected by removal of Nin. More importantly, it was found that removal of Nin caused a premature depletion of cells from the VZ, where radial glia progenitors reside. This effect of Nin shRNAs correlated with their efficacy in suppressing Nin protein expression and was rescued by a shRNA-insensitive Nin plasmid, suggesting that the effect of the Nin shRNA is due to a specific depletion of the endogenous Nin protein. A similar reduction in cells in the VZ was observed when Nin expression was suppressed using small interfering RNA (siRNA) (Wang, 2009).

To characterize further the extent to which removal of Nin leads to a depletion of radial glia progenitors, the fate specification was examined of cells expressing either control or Nin shRNA. When compared with the control, expression of Nin shRNA led to a marked reduction in the percentage of cells positive for Pax6 and glutamate transporter (GLAST), two radial glia progenitor markers, and a significant increase in the percentage of cells positive for TUJ1, a differentiating neuronal marker. These results suggest that removal of Nin leads to a depletion of radial glia progenitors and a concomitant increase in differentiating neurons. Consistent with this, a significant reduction was observed in phospho-histone 3 (P-H3)-labelled mitotic cells at the VZ surface and a marked increase in cell cycle exit. No obvious change in the cleavage plane orientation of late stage mitotic cells at the VZ surface was observed (Wang, 2009).

Previous studies showed that the carboxy-terminus of Nin is responsible for its localization to the centriole and expression of this region displaces endogenous protein at the centriole. Interestingly, it was found that, similar to removal of Nin, expression of the carboxy-terminus of Nin (Nin-Cter) led to a premature depletion of radial glia progenitor cells from the VZ, suggesting that centriolar Nin is critical for maintaining radial glia progenitor cells in the VZ. Taken together, these results indicate that preferential inheritance of a centrosome containing the mature mother centriole is required for the maintenance of radial glia progenitors in the proliferative VZ of the developing neocortex (Wang, 2009).

The results presented in this study suggest that the centrosomes with differently aged centrioles in asymmetrically dividing radial glia progenitors exhibit different behaviour and are differentially inherited by the two daughter cells during the peak phase of mammalian neocortical neurogenesis. Whereas the centrosome with the less mature new mother centriole migrates away from the VZ surface and is largely inherited by differentiating cells, the centrosome with the more mature old mother centriole stays at the VZ surface and is predominantly inherited by renewing radial glia progenitors. Recently, asymmetric behaviour of centrosomes has been observed during asymmetric division of Drosophila male germline stem cells and neuroblasts. The findings of this study suggest that this type of asymmetric centrosome regulation may be a general feature of asymmetric cell division across species. Furthermore, the findings provide new insight into centrosome regulation in the developing mammalian neocortex, which has been linked to the pathogenesis of human microcephaly (Wang, 2009).

Centrosomes with differently aged mother centrioles differ in their protein composition and thereby in their biophysical properties, such as microtubule anchorage activity. In this study, it was found that Nin, an appendage/satellite-specific protein required for centriole maturation, localized differently to the duplicated centrosomes in radial glia progenitors in late mitosis. Notably, another appendage/satellite-specific protein cenexin was recently found to be asymmetrically localized to centrosomes in sister cells after mitosis; moreover, the cell receiving the more mature old mother centriole usually grew a primary cilium first (Anderson, 2009). The asymmetric inheritance of centrosomes with distinct biophysical properties may thereby differentially regulate the behaviour and development of the daughter cells that receive them. For example, given that primary cilia have essential roles in a number of signal transduction pathways, including Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signalling, the asynchrony in cilium formation could differentially influence the ability of the two daughter cells to respond to environmental signals and thereby their behaviour and fate specification. Furthermore, the strong microtubule anchorage activity associated with the centrosome retaining the older mother centriole would facilitate its anchorage to a specific site (for example, the VZ surface), thereby tethering the cell that inherits it. Indeed, it was found that disruption of centriole maturation by removing Nin not only impairs asymmetric segregation of centrosomes, but also depletes radial glia progenitors from the VZ, a proliferative niche in the developing mammalian neocortex. Aside from their participation in microtubule organization and ciliogenesis, centrosomes associate with messenger RNAs (mRNAs) and membrane-bound organelles such as the Golgi and recycling endosomes and regulate protein degradation, thereby raising the possibility that asymmetric centrosome inheritance might contribute to proper segregation of cell fate determinants to the two daughter cells of asymmetrically dividing progenitor/stem cells (Wang, 2009).

Drosophila neuroblasts retain the daughter centrosome

During asymmetric mitosis, both in male Drosophila germline stem cells and in mouse embryo neural progenitors, the mother centrosome is retained by the self-renewed cell; hence suggesting that mother centrosome inheritance might contribute to stemness. This hypothesis was tested in Drosophila neuroblasts (NBs) tracing photo converted centrioles and a daughter-centriole-specific marker generated by cloning the Drosophila homologue of human Centrobin. This study shows that upon asymmetric mitosis, the mother centrosome is inherited by the differentiating daughter cell. These results demonstrate maturation-dependent centrosome fate in Drosophila NBs and that the stemness properties of these cells are not linked to mother centrosome inheritance (Januschke, 2011).

The correlation between mother versus daughter centrosome segregation and asymmetric cell fate was first documented in Drosophila male germline stem cells (mGSCs), in which the mother centrosome is anchored near the niche and is retained by the stem cell, whereas the daughter centrosome migrates to the opposite side of the cell and is segregated into the differentiating gonial cell after mitosis (Yamashita, 2007). More recently, mother centrosome retention has also been demonstrated during asymmetric mitosis in radial glia progenitors in the developing mouse neocortex. In Drosophila mGSCs, the possible functional relevance of such stereotyped centrosome behaviour is still unclear, although tantalizing hypotheses have been put forward. In the developing mouse neocortex, centriole maturation has been shown to be required for maintaining radial glia progenitors (Januschke, 2011 and references therein).

Asymmetric centrosome behaviour has also been documented in Drosophila neuroblasts (NBs). NBs are stem-cell-like precursors that generate the fly's central nervous system through a series of asymmetric divisions giving rise to a self-renewed NB and a differentiating ganglion mother cell (GMC), which typically divides only once into a pair of neurons or glia. NB asymmetric division is largely driven by cortical polarity. It starts by the apical accumulation of a number of protein complexes including the Par complex (Par-3, Par-6, aPKC) as well as Mushroom body defect, recruited by Partner of Inscuteable, which associates with Gαi, Inscuteable and Par-3. Apical complexes control the clustering at the basal cortex of cell fate determinants, such as Numb, Prospero and Brain Tumour, through their adaptor proteins Partner of Numb and Miranda. The apical cortex also controls the orientation of the mitotic spindle along the apico-basal axis, which is essential to position the cytokinesis furrow, such that cleavage segregates the apical and basal sides of the cortex into the newly formed NB and GMC, respectively. NB asymmetric division is also controlled by other auxiliary modulators (Januschke, 2011 and references therein).

Mosaic clones mutant for any of the known cell fate determinants overgrow in the larval brain and develop as malignant neoplasms upon transplantation into wild-type adult flies, showing that correct execution of the asymmetric division programme in Drosophila NBs is crucial to prevent tumour growth. Centrosomes are important in this process and larval brains that have cells without centrosomes or with more than two centrosomes per cell are highly prone to developing tumours (Januschke, 2011 and references therein).

NB asymmetry is not limited to cortical polarity at mitosis. Early in interphase, the NB centrosome splits in two, which display significant structural and functional differences. One of the resulting centrosomes retains most of the pericentriolar material (PCM) and remains at the apical cortex organizing the main microtubule network. The other centrosome has little, if any, PCM and microtubule organizing activity, and migrates extensively across the cytoplasm for most of the interphase. At mitosis, the apical centrosome is retained by the NB and the other centrosome is inherited by the GMC. Remarkably, at the time of splitting, the GMC-fated centrosome does contain a significant amount of PCM, as revealed by centrosomin fused to GFP which is shed off just before it starts to move. These centrosome asymmetries are thought to have a main role in the orientation of cortical polarity and mitotic spindle alignment (Januschke, 2011 and references therein).

Unlike centrioles in vertebrate cells where centriolar satellites, appendages and cartwheel span correlate with centriole maturation, no ultrastructural dimorphism has been reported between mother and daughter centrioles in Drosophila. Mother or daughter centriole-specific molecular markers are also lacking in Drosophila. Consequently, it is still unclear if the conspicuously unequal size, activity and fate of Drosophila NBs' centrosomes correlate with centriole maturation. It is also unclear if at the time of splitting each centrosome contains a full diplosome or a single centriole, as is the case in Drosophila embryos and in some human cell lines (Januschke, 2011 and references therein).

This study shows by tracing photo-converted centrioles that centrosome splitting occurs before centriole duplication and that the centrosome that loses PCM is motile during interphase. This centrosome is also inherited by the differentiating GMC and contains the mother centriole. The cloning of the Drosophila homologue of Centrobin is also reported. In vertebrate cell lines, Centrobin specifically accumulates on the daughter centriole. It was found that at the time of centrosome splitting, a yellow fluorescent protein (YFP) fusion to CNB stays on the centrosome that remains at the apical cortex, which is eventually inherited by the NB, and is excluded from the centrosome that breaks away and is fated to the GMC. From these observations, it is concluded that centrosome maturation and fate are tightly correlated during asymmetric mitosis in Drosophila NBs, in which the cell that retains stemness inherits the daughter centriole. It is also concluded that in spite of the lack of ultrastructural dimorphism, molecular dimorphism exists between mother and daughter Drosophila centrioles (Januschke, 2011).

To investigate if mother-versus-daughter centriole differences determine centriole fate during NB asymmetric division, a direct approach was devised based on photo-conversion of a pancentriolar marker in the apical centrosome. There are two expected outcomes of such an experiment (see Photo-conversion of a centriolar marker in the apical centrosome). If at the time of photo-conversion, once the basal centrosome has moved away, the apical centrosome contains two centrioles, then, in the next cell cycle, both the apical and basal centrosomes will carry the converted signal). Maturation-dependent centrosome fate will become apparent in the following cycle by the consistent segregation of the converted signal, which traces the mother centriole to either the apical centrosome, which is retained in the NB, or the basal centrosome, which is fated to the GMC. Alternatively, if at the time of photo-conversion the apical centrosome contains only one centriole, the hypothetical maturation-dependent centrosome fate will be revealed in the next cell cycle when, upon centrosome splitting, only the apical or the basal centrosome will carry converted signal. In such case, the mother centriole will be systematically fated to either the NB or the GMC, respectively (Januschke, 2011).

A time-lapse series including the most representative time points of a centrosome photo-conversion experiment using the pancentriolar marker PACT-d2Eos is presented. After the basal centrosome has started to move away from the apical centrosome, a pulse of 405 nm light is shed on a manually defined rectangular region of interest which overlaps and slightly exceeds the size of the apical centrosome, causing the photo-conversion of the centriole marker from green to red. The converted signal remains stably associated with the apical centrosome, which is retained by the NB after the ensuing mitosis, and migrates to the apical cortex in early interphase. When the basal centrosome breaks away and starts its characteristic migration through the cytoplasm, the photo-converted signal, which traces the mother centriole, goes with it and is subsequently delivered into the GMC at mitosis. Identical results were obtained in a total of 20 photo-conversion experiments (Januschke, 2011).

Three main conclusions can be derived from these observations. First, at the time when apical and basal centrosomes split in Drosophila NBs, the non-motile apical centrosome contains only one centriole. Second, segregation of mother and daughter centrosomes is highly correlated with asymmetric cell fate. Finally, these results show that NBs retain the daughter rather than the mother centriole (Januschke, 2011).

To independently corroborate these conclusions, it was decided to circumvent the current lack of molecular markers by cloning the Drosophila homologue of the human daughter-centriole-specific protein Centrobin (Jeong, 2007; Zou, 2005). By homology search, open reading frame CG5690, predicted to encode a 78.7 kDa protein of 689 aa (Flybase), was identified as the putative Drosophila centrobin (cnb) gene. Transgenic flies were generated constitutively co-expressing YFP-CNB, and a fusion between mKATE and the pancentriolar marker ASL47, and the localization of both fluorescent probes was followed in larval NBs. Although, as reported for other centriolar proteins, one or two randomly located YFP-CNB aggregates are often found in these cells, a clear pattern of YFP-CNB signal segregation emerges. Centrosome-bound YFP-CNB can be observed without interruption from telophase, up to the time of centrosome splitting, early in the following interphase. Then, when apical and basal centrosomes break apart, YFP-CNB remains bound to the apical non-motile centrosome, and cannot be detected over the migrating basal centrosome. Identical results were obtained in a total of 16 NBs (Januschke, 2011).

Retention of the daughter-centriole marker YFP-CNB by the apical centrosome and the lack of YFP-CNB signal in the motile centrosome corroborates further the conclusions derived from the photo-conversion experiments. In addition, the age-dependent centriole-specific localization of YFP-CNB provides the first instance of molecular asymmetry between mother and daughter Drosophila centrioles (Januschke, 2011).

Tracing mother and daughter centrioles with photo-converted PACT-d2Eos and YFP-CNB, respectively, it was shown that apical and basal Drosophila NB centrosomes do not have fully formed diplosomes when they split, and that mother-versus-daughter centriole segregation tightly correlates with cell fate; contrary to what has been generally assumed, the daughter centrosome is fated to be retained by the NB and the mother centrosome is fated to the GMC (Januschke, 2011).

The functional relevance of specific centrosome retention by the stem cell in Drosophila remains to be ascertained. Both in mGSCs and in NBs, a microtubule-dependent mechanism hooks one centrosome to the region of the cortex, proximal to the hub in the germ line and apical in NBs38; that is, retained by the stem cell after mitosis. Preferential retention of the mother centrosome in the germ line might therefore simply be the inescapable consequence of carrying more PCM and having greater microtubule-organizing activity. This is less likely in NBs where the daughter centriole is retained, while the mother centriole breaks away carrying little, if any, PCM. That implies the removal of PCM from around the mother centriole and the retention of PCM by the daughter centriole. The shedding of PCM by the GMC-fated centrosome as it splits from the apical centrosome has been previously documented (Januschke, 2011).

Precedence for centrosome transmitted cell fate information exist in other species, but not in Drosophila. There are, however, published data on the effect of switching centrosome fate by transient microtubule depolymerization. Upon disassembly of the interphase aster of the NB by treatment with microtubule poisons, the apical centrosome loses connection with the cortex and moves deeper inside the cell so that at mitosis onset, the position of both centrosomes is essentially randomized. Upon recovery of microtubule dynamics, mitosis resumes and asymmetric cell division takes place. In some cases, centrosome fate is switched so that the centrosome originally destined to the GMC is retained by the NB, and vice versa, yet mitosis generates an NB and a GMC that are morphologically normal, strongly suggesting that the identity of the resulting NB and GMC is not severely compromised. It is unknown, however, whether such switch in centriole fate might have long-term developmental consequences (Januschke, 2011).

Centriole-maturation-dependent segregation in Drosophila NBs is somewhat reminiscent of spindle pole body (SPB) segregation in budding yeast. Similar to centrioles in animals, the SPB duplicates once per cell cycle. The 'new' SPB remains connected to the 'old' one by the bridge, which is cleaved in S phase. The two SPBs then separate to form the opposite poles of the spindle. In unperturbed cells, the 'old' SPB always migrates into the bud. 'Old' and 'new' SPBs are also functionally and biochemically distinct. For instance, the Bfa1p-Bub2p GTPase-activating protein complex, which is an integral part of the spindle position checkpoint, specifically binds to the 'old' SPB. The SPC inhibits the mitotic exit network) until the nucleus has migrated into the bud. Also the antigen-presenting cell-related molecule Kar9 localizes only to the 'old' SPB due to the activity of the 'new' SPB-resident Clb4/Cdc28 kinase. Interestingly, SPB fate can also be artificially switched, in which case, Bfa1p still localizes to the SPB that enters the bud independently of whether it is the 'old' or the 'new'. These data show that in budding yeast biochemical differences between the 'old' and 'new' SPBs are functionally relevant (Januschke, 2011 and references therein).

As in yeast where the 'old' SPB is inherited by the long-lived bud, the mother centriole in Drosophila is inherited in testes by the mGSC, which is the most long-lived of the two mGSC daughters, and in larval brains by the GMC whose daughters outlive the NB. Thus, what appears to be a diametrically different behaviour between mGSCs and NBs using stemness as criteria is consistent, when the expected lifespan of each cell type is considered (Januschke, 2011).

Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis

bHLH-O proteins are a subfamily of the basic-helix-loop-helix transcription factors characterized by an 'Orange' protein-protein interaction domain. Typical members are the Hairy/E(spl), or Hes, proteins, well studied in their ability, among others, to suppress neuronal differentiation in both invertebrates and vertebrates. Hes proteins are often effectors of Notch signalling. In vertebrates, another bHLH-O protein group, the Hey proteins, have also been shown to be Notch targets and to interact with Hes. The single Drosophila Hey orthologue is primarily expressed in a subset of newly born neurons that receive Notch signalling during their birth. Unlike in vertebrates, however, Hey is not expressed in precursor cells and does not block neuronal differentiation. It rather promotes one of two alternative fates that sibling neurons adopt at birth. Although in the majority of cases Hey is a Notch target, it is also expressed independently of Notch in some lineages, most notably the larval mushroom body. The availability of Hey as a Notch readout has allowed the study of Notch signalling during the genesis of secondary neurons in the larval central nervous system (Monastirioti, 2010).

Among the superfamily of basic-helix-loop-helix (bHLH) transcription factors, several structurally distinct classes are discerned. One of these, the bHLH-Orange (bHLH-O) class, is characterized by the 'Orange' domain, a protein interaction domain perhaps serving as an extended dimerization surface. bHLH-O proteins are important developmental and physiological regulators in processes ranging from neurogenesis to circadian rhythm control (Monastirioti, 2010).

In a number of invertebrate and vertebrate species, bHLH-O repressors are known to inhibit neural differentiation. In Drosophila, the seven E(spl) bHLH-O proteins are expressed in the neuroectoderm, where they inhibit cells from differentiating as neuroblasts (NBs). In vertebrates, a number of Hes bHLH-O proteins, notably Hes1, Hes3 and Hes5 in the mouse, are also expressed in the neuroectoderm; in this case it is the neural stem cells that express the Hes genes, which are subsequently downregulated in the differentiating neuronal progeny. Triple Hes1, Hes3, Hes5 knock-out causes premature neural differentiation, disruption of the neuroepithelium and a hypoplastic nervous system owing to stem cell depletion. In Drosophila, loss of the entire E(spl) locus results in supernumerary NB specification from the neuroectoderm and a hyperplastic nervous system. Despite these differences, owing to the different mode of neural precursor specification between vertebrates and insects, the generalization can be made that E(spl)/Hes proteins antagonize neuronal differentiation. At most developmental settings across metazoan phylogeny, neural expression of E(spl)/Hes genes is a direct response to Notch signalling (Monastirioti, 2010).

Expression of another subfamily of bHLH-O genes has been detected in the progenitor cell zones of the developing vertebrate central nervous system (CNS). These genes encode the three Hey proteins, so named after a characteristic tyrosine residue in their C-terminal domain (Hairy/enhancer-of-split like with a Y); they are also known as Hrt, Herp, Hesr, Chf or Gridlock. Although neural defects are minor in Hey knock-out mice, overexpression studies have suggested that Hey and Hes proteins might synergize with each other in suppressing neural differentiation and maintaining the neural stem cell fate. Hey1 has even been linked to the pathogenesis and aggressiveness of gliomas. Hey knock-out mice have highlighted their roles in developmental processes outside the nervous system, in particular, heart and vasculature development. In these contexts, all three mammalian Hey genes appear to respond to Notch signalling, similar to E(spl)/Hes genes in neurogenesis. Biochemical data support Hes-Hey heterodimer formation, raising the possibility that these two subclasses of bHLH-O proteins might synergize in some developmental contexts as Notch effectors (Monastirioti, 2010).

The Drosophila genome contains a single Hey orthologue (Kokubo, 1999), which had not been studied to date. This study characterized it in the hope of better understanding the process of neural precursor specification, based on the assumption that, by analogy to vertebrates, Hey might display protein-protein interactions with E(spl). Surprisingly, Hey was not co-expressed with the E(spl) proteins in the neuroectoderm, rather was restricted to differentiating neurons, suggesting a radically different role in neurogenesis than was assumed. Once NBs are specified in Drosophila, they undergo cycles of asymmetric cell divisions that give rise to a secondary precursor, called a ganglion mother cell (GMC), in addition to self-renewing. GMCs divide once to give rise to two neurons or, less often, glia. The majority of GMC divisions are asymmetric, with the fates of the two daughters dictated by unequal levels of Notch signalling. The 'A' sibling neuron requires high Notch signalling, whereas the 'B' sibling neuron downregulates Notch reception, which is usually achieved by asymmetric segregation of a Notch inhibitor, Numb, into the nascent 'B' neuron. This study describes a complex pattern of Hey expression in relation to these divisions during both neurogenic phases of the animal, early embryogenesis and larval life, where thousands of new neurons are added to generate the adult CNS. In all sibling pairs that were identified, Hey was expressed in the 'A' neuron. Genetic analysis confirmed that Hey is a Notch target gene in most instances. These results extend the Hey-Notch relationship to Drosophila in support of an ancient connection between bHLH-O genes and Notch activity and, for the first time, implicate a bHLH-O protein in the process of GMC asymmetric division (Monastirioti, 2010).

A full-length Hey cDNA was amplified from a Drosophila cDNA library, which was used as a probe for in situ hybridization, and for cloning in prokaryotic expression vectors. Bacterially expressed full-length Hey protein was used to raise anti-Hey antibodies. There were no obvious differences between the RNA and protein patterns. Hey protein showed nuclear accumulation, as expected for a transcription factor, and was primarily detected in a segmentally repeated pattern within the CNS starting at stage 10. Later, more Hey-positive cells gradually appear in the CNS. The neuroectodermal epithelium, where the related E(spl) bHLH-O proteins are expressed already starting at stage 8, is devoid of Hey expression, which instead is detected at deeper levels overlapping with the GMC/immature neuron marker Pros. From double-staining with the neuronal antigen Elav it was clear that the vast majority of Hey-positive cells represent neurons rather than GMCs, confirmed as lack of colocalization with the NB/GMC marker Asense. Besides neurons, Hey expression was detected in a subset of Repo-positive glia of the CNS and peripheral nervous system (PNS). Of note, Eve staining, which was used to visualize particular neurons, also marks the dorsally located pericardial cells. No Hey immunoreactivity was detected within or near these heart precursors, contrary to the strong expression of mammalian Hey genes during cardiogenesis. Finally, a few Hey-positive cells per segment were detected in the embryonic PNS. Most of these were also neurons, by virtue of being Elav-positive, but were not characterized further (Monastirioti, 2010).

Lineage-specific markers were used to characterize Hey expression in more detail. One was Even skipped, which marks a subset of neurons: the aCC/pCC sibling pair, the RP2 motoneuron, the cluster of U motoneurons and the cluster of EL interneurons. Another was the AJ96-lacZ enhancer trap, which marks the MP2 precursor and its progeny, the dMP2/vMP2 neurons. With AJ96-lacZ, strong Hey accumulation was detected in vMP2 but not in dMP2. Weak Hey expression was detected shortly before mitosis of the MP2 progenitor during late stage 10. Among the Eve-positive neurons, pCC and the U neurons expressed Hey. aCC, RP2 and the EL neurons were Hey-negative. At stage 11, the sibling of RP2, RP2sib, a smaller cell, which only transiently expresses Eve, was Hey-positive. Hey expression in all these neurons appeared transient. For example, whereas immunoreactivity in vMP2 was strong at stage 12, it was downregulated and barely detectable by stage 14. Similarly, by stage 14 no Hey could be detected in pCC cells, although it was still expressed strongly in some of the later-born U motorneurons. Transient Hey expression was also observed in the two identical progeny of MP1, a midline precursor, which are marked by Odd (Monastirioti, 2010).

Most of the neurons described above belong to well-characterized lineages, in which sibling fates arise through differential Notch signalling. In each of the RP2/RP2sib, aCC/pCC and dMP2/vMP2 pairs, the second cell requires Notch signalling in order to acquire the 'A' fate, distinct from that of its sibling cell ('B' fate). Also in the U lineages, which arise from sequential GMCs from neuroblast NB7-1, the U neurons require Notch, whereas their Eve-negative Usib neurons do not. All Notch-requiring cells, namely RP2sib, pCC, vMP2 and the U cells, robustly express Hey, whereas none of their 'B'-fate siblings do so. This raises the possibility that Hey is expressed in response to Notch (Monastirioti, 2010).

Thus Hey was detected almost exclusively in the CNS in young postmitotic neurons and glia, specifically those that receive a Notch signal at birth. It has long been appreciated that Notch signalling plays an important role in the acquisition of neuronal/glial cell fate after GMC division, with most GMCs producing two different progeny, an 'A' cell with high Notch activity and a 'B' cell with no Notch activity. Still, no Notch target genes had been identified in this process. This study shows that Hey is such a target gene in many, and perhaps all, GMC asymmetric divisions. These conclusions are based on the expression pattern of Hey, its response to Notch pathway perturbation and on the ability of ectopic Hey to block development of RP2 and dMP2, two 'B'-type neurons (Monastirioti, 2010).

Although these is good evidence that Hey expression can recapitulate the effect of Notch signalling, Hey loss-of-function has only a mild phenotype. The trivial possibility that the transposon insertion allele used has residual activity is unlikely as (1) no Hey protein is detectable in homozygous mutants and (2) the Hey^f06656 allele results in recessive lethality. Nevertheless, the issue will be permanently decided with the generation and analysis of more Hey alleles. The alternative hypothesis, which seems more probable, is that one or more additional factors besides Hey can also act as nuclear effectors downstream of Notch in the 'A' GMC progeny. No Hey paralogues exist in the D. melanogaster genome, but structurally divergent proteins, even outside the bHLH-O family, could share similar functional characteristics. At the moment, there are no good candidate for such a factor; however, a number of bHLH-O factors have been excluded that do not seem to be co-expressed with Hey in neurons, namely E(spl)mγ and m8, Hairy and Dpn (Monastirioti, 2010).

Besides GMCs, a number of other neural progenitors, namely NBs, sensory organ precursors (SOPs) and SOP progeny cells, all undergo asymmetric cell divisions with Notch involvement. No Hey expression was detected in either the NB/GMC pair or in the SOP progeny cells of external sensory organs, suggesting that Hey expression is turned on exclusively in GMC asymmetric divisions. Hey-positive glia could also be the progeny of asymmetrically dividing GMCs. It is yet unclear which cells might be the immediate progenitors of the few Hey-positive PNS neurons (Monastirioti, 2010).

Until the present work and the recent paper by Krejci (2009), the only Drosophila bHLH-O genes known to be targets of Notch were the seven of the E(spl) complex. Hey and two other bHLH-O genes, dpn and Her, had been predicted as candidate Notch targets based on nearby clustering of putative Su(H) binding sites, the DNA elements via which activated Notch is tethered to its target genes. Although HES-related (Her) does not seem to be a true Notch target have shown that dpn is a Notch target in the muscle-progenitor-like Drosophila DmD8 cell line; an in vivo context for such a response has yet to be determined. Together with Hey, this makes a total of 9 out of 13 bHLH-O genes in the Drosophila genome which are regulated by Notch. It should be stressed that Notch has a number of additional (non-bHLH-O) targets, depending on the species and cellular context, but few, if any, show such widespread association as the bHLH-O genes. The latter are activated by Notch in a multitude of unrelated contexts, such as neuroectoderm, mesoderm, wing epithelium, leg segmentation and now GMC asymmetric cell divisions in Drosophila, and in neural progenitors, presomitic mesoderm, cardiogenesis and vasculogenesis in vertebrates (Monastirioti, 2010 and references therein).

In addition to its widespread Notch-dependent expression, this study detected a clear instance of Notch-independent expression of Hey within the GMCs and neurons of the MB precursors. Other examples where Hey expression does not correlate with known events of Notch signalling are the MP2 NB and the two MP1 midline neurons. It is also clear that in embryos with severe Notch signalling defects, a small number of Hey-positive cells is still seen in the CNS, suggesting that there are additional neural lineages, where Hey is likely to be expressed independently of Notch. Analysis of the cis regulatory regions of Hey should shed light on Notch-dependent and Notch-independent enhancer elements (Monastirioti, 2010).

The bHLH-O family has undergone considerable diversification during evolution. Although sequence analysis can unambiguously assign genes to this family, it cannot identify orthologues in distantly related species. A classic example is the Drosophila to mammals comparison, where no clear orthologue relationships exist between Hairy, Dpn and the seven E(spl) in Drosophila and Hes1, 2, 3, 5, 6 and 7 in mammals, suggesting that the diversification of these proteins occurred separately after divergence of protostomes and deuterostomes. Hey proteins are the singular exception, being particularly well conserved. The bHLH domain of Drosophila Hey shows 97-98% similarity to that of its mammalian counterparts. This might lead one to expect substantial conservation of Hey function, which, strangely enough, was not observed (Monastirioti, 2010).

First, mammalian Hey genes have a very broad expression pattern, including presomitic mesoderm, embryonic heart, vascular precursors, developing brain and spinal cord, neural crest etc (Kokubo, 1999; Leimeister, 1999). Fly Hey, by contrast, seems confined within the CNS and PNS. Although there is complexity in its expression, as documented in this study with its contextual Notch dependence/independence, the great majority of its expression pattern seems to be in the newly born Notch-dependent 'A'-type neurons. The absence of Hey expression from the developing Drosophila heart is most striking, given the foremost importance of Hey genes in vertebrate cardiogenesis. A second indicator of functional non-conservation comes from comparing the role of Hey within the nervous systems of mammals versus Drosophila. In the former, Hey has been proposed to act in the maintenance of progenitor fate and to antagonize neuronal differentiation, similar to Hes proteins. In fact, it has been proposed that Hey-Hes heterodimers mediate these effects. In the fly, Hey expression was not detected within progenitor cells, with the few rare GMC exceptions, noted above. Hey-E(spl) or Hey-Dpn co-expression could not be detected, although all seven E(spl) genes were not tested for lack of specific reporter lines. To overcome any doubt, functional tests were made by ectopically expressing Hey. Instead of suppressing sensory organ formation, it mildly increased the number of bristles, showing an opposite phenotype from that of E(spl) or hairy ectopic expression. It can therefore be confidently said that Hey does not antagonize neural differentiation in the fly (Monastirioti, 2010).

This leaves a puzzle of why Hey is so strongly conserved. Perhaps some yet uncharacterized molecular aspect of its role in chromatin recognition/transcriptional regulation is conserved, despite considerable diversification in cellular and developmental contexts. These contexts have diverged greatly between insects and vertebrates, the only unifying theme being their regulation by Notch signalling. A homologous function might be that of promoting gliogenesis, as Hey2 was shown to promote Müller glia formation in the murine retina. Further comparative studies encompassing more species will no doubt shed light on the function of this highly conserved bHLH-O protein (Monastirioti, 2010).

Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. This study analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways, including exocyst component Exo84, shibire, sec5, sec6, sec15, rab5, Hrs, Vps25 and erupted/TSG101. Surprisingly, none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner (Halbsgut, 2011).

The data strongly indicate that vesicle trafficking is not involved in polarization of NBs, in contrast to epithelia, where it is essential for polarity. In epithelial cells vesicle trafficking controls cell polarity mainly by regulating the levels of the transmembrane proteins Crumbs and DE-Cad at the membrane. In NBs no asymmetrically localized transmembrane protein has been described so far, except for one: Numb-interacting protein (NIP) is a multipass transmembrane protein that colocalizes with Numb at the basal cortex of dividing NBs. In Drosophila Schneider cells, Numb and NIP colocalize at the plasma membrane, and RNA interference–mediated knockdown of NIP results in a release of Numb from the plasma membrane. Whether NIP is required for proper localization of Numb in dividing NBs is not known since no null mutation in moladietz, the gene encoding NIP, is available. It has also not been studied whether Numb may be required for the asymmetric localization of NIP in NBs (Halbsgut, 2011).

So, how could cell polarity be established and maintained in NBs? Baz, PAR-6, and aPKC are all localized to the apical junctional region of the neuroectodermal epithelium at the time when NBs ingress from the epithelium. Thus the components of the PAR/aPKC complex are already apically enriched in NBs prior to their first division. It is furthermore known that Baz can associate with the plasma membrane by direct binding to phosphoinositide lipids. However, there is no evidence for an asymmetric distribution of phosphoinositides in NBs, which might cause the asymmetric localization of Baz. In analogy to the mechanism that operates in the Caenorhabditis elegans zygote, the hypothesis is favored that the apical localization of Baz is stabilized by a mutual repression mechanism involving phosphorylation of Baz by the basally localized kinase PAR-1 and phosphorylation of PAR-1 by aPKC. Although this mechanism may be sufficient to stably polarize an NB, extrinsic cues from adjacent neuroectodermal cells contribute to the positioning of the Baz crescent to the apical cortex (Halbsgut, 2011).

In conclusion, this work shows for the first time that cortical polarity in NBs can be established even when intracellular vesicular trafficking is blocked, in striking contrast to the situation in epithelia. Although it cannot be completely rule out that the lack of polarity phenotypes in NBs homozygous for the mutations that were analyzed may be due to the perdurance of the respective wild-type protein in the clones, this possibility is considered unlikely. It has been shown that the same mutations that were analyzed in NBs cause strong polarity phenotypes when clones are induced in epithelia. Furthermore, in some of the experiments perdurance cannot be ruled out, for example in the experiments using the shi¹ allele, and these also showed no polarity defects in NBs (Halbsgut, 2011).

These findings imply that the PAR/aPKC complex can function in different ways, to polarize only the cortex, as in NBs or the C. elegans zygote, or the cortex and the plasma membrane, as in epithelia and probably also in neurons. In the future it will be important to dissect these different mechanisms at the molecular level in order to understand the function of the PAR proteins in a specific cellular context (Halbsgut, 2011).

Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo

The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. This study found that the small GTPase Roughened/Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. The current linear model for polarity establishment was tested. Both Bazooka and aPKC regulate Canoe localization despite being 'downstream' of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data suggest that polarity establishment is regulated by a protein network rather than a linear pathway (Choi, 2013).

Polarity is a fundamental property of all cells, from polarized cell divisions in bacteria or fungi to the elaborate polarity of neurons. Among the most intensely studied forms of polarity in animal cells is epithelial apical-basal polarity. Polarity of epithelial sheets is key to their function as barriers between body compartments, and is also critical in collective cell migration and cell shape change during morphogenesis, as cytoskeletal and apical-basal polarity often go hand in hand. Loss of apical-basal polarity is a hallmark of metastasis. Significant advances have been made in defining the machinery required for cell polarity in many settings, but fundamental questions remain unanswered (Choi, 2013).

Cadherin-catenin complexes, which assemble into adherens junctions (AJs) near the apical end of the lateral cell interface, are critical polarity landmarks that define the boundary between apical and basolateral domains. Studies in C.elegans and Drosophila identified other key regulators of apical-basal polarity. In the textbook view, the apical domain is defined by the Par3/Par6/aPKC and Crumbs/Stardust(Pals1)/ PATJ complexes, while Scribble, Dlg, Lgl, and Par1 define the basolateral membrane (Choi, 2013).

Complex cross-regulatory interactions between apical and basolateral proteins maintain these mutually exclusive membrane territories. These proteins also regulate other types of polarity during morphogenesis; e.g., fly Par3 (Bazooka; Baz), aPKC, and AJ proteins are planar-polarized during fly convergent extension, thus regulating polarized cell movements (Choi, 2013).

Polarized cytoskeletal networks also play key roles in establishing and maintaining apical-basal and planar polarity. These networks are thought to be physically linked to apical junctional complexes. The earlier model suggesting that cadherin-catenin complexes link directly to actin via α-catenin is now viewed as over-simplified. Instead, different proteins are thought to mediate this connection in different tissues and at different times (Choi, 2013).

Among the linkers is Canoe (Cno)/Afadin, an actin-binding protein that binds transmembrane nectins via its PDZ domain. While originally hypothesized to be essential for cell adhesion, subsequent work supports a model in which afadin modulates adhesive and cytoskeletal machinery during cell migration in vitro and the complex events of mouse gastrulation. Afadin has two N-terminal Ras association domains for which the small GTPase Rap1 is the major binding partner, and Afadin and Rap1 are functionally linked in both flies and mice. Rap1, Cno, and the Rap1 GEF Dizzy/PDZGEF are all essential for maintaining effective linkage between AJs and the apical actomyosin cytoskeleton during apical constriction of Drosophila mesodermal cells during fly gastrulation. Rap1 regulates Cno localization to the membrane. Cno plays a related role during convergent extension, though its role is planar polarized during this process. Cno also regulates collective cell migration, signaling, and oriented asymmetric divisions. The Rap1/Cno regulatory module is also important in disease, as Afadin and Rap1 are implicated in congenital disorders of the cardiovascular system and cancer metastasis. It remains unclear whether these diverse roles all involve junction-cytoskeletal linkage or whether some are independent functions (Choi, 2013 and references therein).

The small GTPase Rap1 plays diverse cellular roles. Mammalian Rap1 isoforms are perhaps best known for regulating integrin-based cell matrix adhesion, but Rap1 also regulates cell-cell AJs in both Drosophila and mice. In murine endothelial cells, for example, Rap1, its effector Krit1, and VE-cadherin form a complex that regulates endothelial cell junctions and stabilizes apical-basal polarity (Choi, 2013 and references therein).

In Drosophila imaginal disc cells, Rap1 regulates the symmetric distribution of DE-cadherin (DEcad) around the apical circumference of each cell. Rap1 carries out these functions via a diverse set of effector proteins, including Krit1, TIAM, RIAM, and Cno/Afadin. Thus, Rap1 and its effectors are candidate proteins for regulating interactions between AJs, polarity proteins and the cytoskeleton during polarity establishment and maintenance (Choi, 2013).

The early Drosophila embryo provides among the best models of establishing and maintaining apical-basal polarity. Flies start embryogenesis as a syncytium, with 13 rounds of nuclear division without cytokinesis. Membranes then simultaneously invaginate around each nucleus, forming ~6000 cells in a process known as cellularization. Prior to cellularization, the egg membrane is already polarized and serves as a polarity cue for underlying nuclei. This ultimately becomes the apical end of the new cells. Epithelial apical-basal polarity is initiated during cellularization. In the absence of cadherin-catenin complexes, cells form normally but then lose adhesion and polarity as gastrulation begins. These data and earlier work from cell culture suggested AJs are the initial apical cue. However, it was found that Bazooka (Baz)/Par3 acts upstream of AJs in this process. Strikingly, Baz and DEcad apically co-localize in spot AJs from cellularization onset. In the absence of Baz, DEcad loses its apical enrichment and redistributes all along the lateral membrane, while in the absence of AJ proteins, Baz remains apically localized, and a subset of cells retain residual apical-basal polarity, although cell shapes are highly abnormal. Cadherin-catenin and Baz complexes form independently before cellularization, and Baz then helps position DEcad in the apicolateral position where spot AJs will form. This placed Baz atop of the polarization network, raising the question of how it is positioned apically. Two cytoskeletal networks play important roles in initial Baz positioning (Choi, 2013).

Disrupting dynein led to Baz spreading along the lateral membrane, suggesting polarized transport along microtubules (MTs) plays a role. Depolymerizing actin also destabilized apical Baz, as did significantly overexpressing Baz, suggesting an actin-based scaffold with a saturable number of binding sites anchors Baz apically. While both actin and MTs are required for initial Baz polarization, they are not the only cues. Mislocalized Baz is re-recruited or re-stabilized apically at gastrulation onset if either initial cue is disrupted, suggesting a third cue perhaps involving aPKC/Par6 or Par1. Thus, the current model for initial establishment of apical-basal polarity involves a relatively simple pathway in which Baz is positioned apically, and then positions other apical polarity players. However, once initial polarity is established, events become more complex, with a network of mutually reinforcing and inhibitory interactions between apical and basolateral polarity complexes leading to polarity elaboration and maintenance. These were significant advances, but the proteins directing apical accumulation of Baz remained unknown. Work on apical constriction in the fly mesoderm, convergent extension during gastrulation, establishment of anteriorposterior polarity in one cell C. elegans embryos, and on apically constricting Drosophila amnioserosal cells, suggested that a complex network of interactions link AJs, the apical polarity proteins Baz and aPKC, and the actomyosin cytoskeleton. Recent work on Canoe and Rap1's roles in mesoderm apical constriction and convergent elongation (Sawyer, 2011) suggested they also fit into this network. These data led to an exploration of whether Rap1 and Cno play roles in initial apical positioning of AJs and Baz and thus in the establishment and early maintenance of polarity (Choi, 2013).

In regulating polarity establishment, Rap1 and Cno could act by several possible mechanisms. Their role in AJ positioning may be solely due to their effects on Baz localization, or alternatively Rap1 and Cno may independently affect the localization of both Baz and AJs. In the latter case, Cno may directly link AJs to the apical actin scaffold, as it was suggested to act in apical constriction. Rap1 and Cno also clearly regulate Baz positioning. Since Baz apical positioning requires an apical actin scaffold and dynein based MT transport, whether Rap1 and Cno act indirectly by regulating cytoskeletal organization was examined. However, the data suggest this is not the case: both the MT and actomyosin cytoskeletons appear normal in mutants. Thus the most likely model is that Rap1 and Cno are required for anchoring Baz apically. Consistent with this, when Cno was ectopically localized to artificial cell-cell contacts in cultured fly cells, it was able to recruit Baz to that site. This could occur directly, for example, by Cno binding Baz, or indirectly, via unknown intermediaries. Strikingly, however, when Baz was over-expressed in cellularizing embryos, presumably saturating its apical binding sites, it accumulated basolaterally and recruited DEcad but not Cno to these ectopic sites. Thus Cno and Baz do not co-localize obligatorily. It likely that each has multiple binding partners and that when pools are limiting, as Cno may be in this latter experiment, ectopic Baz cannot recruit Cno away from a preferred binding site. Of course, it remains possible that Cno and Rap1 also regulate Baz positioning through effects on MT transport or, given Cno's apical localization, unloading at an apical docking site. It will be important to test these possibilities. As is discussed in more detail below, it will also be important to define the Cno- and Rap1-independent mechanisms that partially restore apical Baz localization after gastrulation onset (Choi, 2013).

Since Rap1 is uniformly distributed along the apical-basal axis during cellularization, the most likely hypothesis is that it is locally activated apically by a GEF. A number of Rap1GEFs exist, many of which are conserved between mammals and flies. Recent work from the Reuter lab demonstrated that, like Cno and Rap1, the Rap1 GEF Dizzy (Dzy/PDZ-GEF) plays an important role in coordinated mesodermal apical constriction, suggesting it is the GEF acting upstream of Cno and Rap1 in that process. They also suggest that Rap1 and Dzy help regulate establishment of AJs. While similar in outline, their analysis of AJs differs from this one in detail, as they see strong effects on DEcad localization without similar effects on Arm. This is surprising, since these two proteins of the cadherin-catenin complex generally localize very similarly at the cortex. However, these differences aside, their data are consistent with Dzy acting with Cno and Rap1 in AJ establishment-it will be important to examine the effects of Dzy on Baz localization. It will also be important to determine how pre-existing egg membrane polarity is translated into localized Rap1 activity (Choi, 2013).

In addition to the parallel roles of Rap1 and Cno in regulating initial apical-basal polarization, this study identified a second role for Rap1 in establishing and maintaining columnar cell shape. The data suggest that this is partially or completely Cno-independent, and thus one of the many other Rap1 effectors may play a role in this process. It will be exciting to examine embryos mutant for other Rap1 effectors, such as Krit1/Bili, TIAM/Still life, RIAM/Pico, or RhoL to see if they are required for establishing columnar cell shape. baz and aPKC mutants also had defects in establishing columnar cell architecture. It is possible that each protein provides an independent mechanistic input into this process. This is consistent with the observed differences in the details of how columnar cell shape is disrupted, with Baz and aPKC primarily regulating apical cell area, while Rap1 affects cell shape at multiple apical-basal positions. A more speculative but perhaps less likely possibility is that Rap1 uses Baz and aPKC as effectors in establishing columnar cell shape. Fly Rap1 can form a complex with aPKC and Par6, and Rap1 acts upstream of cdc42/Par3/aPKC in regulating polarity of cultured neurons (Choi, 2013 and references therein).

Having identified Rap1's direct effector(s) in regulating cell shape, it is necessary to move downstream. Based on analogies with other epithelial tissues in fly development, it is hypothesized establishing columnar cell shape involves regulating apical tension. Other small GTPases play key roles in this; e.g., Rho and cdc42 have striking and opposing roles in apical tension regulation during fly eye development. In that context, Rho acts via separate effectors to maintain AJs and apical tension-it regulates tension via Rok, Diaphanous, and ultimately myosin contractility. It will be interesting to determine whether the defects in apical cell shape in the absence of Rap1, Baz, or aPKC also reflect unbalanced contractility in different nascent cells, and which contractility regulators are involved. However, for now, this is speculative (Choi, 2013).

Previous work has suggested a linear hierarchy regulating polarity establishment, with Baz at the top, positioning AJs and aPKC. The current work extends this hierarchy, positioning Rap1 and Cno upstream of Baz in this process. However, the data further suggest that viewing polarity establishment as a linear process is significantly over-simplified. It is now known that all of the relevant players -- including the AJ proteins, Baz, Cno and aPKC -- are at the cortex in syncytial embryos, prior to cellularization and the initiation of apical-basal polarity. This places them in position to cross-regulate one another. Consistent with this, the data suggest that viewing relationships with an 'upstream-downstream' point of view misses important reciprocal interactions that occur as polarity is established. Two examples point this out most clearly. First, earlier work suggested that localization of aPKC occurs 'downstream' of Baz, as apical positioning of aPKC at gastrulation onset requires Baz function. The new data reveal that Rap1 and Cno are, in turn, 'upstream' of Baz, and thus, if things work in a strictly linear fashion, Rap1 and Cno should be 'upstream' of aPKC. However, in contrast to this simple view, this study found that precise positioning of Cno during cellularization requires aPKC - in its absence, Cno is not cleared from the apical region, and the apical-basal cables of Cno at tricellular junctions are not properly assembled. In a similar fashion, Baz, which in a linear model is 'downstream' of Cno, also regulates precise positioning of Cno during cellularization. aPKC and Baz also play important roles in Cno localization during the early polarity maintenance phase beginning at gastrulation onset. Together, these data suggest that initial positioning of proteins along the apical-basal axis involves a network of protein interactions, similar to that previously suggested to regulate polarity elaboration during the extended germband phase and beyond, as cells develop the full suite of epithelial junctions. It will now be important to define mechanisms by which aPKC and Baz act to precisely position Cno: two broad possibilities are that they act on Cno directly, or that they modulate the fine scale architecture of the actin cytoskeleton, with indirect effects on Cno. It will also be exciting to determine if other polarity determinants, like the basolateral proteins Discs Large, Scribble or Lgl, or the basolateral kinase Par1 also play roles in polarity establishment, as they do in polarity maintenance. Consistent with this possibility, recent work from the Harris lab suggests Par1 is important for the gastrulation onset rescue of Baz localization in embryos in which early cues are disrupted. Finally, it will be interesting to identify the cues that come into play at gastrulation onset, which partially restore apical Baz localization, as part of the increasingly complex network of partially redundant regulatory cues that give polarity its robustness (Choi, 2013).

A NudE/14-3-3 pathway coordinates dynein and the kinesin Khc73 to position the mitotic spindle

Mitotic spindle position is controlled by interactions of cortical molecular motors with astral microtubules. In animal cells, Partner of Inscuteable (Pins) acts at the cortex to coordinate the activity of Dynein and Kinesin-73 (Khc73; KIF13B in mammals) to orient the spindle. Though the two motors move in opposite directions, their synergistic activity is required for robust Pins-mediated spindle orientation. This study identified a physical connection between Dynein and Khc73 that mediates cooperative spindle positioning. Khc73's motor and MBS domains link Pins to microtubule plus ends, while its stalk domain is necessary for Dynein activation and precise positioning of the spindle. A motif in the stalk domain binds, in a phospho-dependent manner, 14-3-3ζ, which dimerizes with 14-3-3ε. The 14-3-3ζ/ε heterodimer binds the Dynein adaptor NudE to complete the Dynein connection. The Khc73 stalk/14-3-3/NudE pathway defines a physical connection that coordinates the activities of multiple motor proteins to precisely position the spindle (Lu, 2013).

Mitotic spindle orientation requires the coordination of several pathways that act on astral microtubules. These pathways may establish cortical-microtubule connections and generate the forces necessary for movement of this large cellular structure with metaphase spindle lengths varying from 2 mm in yeast to 60 mm of a Xenopus single-cell stage. The spindle-orientation protein Pins has a domain that has been thought to capture microtubules (Pins^linker), and another that generates force (Pins^TPR). This study attempted to understand how these two pathways function together by taking advantage of an induced polarity system in cultured S2 cells in which the two pathways can be selectively activated. This system allowed for the identification of the Khc73 stalk domain as a critical element that links Pins^TPR and Pins^linker pathways. This observation was used as a platform for establishing a complete physical connection between the two pathways. This study has also clarified the role of 14-3-3 proteins in spindle orientation, establishing that their interaction with Pins is likely to be indirect (through Dlg and Khc73) (Lu, 2013).

Khc73 performs two functions in Pins-mediated spindle positioning. First, it functions in the Pins^linker pathway to mediate cortical microtubule capture through its MBS and motor domains, respectively. The N-terminal portion of Khc73 is sufficient for linker activity, which is likely occurring through a Dlg^GK/Khc73^MBS interaction at the cortex and a microtubule/ Khc73^motor interaction at the spindle. This suggests that Khc73's motor domain could function at the cortex by itself, however, Ed:Khc73^motor did not have spindle positioning activity, indicating that other factors could be required or the motor domain is not functional in this context (e.g., as a monomer with the coiled-coil stalk). Khc73 must therefore rely on Dlg as an adaptor to target it to the cortex, which is where it can potentially function to facilitate the initial contact of astral microtubules (Lu, 2013).

Although Khc73's MBS domain directly interacts with Dlg, Khc73 is not seen to colocalize with cortical Pins, even though Dlg robustly localizes to Pins crescents. Instead, the motor protein is seen distinctly at the ends of microtubule, suggesting that Khc73 moves to the plus ends where it may be poised for capture by the cortical Pins^linker/Dlg complex. Thus, Khc73's N-terminal domains are likely to facilitate cortical microtubule capture by linking microtubule plus ends to cortical Dlg (Lu, 2013).

In addition to facilitating cortical microtubule capture, this study found that Khc73 also forms a physical connection to the Pins^TPR/Mud/Dynein pathway with its stalk region, which is essential for the synergistic function of the two pathways. Khc73 may activate Dynein by delivering NudE to the cortex, where Dynein is presumably localized by Pins^TPR/Mud. Although it is not possible to observe the localization of Dynein in S2 cells for technical reasons, there is good evidence that it is cortically localized by way of Pins^TPR/Mud. In HeLa cells, Dynein localizes to the cortex with the mammalian homolog of Mud, NuMA, along with mPins, during mitosis (Lu, 2013).

It is proposed that a 14-3-3 motif in Khc73's stalk region activates an 'idling' cortically localized Dynein by cargoing NudE. Interestingly, although the Khc73 14-3-3 motif mutant Khc73^S1374A has a distribution of spindle-orientation angles that isn't random, the distribution is bimodal such that the spindle angles are either fully aligned or orthogonal to the polarity axis. The bimodal phenotype is distinct from the Khc73^motor+MBS fragment, which has a canonical intermediate distribution of spindle angles, suggesting that there may be additional regions or domains in the stalk that are contributing to the bimodal phenotype. It is hypothesized that an element within Khc73's stalk region is required for the proper application of the forces generated from by two motor proteins to properly orient the mitotic spindle. Nevertheless, biochemical and genetic studies demonstrate that the 14-3-3 binding motif is, at the very least, required for proper Pins-mediated spindle positioning and required for Khc73's interaction with the 14-3-3 proteins and NudE (Lu, 2013).

Pins mediates spindle positioning by coordinating two motor proteins that, as a pair, facilitate the cortical capture of microtubules and also provide pulling forces to robustly orient the mitotic spindle. A model is proposed in which orientation occurs through an ordered series of events, beginning with the initial polarization of Pins, followed by recruitment of Mud through its Pins^TPR domain and Dlg through Pins^linker region. Cortical Mud then recruits cytoplasmic Dynein, which is not yet active and will remain inert, but poised at the cortex. Khc73 localizes to the plus ends of microtubules, where it establishes cortical-microtubule contacts through direct binding to Dlg and also delivers NudE to cortical Dynein, thereby activating it. As astral microtubules enter the proximity of the Dynein complex, Dynein can generate specifically timed cortical pulling forces necessary for robust spindle positioning. Future work will be directed at dissecting the precise timing of these synergistic events that underlie differentiation and tissue architecture (Lu, 2013).

The conserved Discs-large binding partner Banderuola regulates asymmetric cell division in Drosophila

Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. To identify additional proteins required for ACD, an in vivo live imaging RNAi screen was carried out for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. Banderuola (Bnd / Wide awake) was identified an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. It is concluded that Bnd is a novel regulator of ACD in different types of cells. The data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution (Mauri, 2014).

Although most cell divisions are symmetric, some cells can divide asymmetrically into two daughter cells that assume different fates. During development, asymmetric cell division (ACD) allows specific cell types to be generated at precise locations relative to surrounding tissues. To achieve this, the axis of ACD needs to be coordinated with the architecture and polarity of the developing organism. Over the past years, a conserved protein machinery for ACD has been identified, but how this machinery connects to the organism architecture is less clear (Mauri, 2014).

The fruit fly Drosophila melanogaster is one of the best-understood model systems for ACD. In particular, the development of the Drosophila CNS and peripheral nervous system relies heavily on ACD and has contributed much to current understanding of this process. In the peripheral nervous system, external sensory (ES) organs are formed by two outer cells (hair and socket) and two inner cells (neuron and sheath). The four cell types arise from a single sensory organ precursor (SOP) cell, which divides asymmetrically into an anterior pIIb cell and a posterior pIIa cell. In a second round of ACD, pIIa and pIIb generate the outer or inner cells of the ES organ, respectively. The difference between pIIa and pIIb cells arises from different levels of Notch signaling in the two daughter cells. This difference is established by the asymmetric segregation of the Notch inhibitor Numb into the pIIb cell. Numb is known to regulate endocytosis, but how it inhibits Notch signaling is not precisely understood (Mauri, 2014).

In SOP cells, the polarity axis is coordinated with the anterior-posterior planar polarity axis of the overlying epithelium. Planar polarity involves the localization of mutually inhibitory components of a well-characterized machinery to the anterior or posterior plasma membrane. In SOP cells, the planar polarity protein Strabismus (Stbm) localizes to the anterior cortex and initiates the reorganization of plasma membrane domains to establish the axis of ACD. One of the most upstream events of this process is the recruitment of the membrane-associated guanylate kinase (MAGUK) Discs-large (Dlg) to the anterior cortex. This may involve a direct interaction of Dlg with the planar polarity protein Stbm. Dlg was originally identified as a tumor suppressor involved in the regulation of epithelial cell polarity and later shown to play a role in ACD and synaptogenesis. Despite its widespread functions, the biochemical pathways regulated by Dlg in those various cell types are not entirely understood (Mauri, 2014).

In SOP cells, Dlg associates with the adaptor protein Pins to direct the protein Bazooka (Baz) to the basal-posterior side of the dividing SOP cell. Together with Par-6 and aPKC, Baz forms the so-called Par protein complex that plays a pivotal role during ACD in many different cell types. Eventually, the kinase aPKC phosphorylates Numb, mediating its release from the posterior plasma membrane and thereby causing its accumulation to the anterior side (Mauri, 2014).

To ensure the asymmetric segregation of Numb to the anterior pIIb cell, the mitotic spindle has to be oriented along the polarity axis. This function is mediated by Pins through the binding of the microtubule binding protein Mushroom body defect (Mud), which forms a cortical attachment site for astral microtubules, aligning the spindle into the correct orientation. The binding to Pins requires the heterotrimeric G protein Gαi, which associates with Pins to mediate its recruitment to the anterior plasma membrane and switches it to an open conformation in which Pins can bind Mud (Mauri, 2014).

The same protein machinery directs ACD in neuroblasts, the stem cell-like progenitors of the Drosophila CNS. Neuroblasts divide asymmetrically into self-renewing daughter neuroblasts and smaller ganglion mother cells (GMCs) that generate two differentiating neurons through a terminal symmetric division. The asymmetric segregation of the cell fate determinants Numb, Prospero (Pros), and Brat into the GMC is required for proper differentiation. The asymmetric partitioning of Pros and Brat is mediated by the adaptor protein Miranda, and the asymmetric localization of both Miranda and Numb depends on phosphorylation by aPKC. Mutations in any of the three segregating determinants lead to the generation of excessive numbers of neuroblasts and ultimately cause the formation of lethal, transplantable brain tumors. As in SOP cells, Pins, Dlg, and Baz are required for ACD in neuroblasts, but they act in a characteristically different manner. First, neuroblast divisions are oriented along the apical-basal axis and not the planar polarity axis. Second, Pins, Dlg, and Baz colocalize apically in neuroblasts while they occupy opposite domains in SOP cells. In part, those differences can be explained by the recruitment of the adaptor protein Inscuteable (Insc) in the apical complex. Insc is not expressed in SOP cells, but in neuroblasts, it coordinates cortical polarity and spindle alignment by connecting Pins to Baz, ensuring the correct segregation of cell fate determinants in the differentiating daughter cell. In addition, Dlg has a neuroblast-specific role in mediating spindle orientation, acting downstream of Pins to align the spindle pole through the interaction with the kinesin motor Khc-73. Pins, Dlg, and Khc-73 also regulate a pathway called 'telophase rescue' that corrects ACD defects during late mitotic stages. This pathway realigns cortical polarity along the spindle axis independently of the Par complex through a Dlg cortical clustering mechanism to ensure that determinants eventually segregate asymmetrically and daughter cell fates are correctly specified. How Dlg performs those seemingly divergent roles in SOPs and neuroblasts is currently unclear (Mauri, 2014).

Because knowledge about ACD is evidently incomplete, several RNAi screens were performed to identify additional players required for the correct establishment of daughter cell fates. This study used the results from one of those screens to identify Banderuola (Bnd; CG45058, FlyBase name: Wide awake, Wake) Banderuola is a a weathervane in the form of a rooster. Bnd is a new key regulator of ACD that acts both in neuroblasts and in SOP cells. Baz, Pins, and Dlg are all mislocalized in bnd mutant SOP cells, placing Bnd at the top of the hierarchy for ACD. In bnd mutant neuroblasts, the asymmetric segregation of cell fate determinants is disrupted because aPKC and Dlg fail to accumulate apically. Importantly, Bnd interacts physically and genetically with Dlg, suggesting that it supports Dlg in performing its divergent functions in various cell types. Because Bnd is conserved in evolution, our data identify a new member of the universal machinery for ACD that might direct cell polarity in vertebrates as well (Mauri, 2014).

These results establish Bnd as a new component of the machinery for asymmetric cell division. bnd RNAi or loss-of-function mutations cause defects in the establishment of polarity and the positioning of the mitotic spindle in mitotic SOP cells. bnd was shown to be required for ACD and continued self-renewal activity in Drosophila larval neuroblasts. Because Bnd interacts both biochemically and genetically with the tumor suppressor protein Dlg, it is proposed that it exerts its function during ACD by regulating the function of Dlg. Moreover, the spindle rotation phenotype that were observed in mitotic SOP cells in bnd mutants is very similar to that of dlg^sw mutants, further strengthening the possibility that the two proteins are functionally connected. Because the mammalian homologs of these two proteins also interact, this function might be conserved in higher organisms as well (Mauri, 2014).

The process of ACD involves the establishment of a polarity axis, the orientation of the mitotic spindle, the polarized distribution of cell fate determinants, and, ultimately, the establishment of different daughter cell fates. In SOP cells, the axis of polarity is established when Dlg and Pins interact with components of the planar polarity pathway to concentrate anteriorly. Because Bnd binds to Dlg and is required for Pins and Dlg localization, but not for planar polarity, the data indicate that it acts at the very top of this hierarchy. Because Dlg is also mislocalized in bnd mutant neuroblasts, the role of Bnd in this tissue appears to be similar. Nevertheless, because the defect in asymmetry establishment is not completely penetrant, it is plausible that bnd function is partially redundant. Alternatively, it might also be that the residual protein derived from maternal contribution is sufficient to maintain, at least partially, the asymmetric partitioning of determinants. Further experiments will be needed to address these issues and clarify the instructive role of Bnd in establishing cell asymmetry (Mauri, 2014).

How could Bnd perform its function on a molecular level? Bnd::GFP localizes at the centrosomes, on the spindle, and, transiently, at the cell cortex. Because Bnd contains both Ankyrin repeats and an FN3 domain, it could mediate protein-protein interactions leading to the anterior localization of Dlg downstream of the PCP pathway. The localization of Dlg and Pins to the anterior side of dividing SOP cells is regulated by Strabismus (Stbm) and Dishevelled (Dsh). It is thought that Dsh excludes Dlg/Pins from the posterior side, whereas Stbm binds Dlg at the anterior cortex, promoting the association with Pins. This hypothesis is reinforced by the fact that Dlg interacts directly with the PDZ binding motif (PBM) of Stbm in Drosophila embryos. However, Pins is localized to the anterior cortex in stbm mutant SOP cells expressing a Stbm protein lacking the PBM domain. Hence, the localization of Dlg/Pins can be regulated independently of a direct binding to Stbm. It is tempting to speculate that Bnd could be a mediator between the PCP pathway and the establishment of the asymmetry axis in mitotic SOP cells (Mauri, 2014).

Alternatively, however, Bnd could also affect the function of Dlg and other cortical proteins through its RA domain. RA domains mediate binding to small GTPases and regulate their activity. Small GTPases are involved in the modification of the actomyosin network, and the establishment of polarity is influenced by myosin activity and by the contractility of the actomyosin mesh. In particular, Cdc42, a small GTPase of the Rho family, plays a central role in the establishment of polarity in a wide variety of biological contexts, including the localization of Par6/aPKC to the apical cortex of neuroblasts. More recent data have also implicated small Ras-like GTPases in regulating cortical polarity and spindle orientation. The Rap1/Rgl/Ral signaling network was shown to mediate those events through the regulation of the PDZ domain protein Canoe, which is a known binding partner of Pins. It is intriguing to hypothesize that Bnd could be part of a similar signaling network impinging on Dlg. Because the RA domain of Banderuola is not conserved in higher organisms, however, the first hypothesis is favored that rests on the conserved domains of the protein (ANK domains, FN3 domain, and Bnd motif). Hence, Bnd could act as an adaptor that mediates protein-protein interactions and regulates the function of binding partners such as Dlg (Mauri, 2014).

Why Bnd is also found at centrosomes and at the spindle is harder to explain. In fact, Bnd is the only known protein apart from Mud that localizes to both the centrosome and the cell cortex during ACD. It could help in promoting the alignment of the spindle through the interaction with the Pins/Gαi/Mud complex, but this cannot explain the entire phenotype because microtubules are not strictly required for polarity establishment during ACD. Although no biochemical interaction were detected between Bnd and Pins, Gαi, or Mud, this interaction could be transient, or it could depend on polymerized microtubules. It will be compelling to verify the localization of the endogenous protein because this would consolidate the data derived from the protein overexpression experiments. Furthermore, this could allow unraveling in detail the dynamics of Bnd cortical localization and its alignment with the SOP polarity axis, which could be concealed in overexpression conditions (Mauri, 2014).

In neuroblasts, Bnd is required for self-renewal and asymmetric protein segregation and has an antioncogenic function that is redundant with Dlg. In bnd mutants, defects were observed leading to neuroblast loss. The remaining neuroblasts are misshapen, displaying abnormalities in the asymmetric protein segregation and reduced mitotic activity. Although the FRT site remaining in the bnd mutants prevents addressing this question through a clonal analysis, the hypothesis is favored that the phenotype is cell autonomous and is due to premature differentiation of neuroblasts. Indeed, this is consistent with the phenotype in the SOP lineage because genetic manipulations resulting in a pIIa to pIIb transformation (like Numb overexpression or Notch loss of function) often cause neuroblasts to divide symmetrically into two differentiating daughter cells (Mauri, 2014).

The localization of both the basal determinants and Dlg itself are affected in bnd mutants. Dlg is known to mediate the basal localization of cell fate determinants in Drosophila neuroblasts. The abnormal localization of aPKC in bnd mutant neuroblasts could also be explained as an effect of dlg LOF because aPKC localization is affected in dlg mutants. Thus, the various protein mislocalization phenotypes in bnd mutant neuroblasts could be explained by a model in which Bnd exerts its function solely by localizing Dlg (Mauri, 2014).

The tumor phenotypes, on the other hand, suggest that the two genes act in parallel. Overproliferation phenotypes are observed only upon LOF of both genes, and bnd LOF enhances the dlg RNAi phenotype. In fact, this type of genetic interaction has been described for pins and lgl before: whereas pins mutant neuroblasts underproliferate due to self-renewal failure, pins lgl double mutants have a massive overproliferation of neuroblasts due to an aberrant self-renewal program triggered by aPKC. A similar mechanism could underlie the overproliferation was observed upon double RNAi of bnd and dlg. An alternative explanation for the double knockdown phenotype is provided by the additional role that Dlg has in the telophase rescue pathway, which might be independent from bnd. This pathway is known to mediate the establishment of Pins/Gαi cortical polarity, even in the absence of the Par complex, through a Dlg-dependent mechanism. The pathway is active in wild-type neuroblasts but becomes essential only when components of the apical Par complex are missing. It is possible that the telophase rescue pathway ensures the asymmetric segregation of cell fate determinants upon bnd RNAi. When dlg is inhibited as well, however, this pathway could be compromised, resulting in overproliferation and tumor formation (Mauri, 2014).

Dlg has four mammalian homologs. Like the Drosophila protein, they localize at the basolateral cortex in epithelia and have been shown to regulate cell polarity in various cell types. During rat astrocyte migration, for example, Dlg1 is required in association with APC for the polarization of the microtubule cytoskeleton at the leading edge of the migrating cell. Dlg-mediated polarity can be also considered a gatekeeper against tumor progression: Dlg1 is a target of oncoviral proteins and is often mislocalized or downregulated in late-stage tumors, implicating a causal connection between Dlg1 and cancer. As the interaction between Bnd and Dlg is conserved, Banderuola could be an evolutionarily conserved regulator of Dlg activity, and these studies may therefore be relevant for a variety of biological processes in higher organisms as well (Mauri, 2014).

Aurora A triggers Lgl cortical release during symmetric division to control planar spindle orientation

Mitotic spindle orientation is essential to control cell-fate specification and epithelial architecture. The tumor suppressor Lgl localizes to the basolateral cortex of epithelial cells, where it acts together with Dlg and Scrib to organize apicobasal polarity. Dlg and Scrib also control planar spindle orientation but how the organization of polarity complexes is adjusted to control symmetric division is largely unknown. Lgl redistribution during epithelial mitosis is reminiscent of asymmetric cell division, where it is proposed that Aurora A promotes aPKC activation to control the localization of Lgl and cell-fate determinants. This study shows that the Dlg complex is remodeled during Drosophila follicular epithelium cell division, when Lgl is released to the cytoplasm. Aurora A controlled Lgl localization directly, triggering its cortical release at early prophase in both epithelial and S2 cells. This relied on double phosphorylation within the putative aPKC phosphorylation site, which was required and sufficient for Lgl cortical release during mitosis and could be achieved by a combination of aPKC and Aurora A activities. Cortical retention of Lgl disrupted planar spindle orientation, but only when Lgl mutants that could bind Dlg were expressed. Taken together, Lgl mitotic cortical release is not specifically linked to the asymmetric segregation of fate determinants, and the study proposes that Aurora A activation breaks the Dlg/Lgl interaction to allow planar spindle orientation during symmetric division via the Pins (LGN)/Dlg pathway (Carvalho, 2015).

Evolutionarily conserved polarity complexes establish distinct membrane domains and the polarized assembly of junctions along the apicobasal axis has been extensively characterized. One general feature is that it relies on mutual antagonism between apical atypical protein kinase C (aPKC) and Crumbs complexes and a basolateral complex formed by Scribble (Scrib), Lethal giant larvae (Lgl), and Discs large (Dlg). This study used the Drosophila follicular epithelium as an epithelial polarity model to address how polarity is coordinated during symmetric division. Dlg and Scrib have been shown to provide a lateral cue for planar spindle orientation. Accordingly, Scrib and Dlg remain at the cortex during follicle cell division. In contrast, Lgl is released from the lateral cortex to the cytoplasm during mitosis. This subcellular reallocation begins during early prophase, since Lgl starts to be excluded from the cortex prior to cell rounding, one of the earliest mitotic events, and is completely cytoplasmic before nuclear envelope breakdown (NEB). Thus, the Dlg complex is remodeled at mitosis onset in epithelia (Carvalho, 2015).

The subcellular localization of Lgl is controlled by aPKC-mediated phosphorylation of a conserved motif, which blocks Lgl interaction with the apical cortex. To address the mechanism of cortical release during mitosis, nonphosphorytable form Lgl^3A-GFP was expressed in the follicular epithelium. Lgl^3A-GFP remains at the cortex throughout mitosis indicating that Lgl dynamics during epithelial mitosis also rely on the aPKC phosphorylation motif. Although the apical aPKC complex depolarizes during follicle cell division, Lgl cortical release precedes aPKC depolarization. Using Par-6-GFP as a marker for the aPKC complex and the Lgl cytoplasmic accumulation as readout of its cortical release, it was found that maximum cytoplasmic accumulation of Lgl occurs when most Par-6 is still apically localized (~70% relative to interphase levels). Thus, Lgl cortical release is the first event of the depolarization that characterizes follicle cell division, indicating that Lgl reallocation does not require extension of aPKC along the lateral cortex (Carvalho, 2015).

Although the major pools of Lgl and aPKC are segregated during interphase, Lgl has a dynamic cytoplasmic pool that rapidly exchanges with the cortex. Thus, further activation of aPKC at mitosis onset would be expected to shift the equilibrium toward cytoplasmic localization. Lgl dynamic redistribution in epithelia is similar to the neuroblast, where activation of Aurora A (AurA) leads to Par-6 phosphorylation and subsequent aPKC activation. To test whether a similar mechanism induced Lgl cortical release during epithelial mitosis, Lgl subcellular localization was analyzed in aPKC mutants and in par-6 mutants unphosphorylatable by AurA. Lgl cytoplasmic accumulation is unaffected in par-6; par-6^S34A mutant cells. Temperature-sensitive aPKC^ts/aPKC^k06403 mutants display strong cytoplasmic accumulation of Lgl during prophase, with a minor delay relatively to the wild-type). Moreover, homozygous mutant clones for null (aPKC^k06403) and kinase-defective (aPKC^psu141) alleles also display Lgl cortical release during mitosis. These results implicate that although aPKC activity may contribute for Lgl mitotic dynamics, the putative aPKC phosphorylation motif is under the control of a different kinase, which triggers Lgl cortical release in the absence of aPKC (Carvalho, 2015).

AurA is a good candidate to induce Lgl cortical release as it controls polarity during asymmetric division. Furthermore, Drosophila AurA is activated at the beginning of prophase, which coincides with the timing of Lgl cytoplasmic reallocation. To examine whether AurA controls Lgl dynamics in the follicular epithelium, homozygous mutant clones were generated for the kinase-defective allele aurA³⁷. In contrast to wild-type cells, only low amounts of cytoplasmic Lgl were detected during prophase in aurA³⁷ mutants, which display a pronounced delay in the cytoplasmic reallocation of Lgl during mitosis. This delayed cortical release of Lgl has been previously reported during asymmetric cell division in aurA³⁷ mutants, possibly resulting from residual kinase activity. Thus, AurA is essential to trigger Lgl cortical exclusion at epithelial mitosis onset (Carvalho, 2015).

The idea that Lgl mitotic reallocation is directly controlled by a mitotic kinase implies that Lgl should display similar dynamics regardless of the polarized status of the cell. Consistently, Lgl-GFP is also released from the cortex before NEB in nonpolarized Drosophila S2 cells. Furthermore, Lgl^3A-GFP is retained in the cortex during mitosis, revealing that Lgl cortical release is also phosphorylation dependent in S2 cells. Treatment with a specific AurA inhibitor (MLN8237), or with aurA RNAi, strongly impairs Lgl cortical release during prophase, as Lgl is present in the cortex at NEB. However, inhibition of AurA still allows later cortical exclusion, which could result from the activity of another kinase. Despite their distinct roles, AurA and Aurora B (AurB) phosphorylate common substrates in vitro. Therefore, whether AurB could act redundantly with AurA was analyzed. Inactivation of AurB with a specific inhibitor, Binucleine 2, enables normal Lgl cytoplasmic accumulation before NEB and still allows later cortical exclusion in cells treated simultaneously with the AurA inhibitor As AurB does not seem to participate on Lgl mitotic dynamics, RNAi directed against aPKC was used to examine whether it could act redundantly with AurA. aPKC depletion did not block Lgl cortical exclusion, but it was slightly delayed. However, simultaneous AurA inhibition and aPKC RNAi produced almost complete cortical retention of Lgl during mitosis. Thus, AurA induces Lgl release during early prophase, but aPKC retains its ability to phosphorylate Lgl during mitosis (Carvalho, 2015).

To address which serine(s) within the phosphorylation motif of Lgl control its dynamics during mitosis, individual and double mutants were enerated. As complete cortical release occurs before NEB, the ratio of cytoplasmic to cortical mean intensity of Lgl-GFP at NEB was quantified to compare each different mutant. All the single mutants displayed similar dynamics to Lgl^WT, exiting to the cytoplasm prior to NEB. In contrast, all double mutants were cortically retained during mitosis, indicating that double phosphorylation is both sufficient and required to efficiently block Lgl cortical localization (Carvalho, 2015).

The ability to doubly phosphorylate Lgl would explain how AurA drives Lgl cortical release. Accordingly, the sequence surrounding S656 perfectly matches AurA phosphorylation consensus, whereas the S664 surrounding sequence shows an exception in the -3 position. In contrast, the sequence surrounding S660 does not resemble AurA phosphorylation consensus, and AurA does not directly phosphorylate S660 in vitro as detected by phosphospecific antibodies against S660. That S656 is directly phosphorylated by recombinant AurA was confirmed in vitro using a phosphospecific antibody for S656. Moreover, AurA inhibition or aurA RNAi results in a similar cortical retention at NEB to Lgl^S656A,S664A, suggesting that AurA also controls S664 phosphorylation during mitosis, whereas aPKC would be the only kinase active on S660. Consistent with this, aPKC RNAi increases the cortical retention of Lgl^S656A,S664A, mimicking the localization of Lgl^3A. Furthermore, whereas S660A mutation does not significantly affect the cytoplasmic accumulation of Lgl in aPKC RNAi, S656A and S664A mutations disrupt Lgl cortical release in aPKC-depleted cells, leading to the degree of cortical retention of Lgl^S656A,S660A and Lgl^S660A,S664A, respectively. Altogether, these results support that AurA controls S656 and S664 and that these phosphorylations are partially redundant with aPKC phosphorylation to produce doubly phosphorylated Lgl, which is released from the cortex (Carvalho, 2015).

RNAi-mediated knockdown of Lgl in vertebrate HEK293 cells results in defective chromosome segregation. Furthermore, overexpressed Lgl-GFP shows a slight enrichment on the mitotic spindle suggesting that relocalization of Lgl could be important to control chromosome segregation. However, lgl mutant follicle cells assemble normal bipolar spindles, and although it was possible to detect minor defects on chromosome segregation, the mitotic timing (time between NEB and anaphase) is indistinguishable between lgl and wild-type cells. Additionally, loss of Lgl activity allows proper chromosome segregation in both Drosophila S2 cells and syncytial embryos. Thus, Lgl does not seem to have a general role in the control of faithful chromosome segregation in Drosophila (Carvalho, 2015).

Nevertheless, Lgl cortical release could per se play a mitotic function, as key mitotic events are controlled at the cortex. In fact, the orientation of cell division requires the precise connection between cortical attachment sites and astral microtubules, which relies on the plasma membrane associated protein Pins (vertebrate LGN). Pins uses its TPR repeat domain to bind Mud (vertebrate NUMA), which recruits the dynein complex to pull on astral microtubules, and its linker domain to interact with Dlg, which participates on the capture of microtubule plus ends. Notably, Pins/LGN localizes apically during interphase in Drosophila and vertebrate epithelia, being reallocated to the lateral cortex to orient cell division. Pins relocalization relies on aPKC in some epithelial tissues, but not in chick neuroepithelium and in the Drosophila follicular epithelium, where Dlg provides a polarity cue to restrict Pins to the lateral cortex. Dlg controls Pins localization during both asymmetric and symmetric division, and a recent study has shown that vertebrate Dlg1 recruits LGN to cortex via a direct interaction. However, Dlg uses the same phosphoserine binding region within its guanylate kinase (GUK) domain to interact with Pins/LGN and Lgl. Thus, maintenance of a cortical Dlg/Lgl complex during mitosis is expected to impair the ability of Dlg to bind Pins and control spindle orientation (Carvalho, 2015).

Interaction between the Dlg's GUK domain and Lgl requires phosphorylation of at least one serine within the aPKC phosphorylation site. Although the phosphorylation-dependent binding of Lgl to Dlg remains to be shown in Drosophila, crystallographic studies revealed that all residues directly involved in the interaction with p-Lgl are evolutionarily conserved from C. elegans to humans. Thus, whereas Lgl^3A does not form a fully functional Dlg/Lgl polarity complex, double mutants should bind Dlg's GUK domain and are significantly retained at the cortex during mitosis due to the inability to be double phosphorylated. This led to an examination of their ability to support epithelial polarization during interphase and to interfere with mitotic spindle orientation. Rescue experiments were performed in mosaic egg chambers containing lgl^27S3 null follicle cell clones. lgl mutant clones display multilayered cells with delocalization of aPKC. This phenotype is rescued by Lgl-GFP, but not by Lgl^3A-GFP. More importantly, in contrast to Lgl^S660A,S664A, which extends to the apical domain in wild-type cells and fails to rescue epithelial polarity in lgl mutant cells, Lgl^S656A,S660A and Lgl^S656A,S664A can rescue epithelial polarity, localizing with Dlg at the lateral cortex and below aPKC. Hence, aPKC-mediated phosphorylation of S660 or S664 is sufficient on its own to control epithelial polarity and to confine Lgl to the lateral cortex (Carvalho, 2015).

Whether exclusion of Lgl from the cortex and the consequent release from Dlg would be functionally relevant for oriented cell division was examined. Expression of Lgl-GFP or Lgl^3A-GFP does not affect planar spindle orientation during follicle cell division. In contrast, Lgl double mutants display metaphasic cells in which the spindle axis, determined by centrosome position, is nearly perpendicular to the epithelial layer. Live imaging revealed that these spindle orientation defects were maintained throughout division as it was possible to follow daughter cells separating along oblique and perpendicular angles to the epithelia. Moreover, equivalent defects on planar spindle orientation were detected upon expression of Lgl^S656A,S664A in the lgl or wild-type background, indicating that cortical retention of Lgl exerts a dominant effect. Interestingly, Lgl^S656A,S660A and Lgl^S656A,S664A induce higher randomization of angles, whereas Lgl^S660A,S664A, which is less efficiently restricted to the lateral cortex, produces a milder phenotype. Altogether, these results indicate that retention of Lgl at the lateral cortex disrupts planar spindle orientation only if Lgl can interact with Dlg (Carvalho, 2015).

Despite the ability of Lgl^S656A,S660A-GFP to rescue epithelial polarity in lgl mutants, strong overexpression of Lgl^S656A,S660A-GFP, but not of other Lgl double mutants, can dominantly disrupt epithelial polarity during the proliferative stages of oogenesis. One interpretation is that Lgl^S656A,S660A forms the most active lateral complex of the mutant transgenes, disrupting the balance between apical and lateral domains. Therefore whether the dominant effect of Lgl cortical retention on spindle orientation could solely result from Dlg mislocalization was assessed. Dlg is properly localized at the lateral cortex in Lgl^S656A,S660A-expressing cells presenting misoriented spindles, but this position does not correlate with the orientation of the centrosomes. Thus, cortical retention of Lgl interferes with Dlg's ability to transmit its lateral cue to instruct spindle orientation, which may result from an impairment of the Dlg/Pins interaction (Carvalho, 2015).

In conclusion, these findings outline a mechanism that explains how the lateral domain is remodeled to accomplish oriented epithelial cell division, unveiling that AurA has a central role in controlling the subcellular distribution of Lgl. AurA regulates the activity of aPKC at mitotic entry during asymmetric division, and these results are consistent with the ability of aPKC to phosphorylate and collaborate in Lgl cortical release. However, in epithelia, aPKC accumulates in the apical side during interphase, where it induces apical exclusion of Lgl, in part by generating a phosphorylated form that binds Dlg. Consequently, aPKC has a reduced access to the cortical pool of Lgl at mitotic entry and would be unable to rapidly induce Lgl cortical exclusion. These data show that cell-cycle-dependent activation of AurA removes Lgl from the lateral cortex through AurA's ability to control Lgl phosphorylation on S656 and S664 independently of aPKC. Thus, AurA and aPKC exert the spatiotemporal control of Lgl distribution to achieve unique cell polarity roles in distinct cell types (Carvalho, 2015).

It is proposed that release of Lgl from the cortex allows Dlg interaction with Pins to promote planar cell division in Drosophila epithelia. Lgl cortical release requires double phosphorylation, indicating that whereas Lgl-Dlg association involves aPKC phosphorylation, multiple phosphorylations break this interaction, acting as an off switch on Lgl-Dlg binding. Triple phosphomimetic Lgl mutants display weak interactions with Dlg, suggesting that multiple phosphorylations could directly block Lgl-Dlg interaction. Alternatively, the negative charge of two phosphate groups may suffice to induce association between the N- and C-terminal domains of Lgl, impairing its ability to interact with the cytoskeleton and plasma membrane as previously proposed. This would reduce the local concentration of Lgl available to interact with Dlg, enabling the interaction of Dlg's GUK domain with the pool of Pins phosphorylated by AurA. Therefore, AurA converts the Lgl/Dlg polarity complex generated upon aPKC phosphorylation into the Pins/Dlg spindle orientation complex. This study, underlines the critical requirement of synchronizing the cell cycle with the reorganization of polarity complexes to achieve precise control of spindle orientation in epithelia (Carvalho, 2015).

The Drosophila mitotic spindle orientation machinery requires activation, not just localization

The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Pins, Gαi, Mud, and Dynein in flies - that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. This study revisits the canonical model and finds that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over-ridden by interaction with another Pins interacting protein, Inscuteable (Neville, 2023).

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate. Previous studies have shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter. This study shows that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. The data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis (Loubery, 2014).

Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis

Apical-basal polarity plays critical roles in the functions of epithelial tissues. However, the mechanisms of epithelial polarity establishment and maintenance remain to be fully elucidated. This study shows that the membrane-associated guanylate kinase (MAGUK) family protein Dlg5 is required for the maintenance of apical polarity of follicle epithelium during Drosophila oogenesis. Dlg5 localizes at the apical membrane and adherens junction (AJ) of follicle epithelium in early stage egg chambers. Specifically, the major function of Dlg5 is to promote apical membrane localization of Crumbs, since overexpression of Crumbs but not other major apical or AJ components can rescue epithelial polarity defects resulting from loss of Dlg5. Furthermore, by performing a structure-function analysis of Dlg5, it was found that the C-terminal PDZ3 and PDZ4 domains are required for all Dlg5's functions as well as its ability to localize to apical membrane. The N-terminal coiled-coil motif can be individually targeted to the apical membrane, while the central linker region can be targeted to AJ. Lastly, the MAGUK core domains of PDZ4-SH3-GUK can be individually targeted to apical, AJ and basolateral membranes (Luo, 2016).

How cell polarity is established and maintained is an important question in the fields of cell and developmental biology. During animal development, polarized cells such as epithelial cells maintain their apical-basal polarity despite undergoing dramatic shape changes and tissue remodeling during morphogenesis. Genetic screens done in developing Drosophila and C. elegans have uncovered a number of highly conserved apical and basolateral regulators essential for the establishment and maintenance of apical-basal polarity. Specifically, the Par3/Par6/aPKC complex and the Crumbs (Crb)/Stardust (Sdt)/Patj complex are thought to define and maintain the identity of apical membrane, and the Discs-large (Dlg)/Lethal giant larvae (Lgl)/Scribble (Scrib) complex delineates the basolateral membrane. Finally, the junctional complex of E-cadherin/β-catenin/α-catenin initiates and maintains the adherens junction (AJ), which divides the cortex into apical and basolateral regions. Decades of research have formed a consensus model, in which the apical-basal polarity is generated and maintained by a mutually antagonistic interaction between the apical regulators and the basolateral regulators. Recently, a mathematic modeling study done in Drosophila follicle epithelia suggested that in addition to the negative feedback between apical regulators and basolateral regulators, a positive feedback loop among apical polarity regulators is required to maintain the apical-basal polarity in epithelia. A central component of this positive feedback loop is the transmembrane protein Crb, and its apical membrane localization was thought to be the key to maintenance of apical-basal polarity (Luo, 2016).

Discs-large 5 (Dlg5) belongs to the MAGUK family, and it is highly conserved across species including human, mouse, chicken, zebra fish and Drosophila. MAGUK members also include Dlg and Sdt, which act as molecular scaffolds and are core components of the basolateral Dlg/Lgl/Scrib complex and the apical Crumb complex respectively. Dlg5 was first identified in human and was found to be expressed in placenta and in prostate gland epithelia. Since then, Dlg5 was found to interact with a variety of junctional, cytoskeletal, trafficking, and receptor molecules, including β-catenin, P55, vinexin, Girdin, Citron kinase, Syntaxin, Smoothened and TGF-β receptors. And its functions vary from inhibiting cancer cell migration to mediating receptor signaling, but most of these functions were obtained from cell culture studies. Detailed genetic analysis of Dlg5 was first done using Dlg5 knockout mice, which displayed failure of epithelial tube maintenance resulting in brain hydrocephalus and kidney cysts. These defects were likely due to disruption of apical polarity and AJ. This study focused on Dlg5's requirement of AJ function and found that Dlg5 physically interacted with the β-catenin/cadherin complex and was found together with β-catenin/cadherin complex in both the Rab11-labeled recycling vesicles and the AJ. A more recent work using the same Dlg5^-/- mice found that Dlg5 was also required for lung morphogenesis. Specifically, deletion of Dlg5 resulted in loss of apical polarity markers such as aPKC, and Dlg5 was partially colocalized with aPKC in the apical membrane of the wild type lung epithelia. But how Dlg5 exerts its functions in the apical membrane and regulate apical polarity is unknown (Luo, 2016 and references therein).

In Drosophila, RNAi knockdown of dlg5 affected the cohesion and morphology of border cell clusters as well as delaying their migration. Recently, genetic analysis of Drosophila Dlg5 revealed that its mutation caused embryonic lethality and loss of germ cells in the embryonic gonad. Moreover, reduction of Dlg5 in the follicle cells in the adult ovary leads to defects in egg chamber budding, stalk cell overgrowth, ectopic polar cell induction and abnormal distribution of E-cadherin. However, detailed analysis of whether and how Drosophila Dlg5 regulates epithelial or apical polarity has not been done. This study reports that a genetic screen for follicle epithelial morphogenesis has identified the Drosophila Dlg5 as an essential player for maintenance of apical polarity by promoting Crb's apical membrane localization (Luo, 2016).

Dlg5 is specifically required for the maintenance of apical polarity and AJ of follicle epithelia during early stages of oogenesis, since both loss-of-function mutation and RNAi knockdown of dlg5 affected only apical polarity regulators and the sub-apical AJ components but not the basolateral regulators. Furthermore, the apical markers (Crb, Sdt, Patj, aPKC and Par6) were more severely reduced than the sub-apical AJ markers (Arm, E-cad and Baz) with the exception of N-cad, suggesting that the loss of apical polarity is the main cause of severe morphological defects in Dlg5-deficient follicle cells. A previous study found that loss of Crb, aPKC and Par6 did not affect the lateral localization of Dlg, whereas loss of Arm caused Dlg spreading to the apical membrane of follicle cells. This is consistent with the result that no apical spreading of Dlg was observed in dlg5 mutant clones, further confirming that loss of Dlg5 affected apical polarity more severely than the AJ function. Indeed, rescue of apical polarity defects by Crb but not Arm expression further validated this notion (Luo, 2016).

Importantly, this study demonstrates that Dlg5 positively regulates apical polarity by specifically promoting Crb's apical membrane localization, based on the following results. First, double staining revealed that Crb reduction was sometimes more severe than reduction of other apical markers (aPKC and Par6) in dlg5 mutant clones. Second, overexpression of Crb but not other apical or AJ regulators (aPKC, Par6, Arm) could completely rescue dlg5's apical polarity defects. Third, the increased membrane localization and membrane spreading of Crb as caused by blocking the Rab5-mediated endocytosis could be dramatically suppressed by dlg5 mutation. Moreover, the apical enrichment of Dlg5 in the early and mid-stage follicle epithelia (stage 1-stage 9) further suggests that Dlg5 could function at the apical region to promote Crb's membrane localization. On the other hand, Crb might conversely enhance Dlg5's localization to the apical membrane, since overexpression of Crb and hence its membrane spreading toward the basolateral region led to stronger localization of Dlg5 in the basolateral membrane. A previous study has reported that deletion of Dlg5 in mouse resulted in loss of aPKC but not Par3 (homologous with Drosophila Baz) in the developing lung epithelia and that Dlg5 was partially colocalized with aPKC, which are similar to the current findings. But how Dlg5 regulated the apical polarity during mouse lung morphogenesis was not understood. Based on the current results, it would be worthwhile to check whether murine Dlg5 promotes apical polarity by primarily regulating one of the three mammalian CRB paralogs (CRB1, CRB2, CRB3) (Luo, 2016).

As a MAGUK family member, Dlg5 is thought to function as a scaffold protein. Previous works have focused on which domains of Dlg5 physically interact with junctional and membrane-bound proteins like β-catenin, vinexin and smoothened, and trafficking regulators like syntaxin 4 in vitro or in cultured cells. But whether such domains are essential for its function and localization in vivo and which domains possess apical or AJ membrane targeting ability have not yet been addressed. Structure-function analysis demonstrates that the C-terminal fragment including MAGUK core (GUK, SH3, PDZ4) and PDZ3 is necessary but not entirely sufficient for Dlg5's functions. Furthermore, deletion of this C-terminal fragment (Δ4) caused most of Dlg5 to re-distribute to the cytoplasm, losing its membrane localization in the apical, AJ, and lateral regions. Interestingly, PDZ3 and PDZ4 (a subset of C terminal fragment) were also required for Dlg5's functions, and their deletion (Δ5) likewise resulted in the loss of apical and lateral (but not AJ) membrane localization. One interesting difference between the localization patterns of the other deletion mutants (that still possessed rescue abilities; Δ1-3, Δ6-8) and the patterns of Δ4 and Δ5 is that other deletions all retained some degree of apical localization, in contrast to the lack of localization in the apical membrane for Δ4 and Δ5. Together, these results suggest that MAGUK core and PDZ3's requirement for Dlg5's membrane localization in general and PDZ3-PDZ4's requirement for Dlg5's apical membrane localization may be critical for Dlg5's functions in the follicle cells. This is consistent with Dlg5's role in promoting Crb's apical localization. Lastly, this study found that the N-terminal coiled coil domain, the middle linker region and the MAGUK core could be individually membrane-targeted to apical, AJ and all (apical, AJ and basolateral) regions respectively (Luo, 2016).

Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly

The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, this study analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. Cysts was demonstrated to be recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, these findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity (Silver, 2019).

Antagonistic interactions between apical and basolateral polarity regulators position AJs at the apico-lateral membrane to form a junctional complex. In turn, AJs are thought to maintain apical-basal polarity through the segregation of the apical and basolateral membrane domains, organization of the cytoskeleton, and direct polarity by acting as signaling centers for polarity complexes. Although a number of Drosophila RhoGEFs and RhoGAPs have been implicated in epithelial polarity and AJ stability, no single RhoGEF or RhoGAP has been found to phenocopy the polarity or junctional defects that are seen in embryos compromised for factors such as Crb, aPKC, or E-cadherin. The current findings suggest that loss of the RhoGEF Cysts causes a polarity phenotype strikingly similar to the loss of core apical polarity proteins. Moreover, this study found that Cyst is recruited to the apico-lateral cortex by the action of polarity proteins and, by activating Rho1, stabilizes AJ-associated actomyosin, which supports junctional and epithelial integrity (Silver, 2019).

In Cysts-compromised embryos, AJ formation is disrupted in early gastrulation, and AJs do not form a circumferential belt. These defects in AJ assembly or stability correlate with reduced and irregular myosin accumulation at the apico-lateral cortex. Given the molecular function of Cysts as a GEF for Rho1, loss of myosin activity is presumably the immediate cause for the defects in AJ formation and the subsequent loss of apicobasal polarity in many epithelial cells. crb-depleted embryos failed to recruit Cysts to apical junctions and showed a similar decline in junctional myosin. Therefore, a major function of the apical Crb polarity complex appears to be the Cysts-mediated support of junctional actomyosin (Silver, 2019).

While many cells in crb or cyst mutants undergo programmed cell death, others retain or recover polarity and form small epithelial cysts, a process seen from mid-embryogenesis (postgastrulation stages) onward. Several polarity proteins such as Crb, Sdt, and Baz are needed for normal epithelial polarization in early embryos but are not essential for polarization in postgastrulation embryos, which explains the ability of some epithelial cells in these mutants to form epithelial cysts with normal polarization. In fact, when programmed cell death is suppressed, cyst formation is shown by all epithelial cells in crb mutants. Formation of epithelial cysts seen in cysts mutant embryos therefore suggests that Cysts is also not essential for epithelial polarity in late embryos. This view is supported by the decline of Cysts protein accumulation at AJs seen in late embryos (Silver, 2019).

Several observations, including the genetic interaction of cysts with genes encoding basolateral polarity proteins, the dependence of the junctional localization of Cysts on the apical polarity proteins Baz and Crb, the physical interactions between Cysts and apical polarity proteins, and the function of Cysts in stabilizing AJs, indicate that Cysts is an integral part of the apical polarity machinery in early Drosophila embryos. A particularly striking finding was the complete suppression of the cysts phenotype by codepletion of the basolateral polarity proteins Scrib or Lgl, seen in double-mutant embryos that showed phenotypes indistinguishable from single scrib or lgl mutants. This mimics previous observations with double mutants of crb or sdt and scrib, lgl or discs large. Moreover, this study found that a reduction of aPKC enhanced Baz mislocalization in Cysts-compromised embryos, suggesting that aPKC cooperates with Cysts and acts upstream or in parallel to Cysts to organize Baz. These findings emphasize that Cysts, similar to Crb and aPKC, is a component of a negative feedback circuit between apical and basolateral regulatory networks that govern epithelial polarity. The dependence of Cysts localization on Crb and Baz suggests that Cysts acts downstream of these two proteins. Once polarized, Cysts appears to maintain polarity and junctional stability through actomyosin remodeling (Silver, 2019).

In vivo structure-function data indicate that the C-terminal region is essential for Cysts activity. Moreover, it was found that the C-terminal region of Cysts can oligomerize, potentially facilitated by the CC domain. It is speculated that clustering of Cyst could enhance its cortical association. The Crb complex protein Patj represents one possible anchor for Cyst clusters at the cortex. Biochemical data show that the Cysts C-terminal region is sufficient for interactions with Patj. Patj has been implicated as a myosin II activator in the embryo. It is proposed therefore that Crb, Patj, and Cyst form a complex that organizes junctional actomyosin. However, as Patj is not essential for embryonic survival, Cysts may interact with additional binding partners within the Crb complex. Another apical binding partner for Cyst is Baz/Par3, which is required for Cyst cortical recruitment, coprecipitates with the Cyst C-terminal region, and coaggregates with Cyst in HeLa cells (Silver, 2019).

A recent independent study also arrived at the conclusion that Cysts activates Rho1 at AJs during germband extension in the Drosophila embryo (de Las Bayonas, 2019). It is further shown that depletion of Cysts acts downstream of a G protein-coupled receptor (GPCR) and the Gβ13F/Gγ1 heterotrimeric G protein in directing cell rearrangements promoting germband extension, and that germband extension is somewhat reduced when Cysts is depleted. Loss of Gγ1 causes an ~20% reduction in Cyst junctional enrichment (de Las Bayonas et al., 2019). These and the current data suggest that the normal junctional recruitment of Cyst requires at least three distinct inputs: interactions with Baz/Par3 and the Crb complex, and heterotrimeric G protein signaling (Silver, 2019).

This study found that Cysts becomes enriched at the apico-lateral cortex after the mesoderm and endoderm have invaginated and the germband starts to elongate. This localization coincides with the assembly of the apical-cortical actomyosin network. Rho-Rho kinase signaling plays a critical role in the activation of myosin II in this process. Structure-function analysis showed that Cyst contains an essential RhoGEF domain as predicted, and the use of Rho activity probes, genetic interactions, and biochemical assays showed that Cysts preferentially targets Rho1. Although the biochemical assay also revealed stimulation of Rac1 activity by Cysts, all other data point to Rho1 as the primary target of Cyst. It is proposed therefore that Cyst activates Rho1 to organize actomyosin at the cortex at a time when AJs assemble into a circumferential belt (stages 6/7). Consistent with this, it was found that Cysts is important for maintaining normal cortical levels of myosin II. A similar loss in junctional myosin was also observed in Crb-compromised embryos in line with the finding that Crb is required for Cysts junctional recruitment. The cysts mutant phenotype suggests that Cysts is the key RhoGEF that activates Rho1 at ectodermal AJs. In contrast, RhoGEF2 functions in the mesoderm and ectoderm, where it becomes apico-cortically enriched and activates Rho1 to recruit myosin II to the apical-medial cortex. Thus, RhoGEF and Cysts act in parallel on Rho1 to orchestrate the balance of cortical and medial actomyosin dynamics (Silver, 2019).

Cysts is the single orthologue of a group of four mammalian RhoGEFs that target RhoA in cell culture . One of the mammalian orthologues (p114RhoGEF) stabilizes tight junctions and AJs through organization of the actin cytoskeleton associated with cellular junctions (Nakajima and Tanoue, 2011; Terry, 2011; Acharya, 2018). p114RhoGEF is recruited to apical junctions through a mechanism involving CRB3A, Ehm2/Lulu2, Par3, Patj, the heterotrimeric G protein Gα12, and the GPCR Sphingosine-1 phosphate receptor 2 (Acharya, 2018). p114RhoGEF requires the polarity regulator Ehm2/Lulu2 (a homologue of Drosophila Yrt) to activate RhoA. In contrast, this study did not detect genetic or biochemical interactions between Cyst and Yrt in Drosophila. Recently, ARHGEF18, the human orthologue of p114RhoGEF, was identified as a gene associated with retinal degeneration, and a fish orthologue is required to maintain epithelial integrity of the retina. ARHGEF18 mutant retinal defects closely resemble those found in patients carrying mutations in the crb homologue CRB1. It is concluded that the function of Cyst and p114RhoGEF/ARHGEF18 in coupling apical polarity proteins and GPCR signaling to junctional Rho activity and actomyosin function is conserved between flies and vertebrates and likely contributes to retinal health in humans, although some of the molecular interactions may have shifted in relative importance (Silver, 2019).

The other mammalian orthologues of Cyst, p190RhoGEF, AKAP-13, and GEF-H1 have not been implicated as regulators of epithelial polarity. GEF-H1 (also known as ARHGEF2 and Lfc) was shown to be inactive at mature tight junctions. In this case, the tight junction protein Cingulin forms a complex with GEF-H1, preventing it from activating RhoA. Instead, GEF-H1 is thought to promote junction disassembly and cell proliferation, presumably through an association with the mitotic spindle. GEF-H1 was also implicated in the morphogenesis of the vertebrate neural tube, and in the regulation of RhoA activity during cytokinesis. Like GEF-H1, p190RhoGEF has been shown to associate with microtubules. GEF-H1 and AKAP-13 were also found to serve additional functions independent of their RhoGEF activity. Whether and how Cyst might consolidate the functions of its various mammalian orthologues remains to be explored (Silver, 2019 and references therein).

Crumbs complex-directed apical membrane dynamics in epithelial cell ingression

Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cells lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. This study shows that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. These findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer (Simoes, 2022).

Drosophila NBs are an outstanding model for scrutinizing the cellular machineries underpinning an EMT-like process with high temporal and spatial resolution. While ingressing, a single NB sequentially loses AJs responding to tensile forces exerted by two pools of actomyosin: a planar polarized pool enriched at anterior-posterior junctions, which disassembles first, and a pulsatile pool at the free apical cortex, which further tugs on shrinking junctions in a ratchet-like manner. However, while actomyosin forces reduce the apical perimeter, it remained unclear how cells lose their apical domain and how polarity regulators contribute to the dynamics of apical domain loss. This study demonstrates that regulation of the Crb complex plays a key role in orchestrating apical domain loss during ingression. During early ingression, cells shrink their apical domain while retaining total levels of Crb, which is crucial for maintaining normal actomyosin in NBs and NICs (neighboring non-ingressing cells) to generate the tension balance required for normal ingression dynamics. During late ingression, Crb is rapidly lost from the apical domain, a process initiated by an interaction between Neur and Sdt, which causes the disassembly of the Crb complex. The loss of the Crb complex then precipitates the concurrent loss of the apical membrane and AJs. A similar regulatory interplay between Crb, Sdt, and Neur was also observed during early neurogenesis in the Drosophila optic lobes. Here, neural stem cells emerge from a wave front in the optic lobes rather than ingress as individual cells. Despite these topological differences, the Crb/Sdt/Neur module appears to be a common cell biological regulator of EMT during early neurogenesis (Simoes, 2022).

Endocytosis and endocytic degradation and recycling are requirements for normal NB ingression dynamics. The apical membrane of neuroepithelial cells is much more active endocytically than the basolateral membrane. Notably, Crb is endocytosed during apical contractions and re-secreted during expansions, suggesting that myosin-driven cell contact contraction promotes endocytosis, consistent with recent data from mammalian cells, whereas expansions allow for enhanced secretion. Blocking endocytosis increases surface levels of Crb and Ecad as expected, and prevents NB ingression, whereas enhancing endocytosis accelerates ingression. Moreover, endocytic trafficking plays a key role in determining ingression speed. Loss of ESCRT complex-mediated degradation appears to enhance apical Crb and slow ingression, whereas loss of Retromer-mediated recycling dramatically reduced surface Crb and accelerated ingression. In fact, Retromer-compromised embryos showed the fastest ingression speed of any condition examined, suggesting that the Retromer not only recycles Crb but also other factors that counteract apical domain loss in NBs. Crb turnover during early ingression maintains a steady Crb surface abundance. During late ingression, Crb endocytosis is enhanced during both apical contraction and expansion as a result of the disruption of the Crb-Sdt interaction by Neur, which likely makes the Crb cytoplasmic tail accessible to the Clathrin adapter AP2. AP2 binds to Crb competitively with Sdt, facilitating the rapid endocytic removal of Crb and the apical membrane (Simoes, 2022).

The relationship between actomyosin contraction, endocytosis, Crb protein levels, and their function in NB ingression illuminates the complexity and robustness of morphogenesis. Disrupting several individual molecular processes, while changing the dynamics of ingression, rarely abrogates ingression entirely. First, although endocytosis is essential for ingression, strong depletion of endocytotic regulator delayed ingression but did not prevent delamination in most cases. Similarly, a dramatic depletion of myosin, which is essential for apical constriction of NBs, extended the ingression period but did not block delamination. Evidence of the mutual dependency of endocytosis and myosin-driven contractions in NBs. Contractions foster endocytosis, whereas endocytosis promotes contractions, a co-dependency that likely finetunes ingression dynamics. Second, maintaining Crb in the apical membrane (through overexpression or expression of a non-endocytosable form of Crb) delayed ingression but did not prevent it in most cases, suggesting that Crb membrane persistence is overcome by other mechanisms such as the Neur-dependent disassembly of the Crb complex. Moreover, failure to resolve the Crb complex in late ingression caused part of the apical membrane to persist as apical plugs. Nevertheless, NB delaminate, detach from apical plugs and animals are viable, suggesting that nervous system development proceeds rather normally. Crb surface abundance is dependent on endocytosis and versicle trafficking, and Crb regulates junctional myosin which contributes to apical contraction. Together, these findings highlight the multilayered regulation of ingression through the co-dependent interactions between apical polarity, vesicle trafficking, and actomyosin contractions (Simoes, 2022).

EMT is thought to be initiated by the expression of EMT transcription factors (EMT-TFs) of the Snail, Zeb, or bHLH families that downregulate key adhesion or polarity proteins such as Ecad and Crb. NBs are specified through the combined action of proneural genes that include bHLH proteins of the Achaete-Scute complex, the Snail family protein Worniu, and the SoxB family protein SoxNeuro. However, although genes that encode Ecad and Crb are transcriptionally downregulated in NBs, this repression does not appear relevant for NB ingression. Replacing endogenous Ecad with a transgene expressing Ecad under the control of a ubiquitous promoter had no impact on NB ingression dynamics. This study shows that surface levels of Crb remained high in NBs during early ingression before Crb is rapidly removed by endocytosis during late ingression. This raises the question of how the upregulation of proneural genes in presumptive NBs elicits enhanced actomyosin contractility and endocytic removal of apical membrane and junctions (Simoes, 2022).

One proneural gene target is neur. Neur is found throughout the neuroepithelium participating in Delta-Notch-mediated lateral inhibition to select the NB from an equivalence group of 5-7 cells. Neur upregulation in ingressing NBs is thought to be part of a positive feedback that stabilizes NB fate through persistent asymmetric Delta-Notch signaling. The increase in Neur may also be important for the effective disruption of the Crb complex to destabilize the apical domain. Neur can disrupt the Crb complex across the epithelium but is normally prevented from doing so by Bearded proteins that act as inhibitors of Neur. Increasing Neur concentration may overcome this inhibition in NBs. This raises the possibility that the proneural gene-dependent upregulation of neur contributes to the timing of ingression, consistent with the observation that in Neur-depleted embryos ingression is prolonged. Furthermore, Neur may enhance actomyosin contractility in NBs seen in late ingression as was reported for Neur in the Drosophila mesoderm. It is hypothesized therefore that Neur could be a central regulator of NB selection and ingression, stabilizing NB fate, driving apical membrane constriction through actomyosin contraction, and disrupting the Crb complex to remove apical membrane and junctions. Interestingly, it was also noted that during ingression, the number of alternative isoforms of Sdt is limited to Sdt3, the isoform susceptible to Neur. Hence, NBs appear to develop the molecular competence for apical membrane removal at least in part through rebalancing Sdt splice forms (Simoes, 2022).

The loss of apical-basal polarity is an early event during EMT marked by the loss of epithelial AJs that can trigger expression of EMT-TFs and the disassembly of cell junctions. However, the findings of this study indicate that the loss of apical-basal polarity in NBs is preceded by a period (~20 min; early ingression) of ratcheted apical contractions that reduce the apical area of delaminating cells. The maintenance of normal Crb levels during early ingression is crucial for normal ingression dynamics. Crb stabilizes junctional myosin through its effector, Cyst, which is recruited to the junctional domain by the Crb complex (Silver, 2019). Although the loss of Crb or loss of Cyst causes similar reductions of junctional myosin in the neuroepithelium (Silver, 2019), NB ingression was consistently faster in Cyst-compromised embryos than in controls. In contrast, NBs in Crb-compromised embryos showed much larger variability of ingression speeds, with a small fraction of NBs ingressing rapidly while the majority was slower than controls. Thus, it is likely that Crb makes other contributions to regulating NB ingression in addition to its Cyst-mediated function in supporting junctional actomyosin. Interestingly, the mouse Crb homolog Crb2 is required for myosin organization and ingression during gastrulation. The predominant defect in Crb2-compromised mice appears to be a failure of ingression, which may be similar to the fraction of NBs showing slower than normal ingression seen with the loss of Drosophila Crb. To what extent the differences in cell behavior caused by the loss of Crb and Crb2 depend on the biomechanical specifics of the tissue context or result from differences in molecular pathways in which Crb and Crb2 operate remains to be explored (Simoes, 2022).

Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis

Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Tau, a microtubule associated protein, was analyzed in this study to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. Tau was found to have a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation (Nam, 2016).

The endoplasmic reticulum is partitioned asymmetrically during mitosis prior to cell fate selection in proneuronal cells in the early Drosophila embryo

Asymmetric cell division is the primary mechanism to generate cellular diversity and relies on the correct partitioning of cell fate determinants. However, the mechanism by which these determinants are delivered and positioned is poorly understood and the upstream signal to initiate asymmetric cell division is currently unknown. This study reports that the Endoplasmic Reticulum (ER) is asymmetrically partitioned during mitosis in epithelial cells just prior to delamination and selection of a proneural cell fate in the early Drosophila embryo. At the start of gastrulation, the ER divides asymmetrically in a population of asynchronously dividing cells at the anterior end of the embryo. This asymmetric division of the ER is dependent on the highly conserved ER membrane protein Jagunal (Jagn). RNA inhibition of jagn, just prior to the start of gastrulation, disrupts this asymmetric division of the ER. In addition, jagn deficient embryos display defects in apical-basal spindle orientation in delaminated embryonic neuroblasts (NB). The results presented in this study describe a striking model in which an organelle is partitioned asymmetrically in an otherwise symmetrically dividing cell population just upstream of cell fate determination, and updates previous models of spindle-based selection of cell fate during mitosis (Eritano, 2017).

Prefoldin and Pins synergistically regulate asymmetric division and suppress dedifferentiation

Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. This study shows that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. This study shows that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or β-tubulin in pins^- mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. These data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells (Zhang, 2016).

Control of tissue homeostasis is a central issue during development. The neural stem cells, or neuroblasts, of the Drosophila larval brain is an excellent model for studying stem cell homeostasis. Asymmetric division of neuroblasts generates a self-renewing neuroblast and a different daughter cell that undergoes differentiation pathway to produce neurons or glia. Following each asymmetric division, apical proteins such as aPKC are segregated into the neuroblast daughter and function as 'proliferation factor', while basal proteins are segregated into a smaller daughter cell to act as 'differentiation factors'. At the onset of mitosis, the Partitioning defective (Par) protein complex that is composed of Bazooka (Baz)/Par3, Par6 and atypical protein kinase C (aPKC) is asymmetrically localized at the apical cortex of the neuroblast. Other apical proteins including Partner of Inscuteable (Pins), the heterotrimeric G protein Gαi, and Mushroom body defect (Mud) also accumulate at the apical cortex through an interaction of Inscuteable (Insc) with Par protein complex. Apical proteins control basal localization of cell fate determinants Numb, Prospero (Pros), Brain tumor (Brat) and their adaptor proteins Miranda (Mira) and Partner of Numb (Pon) that are segregated into the ganglion mother cell (GMC) following divisions. Apical proteins and their regulators also control mitotic spindle orientation to ensure correct asymmetric protein segregation at telophase. Several centrosomal proteins, Aurora A, Polo and Centrosomin, regulate mitotic spindle orientation (Zhang, 2016).

There are at least two different types of neuroblasts that undergo asymmetric division in the larval central brain. Perturbation of asymmetric division in either type of neuroblast can trigger neuroblast overproliferation and/or the induction of brain tumors. The majority of neuroblasts are type I neuroblasts that generate a neuroblast and a GMC in each division, while type II neuroblasts generate a neuroblast and an intermediate neural progenitor (INP), which undergoes three to five rounds of asymmetric division to produce GMCs. Ets transcription factor Pointed (PntP1 isoform), exclusively expressed in type II neuroblast lineages, promotes the formation of INPs. Failure to restrict the self-renewal potential of INPs can lead to dedifferentiation, allowing INPs to revert back into 'ectopic neuroblasts'. Notch antagonist Numb and Brat function cooperatively to promote the INP fate. Loss of brat or numb leads to 'ectopic type II neuroblasts' originating from uncommitted immature INPs that failed to undergo maturation. A zinc-finger transcription factor Earmuff functions after Brat and Numb in immature INPs to prevent their dedifferentiation. Earmuff also associates with Brahma and HDAC3, which are involved in chromatin remodeling, to prevent INP dedifferentiation. However, the underlying mechanism by which INPs possess limited developmental potential is largely unknown (Zhang, 2016).

Prefoldin (Pfdn) was first identified as a hetero-hexameric chaperone consisting of two α-like (PFDN3 and 5) and four β-like (PFDN 1, 2, 4 and 6) subunits, based on its ability to capture unfolded actin (Vainberg, 1998). Prefoldin promotes folding of proteins such as tubulin and actin by binding specifically to cytosolic chaperonin containing TCP-1 (CCT) and by directing target proteins to it. The yeast homologs of Prefoldin 2–6, named GIM1-5 (genes involved in microtubule biogenesis) are present in a complex that facilitates proper folding of α-tubulin and γ-tubulin. All Prefoldin subunits are phylogenetically conserved from Archaea to Eukarya. Structural study of the Prefoldin hexamer from the archaeum M. thermoautotrophicum showed that Prefoldin forms a jellyfish-like shape consisting of a double β barrel assembly with six long tentacle-like coiled coils that participate in substrate binding. The function of Prefoldin as a chaperone has also been illustrated in lower eukaryotes like C. elegans, in which loss of prefoldin resulted in defects in cell division due to reduced microtubule growth rate. Depletion of PFDN1 in mice displayed cytoskeleton-related defects, including neuronal loss and lymphocyte development defects. The only Prefoldin subunit in Drosophila that has been characterized to date, Merry-go-round (Mgr), the Pfdn3 subunit, cooperates with the tumor suppressor Von Hippel Lindau (VHL) to regulate tubulin stability (Delgehyr, 2012). However, the functions of Prefoldin in the nervous system remain elusive (Zhang, 2016).

This study describes the critical role of evolutionarily-conserved Prefoldin complex in regulating neuroblast and INP asymmetric division and suppressing INP dedifferentiation. Mutants for two Prefoldin subunits, Mgr and Pfdn2, displayed neuroblast overgrowth with defects in cortical polarity of Par proteins and microtubule-related abnormalities. Interestingly, co-depletion of Pins in mgr or pfdn2 mutants led to massive neuroblast overgrowth. Prefoldin and Pins synergistically regulate asymmetric division of both neuroblasts and INPs. Surprisingly, they also synergistically suppress dedifferentiation of INPs back into neuroblasts. Knocking down tubulins in pins mutant background resulted in severe neuroblasts overgrowth, mimicking that caused by co-depletion of Prefoldin and Pins. These data provide a new mechanism by which Prefoldin and Pins regulates neural stem cell homeostasis through regulating tubulin stability in both neuroblasts and INPs (Zhang, 2016).

pfdn2/CG6302, encoding a Prefoldin β-like subunit, was identified from a RNA interference (RNAi) screen in larval brains. Ectopic neuroblasts labeled by a neuroblast marker, Deadpan (Dpn), were formed upon knocking down pfdn2 under a neuroblast driver insc-Gal4. Only one neuroblast was observed in control type I neuroblast lineages using insc-Gal4 and type II neuroblast lineages using worniu-Gal4 with asense (ase)-Gal80. In contrast, upon pfdn2 RNAi excess neuroblasts were observed in both type I neuroblast lineages and type II neuroblast lineages, respectively. To verify the function of Pfdn2 in neuroblasts, a putative hypomorphic allele of pfdn2, pfdn2⁰¹²³⁹, was analyzed that has a P element inserted at the 5′ untranslated region (UTR) of pfdn2. Hemizygous larval brains of pfdn2⁰¹²³⁹ over Df(3L)BSC457 (referred to as pfdn2⁻ thereafter) displayed 235.3 ± 31.7 neuroblasts per brain hemisphere, suggesting that Pfdn2 inhibits the formation of ectopic neuroblasts in larval brains. Consistently, an increase of EdU (5-ethynyl-2′-deoxyuridine)-incorporation was also observed in pfdn2⁻ mutants compared to the control. To generate pfdn2 null alleles, a P element, EY06124, was mobilized. Its imprecise excision yielded two loss-of-function alleles, pfdn2^Δ10 and pfdn2^Δ17, both deleting the entire opening reading frame (ORF) of pfdn2. pfdn2^Δ10 and pfdn2^Δ17 mutants survive to pupal stage and display strong phenotypes with ectopic neuroblasts labeled by Dpn. These phenotypes in pfdn2^Δ10 and pfdn2^Δ17 mutant brains can be fully rescued by overexpression of wild-type pfdn2 or pfdn2-Venus transgene. Pfdn2 is abundantly expressed in neuroblasts, INPs and their immediate neural progeny- GMCs, detected by a specific antibody generated against Pfdn2 full length and a transgenic Pfdn2 with a Venus tag at the C-terminus. In addition, Pfdn2 expression under the tubulin-Gal4 fully rescued the lethality of both pfdn2^Δ10 and pfdn2^Δ17 mutants. Pfdn2 protein was undetectable in pfdn2^Δ10 zygotic mutants, further supporting that it is a null allele. Both type I and type II MARCM (Mosaic Analysis with Repressible Cell Marker) clones of pfdn2^Δ10 generated excess neuroblasts. These phenotypes were slightly weaker than pfdn2^Δ10 zygotic mutants, likely due to residual Pfdn2 protein in the clones. These data indicate that Pfdn2 is required in both type I and type II neuroblast lineages to prevent the formation of ectopic neuroblasts (Zhang, 2016).

This study has identified an unexpected synergism between Prefoldin and Pins in suppressing neuroblasts overgrowth. Barious subunits of Prefoldin complex are implicated in asymmetric division of neuroblasts, especially during asymmetric protein segregation at telophase. It is known that depletion of Pins results in the formation of smaller larval brains, despite partial loss of neuroblasts polarity. Interestingly, co-depletion of Pfdn2 and Pins results in severe neuroblasts overgrowth, while Pfdn2 depletion alone only causes mild brain overgrowth. This phenotype is contributed by a combination of loss of neuroblast polarity, defects of asymmetric division of INPs, as well as INP dedifferentiation. Knocking down tubulins in pins mutant background mimics the co-depletion of Prefoldin and Pins, suggesting that tubulin stability appears to be critical for the suppression of neuroblast overgrowth in the absence of Pins function. The data also suggest that Prefoldin function and tubulin stability in INPs are important to suppress their dedifferentiation back into neuroblasts (Zhang, 2016).

How microtubules induce cortical polarity is poorly understood in Drosophila neuroblasts. Previously, one report showed that kinesin Khc-73, which localized at the plus end of astral microtubules, and Discs large (Dlg) induced cortical polarization of Pins/Gαi in neuroblasts. However, microtubules are considered not essential for neuroblast polarity. This study shows that Drosophila Prefoldin regulates asymmetric division of both neuroblasts and INPs through tubulins, suggesting an important role of microtubules in neuroblast polarity. The essential role of microtubules directly regulating cell polarity is found in various systems. During C. elegans meiosis, a microtubule-organizing center is necessary and sufficient for the establishment of the anterior-posterior polarity. In the fission yeast Schizosaccharomyces pombe, interphase microtubules directly regulate cell polarity through proteins such as tea1p. In mammalian airway cilia, microtubules are required for asymmetric localization of planer cell polarity proteins (Zhang, 2016).

This study shows that the role of Drosophila Prefoldin complex in regulating asymmetric division is very likely dependent on microtubules. This is consistent with the known essential role of Prefoldin for maintaining tubulin levels in various organisms such as yeast, C. elegans, plants and mammals. In yeast, Gim (Prefoldin) null mutants become super-sensitive to the microtubule-depolymerizing drug benomyl as a result of a reduced level of α-tubulin. In the absence of Prefoldin, the function of the chaperone pathway is damaged and unable to fold sufficient amount of tubulins for normal yeast growth. In C. elegans, reducing Prefoldin function causes defects in cell division presumably due to the reduction of tubulin levels and microtubule growth rate. Genetic analysis of mammalian Prefoldin also suggests that cytoskeletal proteins like actin and tubulin make up the major substrate of Prefoldin in mammals. These studies in different organisms together suggest that Prefoldin complex plays a conserved central role in tubulin folding (Zhang, 2016).

'Telophase rescue', a term refers to the phenomenon that protein mis-localization at metaphase is completely restored at telophase, is observed in many mutants that affect neuroblast asymmetric division. However, both apical and basal proteins are still mis-segregated in pfdn2 and mgr mutants, suggesting that 'telophase rescue' is defective in these mutants. Telophase rescue is regulated by TNF receptor-associated factor (DTRAF1), which binds to Baz and acts downstream of Egr/TNF. Telophase rescue also depends on Worniu/Escargot/Snail family proteins and a microtubule-dependent Khc-73/Dlg pathway. Pins did not form a protein complex with Mgr, α-tubulin or β-tubulin in co-immunoprecipitation assay. Given that Dlg is a Pins-interacting protein, Prefoldin appears to function in a different pathway with Dlg or Khc-73 during asymmetric division (Zhang, 2016).

Recently, merry-go-round (mgr), encoding Prefoldin 3 (Pfdn3)/VBP1/Gim2 subunit, was reported to regulate spindle assembly. Loss of mgr led to formation of monopolar mitotic spindles and loss of centrosomes because of improper folding and destabilization of tubulins. The current analysis on Pfdn2 indicates that pfdn2 mutants displayed similar spindle and centrosome abnormalities. In addition, the incorrectly folded tubulin due to loss of mgr may be eliminated by Drosophila von Hippel Lindau protein (Vhl), an E3 ubiquitin-protein ligase. Interestingly, the data suggest that Prefoldin has a tumor-suppressor like function in preventing neuroblast overgrowth. However, Drosophila Vhl is not important for brain tumor suppression, as its loss-of-function neither affects number of neuroblasts nor suppresses overgrowth observed in pfdn2 RNAi or mgr RNAi (Zhang, 2016).

This study shows a novel synergism between Prefoldin and Pins in suppressing dedifferentiation of INPs back into neuroblasts. Prefoldin and Pins apparently suppress dedifferentiation through regulating tubulin levels. It is likely that appropriate tubulin levels in INPs are important for their differentiation, while reducing tubulin levels can increase the risk of INP dedifferentiation. Currently, several cell fate determinants such as Brat, Numb and the SWI/SNF chromatin remodeling complex with its cofactors Erm and Hdac3 are critical to suppress INP dedifferentiation back into neuroblast. It is currently unknown whether or how Prefoldin/Pins are linked to these known suppressors of dedifferentiation. It is possible that symmetric division of INPs causes reduced levels of Brat and Numb in these abnormal INP daughters, leading to their dedifferentiation. Alternatively, Prefoldin might regulate transcription of genes within INPs to suppress dedifferentiation. It was reported that the human homolog of Pfdn5, MM-1, has a role in transcriptional regulation by binding to the E-box domain of c-Myc and represses E-box-dependent transcriptional activity. Interestingly, Prefoldin Subunit 5 gene is deleted in Canine mammary tumors, suggesting that it may be a tumor suppressor gene. This study has revealed a novel mechanism by which Prefoldin and Pins function through tubulin stability to suppress stem cell overgrowth. It is expected to contribute to the understanding of mammalian/human Prefoldin function in tumorigenesis (Zhang, 2016).

Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division

Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. This study shows that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery (Banerjee, 2017).

Polarity is a fundamental feature of most cells and tissues. It is evident both at the level of individual cells and groups of cells (e.g. planar cell polarity (PCP) in epithelia. However, despite the fact that different cell types use a common set of molecules to establish and maintain polarity (Par complexes, Fz-PCP pathway), the organization of polarized cells and cell assemblies varies dramatically across different species and tissues. This implies the existence of factors that act in a cell or tissue-specific manner to modulate/rewire the core polarity machinery into the appropriate organization. Despite many advances in understanding of polarity in unicellular and multicellular contexts, little is known about the identity or function of such factors (Banerjee, 2017).

An example of polarity remodeling is the process of asymmetric cell division (ACD), where cells need to rearrange their polarity determinants into a machinery capable of asymmetrically segregating cell fate determinants, vesicles and organelles, as well as controlling the orientation of the mitotic spindle. ACDs result in two daughter cells of different fates and occur in numerous cell types and across species. Well-studied examples include budding in Saccharomyces cerevisiae, ACD in the early embryo of Caenorhabditis elegans, or ACD of progenitor cells in the mammalian stratified epidermis and neural stem cells in the mammalian neocortex. In Drosophila melanogaster, the study of germline stem cells, neuroblasts (neural stem cells) and sensory organ precursors (SOPs) has greatly contributed to understanding of the cell biology and molecular mechanisms of ACD (Banerjee, 2017).

SOPs (or pI cells) divide asymmetrically within the plane of the epithelium into pIIa and pIIb daughter cells. pIIa and pIIb themselves divide asymmetrically to give rise to the different cell types of the external sensory organs (bristles), which are part of the peripheral nervous system and allow the adult fly to sense mechanical or chemical stimuli. Individual SOPs are selected by Notch-dependent lateral inhibition from multicellular clusters of epithelial cells expressing proneural genes (proneural clusters) (Banerjee, 2017).

The unequal segregation of cell fate determinants (the Notch pathway modulators Numb and Neuralized), which specifies the different fates of the daughter cells, requires their asymmetric localization on one side of the cell cortex prior to mitosis. This is achieved by remodeling the PCP and apical-basal polarity systems in the SOP, and by orienting the spindle relative to the tissue axis. The epithelial sheet that forms the pupal notum (dorsal thorax), where the best-studied SOPs are located, is planar polarized along the anterior-posterior tissue axis, with the transmembrane receptor Frizzled (Fz) and its effector Dishevelled (Dsh) localizing to the posterior side of the cell cortex, while the transmembrane protein Van Gogh (Vang, also known as Strabismus) and its interactor Prickle (Pk) are found anteriorly. The apical-basal polarity determinants central to SOP polarity are the PDZ domain-containing scaffold protein Bazooka (Baz, or Par3), atypical Protein Kinase C (aPKC) and Partitioning defective 6 (Par6), which localize apically in epithelial cells and the basolaterally localized membrane-associated guanylate kinase homologues (MAGUK) protein Discs-large (Dlg). In most epithelial cells, these proteins localize uniformly around the cell cortex, whereas in SOPs they show a striking asymmetric localization during mitosis: the Baz-aPKC-Par6 complex is found at the posterior cell cortex, opposite an anterior complex consisting of Dlg, Partner of Inscuteable (Pins) and the G-protein subunit Gαi. The Fz-Dsh complex provides the spatial information for the Baz-aPKC-Par6 complex, while Vang-Pk positions the Dlg-Pins-Gαi complex (likely through direct interaction between Vang and Dlg). The asymmetric distribution of the polarity determinants then directs the positioning of cell fate determinants at the anterior cell cortex. Additionally, Fz-Dsh and Pins orient the spindle along the anterior-posterior axis by anchoring it on both sides of the cell via Mushroom body defective (Mud, mammalian NuMA) and Dynein (Banerjee, 2017).

The planar symmetry of the Baz-aPKC-Par6 complex in SOPs is initially broken in interphase via Fz-Dsh, and is independent of the Dlg-Pins-Gαi complex. Once this initial asymmetry is established, the core PCP components become dispensable for Par complex polarization at metaphase due to the mutual antagonism between the opposing polarity complexes, which then maintains asymmetry during cell division. Indeed, Baz is still polarized in fz mutants during mitosis, but losing both pins and fz results in Baz spreading uniformly around the cortex. Crucially, it is unclear how Fz-Dsh can transmit planar information to the Baz-aPKC-Par6 complex in SOPs but not in neighboring epithelial cells. The cell-type dependent coupling between PCP and apical-basal polarity suggests the involvement of unknown SOP-specific factors in this process (Banerjee, 2017).

The four N-terminal RASSFs (Ras association domain family) in humans (RASSF7-10) have been associated with various forms of cancer, but the exact processes in which these scaffolding proteins act remain mostly elusive. Drosophila RASSF8, the homologue of human RASSF7 and RASSF8, is required for junctional integrity via Baz. Interestingly, human RASSF9 and RASSF10 were found in an interaction network with Par3 (the mammalian Baz homologue) and with several PCP proteins. The Drosophila genes CG13875 and CG32150 are believed to be homologues of human RASSF9 and RASSF10, respectively and remarkably, CG32150 mRNA is highly enriched in SOPs (Banerjee, 2017).

This study shows that Meru, encoded by CG32150, is an SOP-specific factor, capable of linking PCP and apical-basal polarity. Meru localizes asymmetrically in SOPs based on the polarity information provided by Fz/Dsh, and is able to recruit Baz to the posterior cortex (Banerjee, 2017).

PCP provides the spatial information for the initial polarization of SOPs at interphase, resulting in the planar polarization of Baz, which is uniformly localized prior to SOP differentiation. How Fz/Dsh communicate with Baz and enable its asymmetric enrichment was unknown. Based on the current results and previous findings, the following model is proposed for the role of Meru in SOP polarization. Upon selection and specification of SOPs, Meru expression is transcriptionally activated by the AS-C transcription factors (Reeves and Posakony, 2005). At interphase, planar-polarized Fz/Dsh recruit Meru to the membrane and hence direct its polarization. Meru in turn positions and asymmetrically enriches Baz, promoting the asymmetry of aPKC-Par6. Upon entry into mitosis, Meru is also required to retain laterally localized Baz, thus supporting the antagonism between the opposing Dlg-Pins-Gα_i and Baz-aPKC-Par6 complexes, ultimately enabling the correct positioning of cell fate determinants (Banerjee, 2017).

The meru mutant cell fate phenotype (bristle duplication or loss) is weaker than the baz loss-of-function phenotype, which results in loss of entire SOPs. This is likely due to two factors: (1) unlike meru mutants, the full baz mutant phenotype is the result of a complete loss of Baz in all cells of the SOP lineage, which is known to cause multiple defects including apoptosis of many sensory organ cells as well as cell fate transformations; (2) since a small amount of Baz is retained at the cortex of some meru mutant cells, it is likely that this residual Baz can still be polarized through the antagonistic activity of Pins at metaphase and thus partially rescues SOP polarization. Indeed, it was observed that reduction of pins or baz levels by RNAi strongly enhanced the meru cell specification phenotype. Conversely, supplying excess levels of Baz in a meru mutant background presumably restores sufficient Baz at the cortex to rescue the meru specification defect, as long as Pins is present to drive asymmetry at mitosis. (Banerjee, 2017).

While a decrease in cortical Baz can account for the cell specification defects in meru mutants, it does not explain the spindle orientation phenotypee. This abnormal spindle alignment could either be due to a decrease in Fz/Dsh levels/activity, or a decrease in the ability of Dsh to recruit the spindle-tethering factor Mud. No gross abnormalities were detected in Fz levels in meru mutants, though the presence of Fz in all neighboring cells would make it difficult to detect subtle decreases in SOPs. Further work will be required to understand Meru's role in spindle orientation (Banerjee, 2017).

Analysis of Meru in Drosophila is in agreement with the association of human RASSF9 and RASSF10 with both Par3 and PCP proteins previously reported. However, while the interaction with Dsh is conserved between the fly and human proteins, the transmembrane protein Vangl1 (the mammalian homologue of Vang), rather than its antagonist Fz was recovered in the mammalian proteomic analysis. This could reflect species-specific differences or altered polarity in the transformed human embryonic kidney 293 cells used for the mammalian work. Although Meru (CG32150) was classified as a potential homologue of RASSF10, alignment of the protein sequences showed similar sequence identities for both human RASSF9 (31%) and RASSF10 (26%). Thus, further functional work on Meru, its Drosophila paralogue CG13875, as well as mammalian RASSF9 and RASSF10 is required to understand the evolutionary and functional relationships between these proteins (Banerjee, 2017).

Little is known about the in vivo functions of either RASSF9 or RASSF10 in other species. Xenopus RASSF10 is prominently expressed in the brain and other neural tissues of tadpoles, potentially indicating a function in neurogenesis, a process where ACDs are known to take place. Interestingly, mouse RASSF9 shows a cell-specific expression in keratinocytes of the skin and loss of RASSF9 results in differentiation defects of the stratified epidermis. Considering that Par3 is required for ACD of basal layer progenitors of the stratified epidermis this raises the exciting prospect that RASSF9 might regulate ACD in the mammalian skin (Banerjee, 2017).

The polarization of cells and tissues is essential for their architecture and ultimately allows them to fulfill their function. The polarity machinery can be considered as a series of modules that are combined in a cell or tissue-specific manner, and hence requires specific factors that can create a polarity network appropriate to each tissue and cell type. This study has identified Meru as an SOP-specific factor, which is able to link PCP (Fz-Dsh) with apical-basal polarity (Baz). The PCP proteins Vang and Pk promote the positioning of the opposing Dlg-Pins-Gα_i complex. Although Vang can directly bind to Dlg, the SOP and neuroblast-specific factor, Banderuola (aka Wide Awake) was recently shown to be required for Dlg localization and could thus constitute a link between the two polarity systems on the opposite side of the cortex (Banerjee, 2017).

There is increasing evidence that cell-type specific rewiring of the polarity modules may be a widespread phenomenon. For instance, in different parts of the embryonic epidermis, Baz is planar polarized by Rho-kinase or by the Fat-PCP pathway, while in the retina, Vang is responsible for Baz polarization. Apical-basal polarity can also operate upstream of PCP in some systems, as in Drosophila photoreceptor specification, where aPKC restricts Fz activity by inhibitory phosphorylation in a subset of photoreceptor precursors. Thus, tissue-specific factors are likely to operate in a number of different contexts (Banerjee, 2017).

The interplay between PCP and apical-basal polarity is also evident in other species, as Dishevelled has been reported to promote axon differentiation in rat hippocampal neurons by stabilizing aPKC, while Xenopus Dishevelled is required for Lethal giant larvae (Lgl) basal localization in the ectoderm. Interestingly, both mammalian Par3 and the Vang homologue Vangl2 are required for progenitor cell ACD in the developing mouse neocortex, raising the question as to whether PCP and apical-basal polarity are also connected in mammalian ACDs. It is therefore proposed that tissue-specific factors such as Meru might enable the diversity and plasticity observed across different polarized cells and tissues by rewiring the core polarity systems (Banerjee, 2017).

Magi is associated with the Par complex and functions antagonistically with Bazooka to regulate the apical polarity complex

The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. This study investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. A Magi null mutant was generated and found to be viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi results in the displacement of Baz/Par3 and aPKC and leads to an increase in the level of PIP3. Interestingly, it was found that Magi and Baz function in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex (Padash Barmchi, 2016).

A common component of junctional and polarity complexes is modular scaffolding proteins that are capable of binding to each other as well as recruiting other proteins to the complex. Magi proteins are evolutionarily conserved scaffolding proteins and contain multiple domains including a N-terminal catalytically inactive GUK domain, two WW domains and five to six PDZ (PSD95/Dlg/ZO-1) domains (Dobrosotskaya, 1997). There are three MAGI proteins in vertebrates (MAGI-1,2,3) all with multiple splice isoforms. MAGI-1 and MAGI-3 are relatively ubiquitously expressed and localize to a range of junctions including epithelial tight junctions. MAGI-2 (also known as AIP1/S-SCAM/ARIP1) is expressed in the nervous system as a synaptic protein and within glomerular podocytes in the kidney and plays important role in scaffolding synaptic proteins such as NMDA receptors and Neuroligin, the tip-link protocadherin Cadherin23, the Kir4.1 K(+) channel, as well as kidney proteins such as nephrin and JAM4 (Padash Barmchi, 2016).

Within epithelia and endothelia, MAGI-1 and -3 are localized at tight junctions and form a structural scaffold for the assembly of junctional complexes. MAGI-1 also localizes and plays a role in modulating adherens junction adhesion through scaffolding beta-catenin and PTEN. MAGI-1 overexpression stabilizes adherens junctions and epithelial cell morphology through increased E-cadherin and β-catenin recruitment. Silencing of MAGI-1 has the opposite effect with decreased adherens junction adhesion and reduced focal adhesion formation leading to anchorage-independent growth and migration in vitro. MAGI-1 overexpression suppresses the invasiveness of MDCK cells, as well as suppresses tumor growth and spontaneous lung metastasis through the increased recruitment of PTEN or β-catenin and E-cadherin (Padash Barmchi, 2016).

Overall, MAGI proteins play important roles in the stabilization of cell-cell interactions and as such Magi is a key target in polarized epithelia during cell death and viral infection. For instance, MAGI-1 is cleaved by activated caspases during apoptosis, a process thought to mediate the disassembly of cell-cell contacts (Gregorc, 2007). MAGI proteins are also targeted by a number of oncogenic viruses: it is aberrantly sequestered in the cytoplasm by Adenovirus E4orf1, and is targeted for degradation by the E6 oncoprotein of high-risk human papillomavirus. E6-mediated degradation of MAGI-1 in cultured epithelial cells leads to loss of tight-junction integrity (Padash Barmchi, 2016 and references therein).

There is a high degree of conservation of protein structure and function in the invertebrate homologues of Magi in particular with regards to epithelial junction formation and maintenance. In C. elegans, Magi-1 plays a role in the segregation of different cell adhesion complexes into distinct membrane domains along the lateral plasma membrane. In Drosophila, Magi binds Ras association domain protein 8 (RASSF8) and modulates adherens junctions remodeling in late eye development during interommatidial cell (IOC) rearrangements. In this context Magi function is necessary to recruit the polarity protein Par-3 (Drosophila Bazooka, Baz) to the remodeling adherens junction. However, the association of Drosophila Magi or any Magi homologue with any components of the Par polarity complex in stable epithelia has not been determined (Padash Barmchi, 2016).

The Par complex consisting of Par-3/Par-6/aPKC localizes to tight junctions where MAGI is present in vertebrate epithelial cells and is necessary for assembly of this junctional complex as well as for separation of the apical region of the plasma membrane from the basolateral domain. In Drosophila epithelial cells, the Par complex localizes to the apicolateral membrane and demarcates the boundary between the apical and basolateral membrane regions. Mutant embryos for any member of this complex show loss of apicobasal polarity and disruption in the integrity of epithelia. Although the members of the Par complex are important for the establishment of cell polarity, some of the core components of this complex such as Baz are dispensable for the maintenance of cell polarity during later stages of development. Baz localizes to adherens junction and mutant clones of baz in wing imaginal discs are fully viable with no polarity or adherens junction defects. Similarly, Magi function in AJ stability has been determined in many systems, but surprisingly loss of Drosophila Magi has no effect on established, stable AJs (Zaessinger, 2015). Little is known about the convergence of Magi and Par complex function at the adherens junctions and it is possible that Baz and Magi function in established epithelia are redundant. Therefore this study investigated the role of Magi in the established and stable epithelia of the Drosophila wing imaginal disc to test the potential interactions between Magi and members of the Par complex (Padash Barmchi, 2016).

Drosophila Magi was found associated with the PAR polarity complex and is localized at the adherens junction with Baz, Par-6, and aPKC. Overexpression of Magi resulted in the reduction of apical polarity proteins from the membrane and these interactions required the second half of the Magi protein containing the four PDZ domains. Overexpression of Baz resulted in a reduction of Magi from the membrane but an increase in aPKC and Par-6. While Magi mutants were viable with no polarity defects, Magi levels were found to be antagonistic with Baz, and a balance between the two was found to be necessary to regulate the level and localization of Par complex (Padash Barmchi, 2016).

PDZ domain-containing proteins form scaffolding protein complexes with a wide range of roles including cell polarity and signaling. As a MAGUK protein, Magi is part of a scaffold that interacts with members of the polarity complex at the adherens junctions in the epithelia of the imaginal disc. The scaffolding function of Magi has been well established in other systems. In vertebrates epithelial cells MAGI-1 has been shown to act as structural scaffold at tight junctions and adherens junctions. In C. elegans, Magi-1 localizes apical to adherens junction and functions as an organizer to ensure that different cell adhesion complexes are segregated into distinct membrane domains along the lateral plasma membrane. In neuronal cells MAGI-2/S-SCAM was also shown to cluster the cell adhesion molecule Sidekick, and the AMPA and NMDA glutamate receptors at the synapse (Padash Barmchi, 2016).

Given the strong conservation of the Magi protein it is surprising that null mutants of Drosophila Magi exhibit no lasting cellular defects (other than transient defects in the interommatidial cells of the pupal eye and null animals are fully viable. Similarly in C. elegans, magi-1 null worms are healthy with only a few embryos (1.3%) with defects during the ventral enclosure stage. As Magi is highly conserved, it is plausible that Magi may only act in response to cell stress, DNA damage or some other trigger. For example, loss of p53 does not disrupt cellular function under normal conditions and p53 null flies or mice are viable with no cellular defects. However, the role of p53 in response to DNA damage is well established and when these animals are exposed to irradiation apoptosis is not induced. Alternatively, Magi function might be redundant with other components of the apical polarity complex or another protein and that loss of both is necessary for the disruption of cellular function. Core scaffolding components of the apicobasal polarity complex are dispensable for maintaining polarity in the wing imaginal disc epithelia supporting the idea of redundancy in this system. For instance, somatic clones of loss of function mutations in crb, sdt and baz have no effect on the polarity in the wing disc epithelia of the 3rd instar larvae. Baz is a strong candidate for redundancy with Magi given the localization to the adherens junction and function as a PDZ scaffolding protein. As loss of baz in the wing imaginal disc does not disrupt the polarity of wing disc epithelia this leads to the hypothesis that Baz and Magi are redundant. However, somatic clones of a baz null mutant in a Magi mutant background did not lead to a loss of cell polarity or apoptosis. While the two scaffolding proteins do not appear to functionally interact, it was observed that Magi and Baz are in a protein complex and their close proximity within the wing columnar epithelia also suggests a common complex. Overexpression of Magi displaces Baz and aPKC from the apical membrane and, likewise overexpression of Baz displaces Magi from the membrane. The simultaneous over-expression of Magi and Baz suppresses the changes caused by their individual expression, suggesting a balance or competition between the two proteins. The maintenance of a balance between Magi and Baz might be due to a direct physical competition between these two proteins or opposite effects on a common mediator or interactor (Padash Barmchi, 2016).

Baz and vertebrate MAGI proteins bind the lipid phosphatase PTEN and thus the Magi-Baz interaction and balance could be influenced by changes in the level of phosphoinositides such as PtdIns(4,5)P2 (PIP2) or PtdIns(3,4,5)P3 (PIP3). In polarized epithelia, PIP2 is found within the apical domain and PIP3 restricted to the basal-lateral domain. Baz localization in polarized epithelia depends on PIP2 and on the PI4P5 kinase Skittles. Baz in turn can be a positive regulator of PIP2 levels at the plasma membrane by local recruitment of the lipid phosphatase PTEN. This study observed an increase in PIP3 levels with increased expression of Magi, which may reflect the loss of Baz and a loss of PTEN recruitment to the membrane. This study was not able to assess changes in PTEN levels at the membrane with available antibodies. However it was observe that the recruitment of Magi or Baz was not affected in Pten mutant cells. Similarly the changes in PIP3 levels are unlikely to be the cause of Baz loss in the presence of increased Magi as co-expression of PTEN and Magi still resulted in the loss of Baz from the membrane. Prior studies on Magi in Drosophila in the pupal eye did not detect any physical interaction between Drosophila Magi and Pten, and the phenotypes generated by overexpression of Magi in the Drosophila eye were not affected by Pten mutants. Therefore it is likely that loss of Baz in the presence of increased Magi in the wing imaginal disc and vice versa is through competition for a protein component (Padash Barmchi, 2016).

In the developing eye Magi forms a protein complex with RASSF8 (the N-terminal Ras association domain-containing protein) and ASPP (Ankyrin-repeat, SH3-domain, and proline-rich-region containing protein), and this complex plays a role during remodeling of the adherens junctions in the interommatidial cells (IOCs) (Zaessinger, 2015). When IOCs rearrange to create the pupal lattice, this process requires regulation of the E-Cadherin complex where RASSF8 and ASPP regulate adherens junction remodeling and integrity through regulation of Src kinase activity. Magi recruits the RASSF8-ASPP complex in the process of adherens junction remodeling and there are defects in IOC rearrangement in Magi mutants where AJs are frequently interrupted. In the eye the Magi-RASSF8-ASPP complex is necessary for the cortical recruitment of Baz and of the adherens junction proteins α- and β-catenin. A model has been proposed where Magi-RASSF8-ASPP complex functions to localize Baz to remodeling junctions to promote the recruitment or stabilization of E-Cad complexes (Zaessinger, 2015). However, it is not thought that the RASSF8-ASPP complex is the point of competition between Magi and Baz within the wing imaginal disc. In the wing imaginal disc Magi and the RASSF8-ASPP complex are localized to the adherens junction domain independently (Zaessinger, 2015) and while RASSF8 mutants have a wing rounding phenotype, Magi mutants do not. Furthermore no differences were observed in Baz, Ecad or Arm distribution in Magi somatic loss of function clones in the wing imaginal disc. Finally the Magi WW domains are required for the interaction with RASSF8 (Zaessinger, 2015), while the overexpression of the Magi transgene that contains the PDZ domains led to a reduction in Baz suggesting that second half of the Magi protein containing the PDZ domains contains the important sites for this competition (Padash Barmchi, 2016).

Therefore, a strong possibility to explain the reciprocal effects of overexpression is that Baz and Magi compete for a common binding site. Magi was found to interacte with both Baz and aPKC; the latter two are known to interact directly. However, it is unlikely that the shared site is through physical scaffolding of aPKC, as high levels of wild type aPKC had no effect on either Magi or Baz and was not able rescue the changes in Baz levels and localization caused by Magi overexpression. In addition the overexpression of Magi also led to a reduction in aPKC. It is unlikely that the loss of Baz is responsible for this displacement as aPKC is not mislocalized in Baz clones and Baz is not mislocalized in Par-6, aPKC or Cdc42 null clones. Further investigation is required to explore the mechanisms that underlie Magi interactions with components of the apical polarity complex and the adherens junction complex (Padash Barmchi, 2016).

Cell polarity regulates biased myosin activity and dynamics during asymmetric cell division via Drosophila Rho kinase and Protein kinase N

Cell and tissue morphogenesis depends on the correct regulation of non-muscle Myosin II, but how this motor protein is spatiotemporally controlled is incompletely understood. This study shows that in asymmetrically dividing Drosophila neural stem cells, cell intrinsic polarity cues provide spatial and temporal information to regulate biased Myosin activity. Using live cell imaging and a genetically encoded Myosin activity sensor, Drosophila Rho kinase (Rok) was found to enrich for activated Myosin on the neuroblast cortex prior to nuclear envelope breakdown (NEB). After NEB, the conserved polarity protein Partner of Inscuteable (Pins) sequentially enriches Rok and Protein Kinase N (Pkn) on the apical neuroblast cortex. These data suggest that apical Rok first increases phospho-Myosin, followed by Pkn-mediated Myosin downregulation, possibly through Rok inhibition. It is proposed that polarity-induced spatiotemporal control of Rok and Pkn is important for unequal cortical expansion, ensuring correct cleavage furrow positioning and the establishment of physical asymmetry (Tsankova, 2017).

In asymmetrically dividing fly neural stem cells the protein kinases Rok and Pkn respond to cell polarity cues to regulate Myosin activity and dynamics in a stereotypical, spatiotemporal manner. It is proposed that the sequential regulation mediated by these two kinases is necessary to control Myosin activity and actomyosin dynamics, triggering stereotypic cell shape changes at various steps in the neuroblast cell cycle; first to induce cell rounding as neuroblasts enter mitosis, to permit cell elongation and unequal cortical expansion during anaphase, and finally to complete cytokinesis and the establishment of physical asymmetry (Tsankova, 2017).

Myosin recruitment before NEB is mediated by Rok. This kinase, implicated in Myosin phosphorylation, is already localized at the neuroblast cortex before NEB and in rok mutants, Myosin remains cytoplasmic. At NEB both Rok and Myosin enrich on the apical neuroblast cortex. This apical enrichment, but not cortical localization, depends on the polarity protein Pins since only apical Rok and Myosin enrichment is lost if Pins localization is compromised. Based on these data it is proposed that Rok responds to cell cycle cues, presumably through the small GTPase Rho1, to phosphorylate Myosin's regulatory subunit, enabling activated Myosin to engage with F-actin at the cell cortex prior to NEB. Subsequently, polarity cues enhance Rok on the apical cortex, resulting in the elevation of phosphorylated and, thus, activated Myosin on the apical neuroblast cortex at NEB (Tsankova, 2017).

With Pkn a second kinase has been identified, responding to polarity cues since its apical localization, starting at NEB and peaking by the end of metaphase, is dependent on Pins. Pkn is not absolutely necessary for cortical Myosin enrichment; pkn mutant neuroblasts still retain apical Myosin, although elsewhere on the cortex its localization is dramatically reduced. However, Pkn is required for Myosin's timely relocalization from the apical cortex. Wild-type neuroblasts clear Myosin from the apical cortex in early anaphase, creating an asymmetric distribution that is necessary for the unequal cortical expansion. In pkn mutants, however, both Rok and Myosin dynamics are changed, retaining both on the apical neuroblast cortex, causing aberrant cortical constrictions and concomitantly inverted polar expansion (Tsankova, 2017).

Based on these results, the following model is proposed. (1) Rok triggers cortical Myosin accumulation before NEB. (2) At NEB, apically localized Pins enriches Rok on the apical neuroblast cortex and concomitantly increases phospho-Myosin apically. (3) Pins also induces the apical enrichment of Pkn, which is necessary for the timely relocalization of Myosin from the apical neuroblast during metaphase. It is further proposed that Pkn is downregulating Myosin activity through inhibiting or downregulating apical Rok activity. Whether Pkn downregulates Rok activity by direct phosphorylation remains an attractive hypothesis, since vertebrate Rock2 has recently been identified as a Pkn target. Alternatively, Rok activity could be regulated independently of phosphorylation but governed by the length of its coiled-coil tether, linking the kinase domain with the membrane binding domain (Tsankova, 2017).

This sequential regulation of Myosin dynamics seems to be a key regulatory mechanism underlying physical asymmetric cell divisions. For instance, apical Myosin relocalization always precedes basal Myosin clearing in wild-type neuroblasts. Similarly, biasing the localization of activated Myosin affects cleavage furrow positioning and physical asymmetry (Tsankova, 2017).

It is hypothesized that polarity cues provide a cell-intrinsic timer, priming Myosin relocalization on the apical cortex, thereby ensuring the generation of physical asymmetry through unequal cortical extension. Polarity-induced enrichment of activated Myosin on the apical cortex could thus provide a symmetry breaking event, necessary for the subsequent induction of apical Myosin clearing. Consistent with this model is the finding that pins mutants, or uniform cortical localization of Pins, cause Myosin to clear from both poles at the same time and divide symmetrically by size (Tsankova, 2017).

Tissue and organ growth critically depends on the correct spatiotemporal regulation of cell division. This study provides a conceptual framework of how Rok and Pkn respond to both cell cycle and polarity cues. These cues, in conjunction with spindle-dependent signals, ensure correct physical asymmetric cell division that is necessary for stem cell homeostasis and cell differentiation. Spatial and temporal regulation of Myosin activity has also been shown to be important for pulsatile cell shape changes in the Drosophila embryo. Rok and Pkn play important roles during vertebrate development and morphogenesis , and it will be interesting to see how spatiotemporal cues, affecting local cell shape changes, are coordinated with overall tissue morphogenesis in flies and beyond (Tsankova, 2017).

Intra-lineage fate decisions involve activation of Notch receptors basal to the midbody in Drosophila sensory organ precursor cells

Notch receptors regulate cell fate decisions during embryogenesis and throughout adult life. In many cell lineages, binary fate decisions are mediated by directional Notch signaling between the two sister cells produced by cell division. How Notch signaling is restricted to sister cells after division to regulate intra-lineage decision is poorly understood. More generally, where ligand-dependent activation of Notch occurs at the cell surface is not known, as methods to detect receptor activation in vivo are lacking. In Drosophila pupae, Notch signals during cytokinesis to regulate the intra-lineage pIIa/pIIb decision in the sensory organ lineage. This study identified two pools of Notch along the pIIa-pIIb interface, apical and basal to the midbody. Analysis of the dynamics of Notch, Delta, and Neuralized distribution in living pupae suggests that ligand endocytosis and receptor activation occur basal to the midbody. Using selective photo-bleaching of GFP-tagged Notch and photo-tracking of photo-convertible Notch, this study showed that nuclear Notch is indeed produced by receptors located basal to the midbody. Thus, only a specific subset of receptors, located basal to the midbody, contributes to signaling in pIIa. This is the first in vivo characterization of the pool of Notch contributing to signaling. A simple mechanism of cell fate decision based on intra-lineage signaling is proposed: ligands and receptors localize during cytokinesis to the new cell-cell interface, thereby ensuring signaling between sister cells, hence intra-lineage fate decision (Trylinski, 2017).

Several methods are currently available to monitor in vivo the signaling activity of Notch by measuring the level and/or activity of NICD. By contrast, in vivo reporters for ligand-receptor interaction, conformational change of Notch in response to mechanical force, and S2 cleavage of Notch are lacking. Consequently, the subcellular location of Notch receptor activation in vivo and the relative contribution of the different pools of Notch to signaling remain unknown. Two complementary fluorescent-based approaches have been developed in this study to track where NICD comes from. Notch receptors present basal to the midbody along the pIIa-pIIb interface were shown to contribute to the accumulation of NICD, whereas receptors located apical to the midbody did not significantly contribute to NICD production. This study provides the first in vivo analysis of ligand-dependent Notch receptor activation at the cell surface. Moreover, the photo-bleaching and photo-conversion approaches used in this study should be broadly applicable in model organisms that can be genetically engineered and easily imaged (Trylinski, 2017).

Other sites of Notch activation had previously been proposed in pIIa. In one model, based on the specific requirements for Arp2/3 and WASp activities for both Notch signaling and actin organization, Dl at apical microvilli in pIIb would activate Notch located apically in pIIa. However, loss of Arp2/3 activity also disrupted cortical actin along the basal pIIa-pIIb interface, suggesting that regulation of the actin cytoskeleton at this location, rather than at microvilli, may be key for receptor activation. In a second model, Dl-Notch signaling was proposed to occur at the new apical pIIa-pIIb junction. This model was largely based on the detection of Notch at this location. The current study, however, indicated that this pool of Notch did not significantly contribute to the production of NICD in pIIa. In a third model, Notch activation was proposed to occur in specific Sara-positive endosomes in pIIa. Whereas the possible contribution of these endosomes to NICD production could not be directly addressed by photo-tracking, two lines of evidence suggest that their contribution can only be minor. First, live imaging of Notch failed to detect this pool indicating that this pool represents a minor fraction of Notch in pIIa. Second, symmetric partitioning of Sara endosomes did not affect the pIIa-pIIb decision, indicating that this proposed pool is not essential for fate asymmetry. Finally, the nature of the mechanical force acting on Notch at the limiting membrane of the Sara-positive endosomes remains to be addressed. In summary, all available data are fully consistent with the conclusion that receptor activation occurs mostly basal to the midbody (Trylinski, 2017).

Whereas these experiments identified the signaling pool of Notch along the pIIa-pIIb, they did not, however, address whether S3 cleavage takes place at the cell surface or intracellularly following endocytosis. Indeed, the photo-tracking approach used in this study did not inform whether the activation of Notch by Delta, i.e., s2 cleavage, is followed by S3 cleavage at the same location or whether S2-processed Notch is internalized to be further processed in signaling endosomes. It is noted, however, that the accumulation of lateral Notch observed in Psn mutant cells is consistent with S3 cleavage taking place, at least in part, at the cell surface (Trylinski, 2017).

This work also sheds new light on the general mechanism whereby Notch signaling is specifically restricted to sister cells within a lineage. In several tissues, including the gut, lung, and CNS, Notch regulates intra-lineage decisions between sister cells soon after mitosis. In this study it is proposed that Notch-mediated intra-lineage decisions are directly linked to division. Indeed, it is suggested that ligands and receptors localize to the lateral membranes that separate the two sister cells at cytokinesis so that Dl-Notch signaling is primarily restricted to sister cells. Thus, neighboring cells - belonging to other cell lineages - would not interfere with intra-lineage fate decisions. The current data indicating that Neur-dependent activation of Notch by Dl predominantly occurs along the pIIa-pIIb lateral interface, basal to the midbody during cytokinesis, fully support this model. Also, the observation that core components of the secretory machinery, e.g., Sec15, are specifically required for Notch signaling in the context of intra-lineage decisions is also consistent with this view. Thus, targeting both receptors and ligands along the newly formed interface during cytokinesis provides an elegant mechanism to restrict signaling between sister cells, thereby ensuring that intra-lineage signaling regulates intra-lineage fate decision. Because Notch generates fate diversity within neural lineages in both vertebrates and invertebrates, this mechanism of intra-lineage signaling may be conserved (Trylinski, 2017).

Drosophila nucleostemin 3 is required to maintain larval neuroblast proliferation

Stem cells must maintain proliferation during tissue development, repair and homeostasis, yet avoid tumor formation. In Drosophila, neural stem cells (neuroblasts) maintain proliferation during embryonic and larval development and terminate cell cycle during metamorphosis. An important question for understanding how tissues are generated and maintained is: what regulates stem cell proliferation versus differentiation? A genetic screen was performed that identified nucleostemin 3 (ns3) as a gene required to maintain neuroblast proliferation. ns3 is evolutionarily conserved with yeast and human Lsg1, which encode putative GTPases and are essential for organism growth and viability. NS3 is cytoplasmic and it is required to retain the cell cycle repressor Prospero in neuroblast cytoplasm via a Ran-independent pathway. NS3 is also required for proper neuroblast cell polarity and asymmetric cell division. Structure-function analysis further shows that the GTP-binding domain and acidic domain are required for NS3 function in neuroblast proliferation. It is concluded NS3 has novel roles in regulating neuroblast cell polarity and proliferation (Johnson, 2018).

aaquetzalli is required for epithelial cell polarity and neural tissue formation in Drosophila

Morphogenetic movements during embryogenesis require dynamic changes in epithelial cell polarity and cytoskeletal reorganization. Such changes involve, among others, rearrangements of cell-cell contacts and protein traffic. In Drosophila, neuroblast delamination is regulated by the Notch signaling pathway. Maintenance of epithelial cell polarity ensues proper Notch pathway activation during neurogenesis. This study characterized aaquetzalli (aqz), a gene whose mutations affect cell polarity and nervous system specification. The aqz locus encodes a protein that harbors a domain with significant homology to a proline-rich conserved domain of nuclear receptor co-activators. aqz expression occurs at all stages of the fly life cycle, and is dynamic. aqz mutants are lethal, showing a disruption of cell polarity during embryonic ventral neuroepithelium differentiation resulting in loss of epithelial integrity and mislocalization of membrane proteins (shown by mislocalization of Crumbs, DE-Cadherin, and Delta). As a consequence, aqz mutant embryos with compromised apical-basal cell polarity develop spotty changes of neuronal and epithelial numbers of cells(Mendoza-Ortiz, 2018).

Asymmetric nuclear division in neural stem cells generates sibling nuclei that differ in size, envelope composition, and chromatin organization

Although nuclei are the defining features of eukaryotes, how the nuclear compartment is duplicated and partitioned during division is still do not fully understand. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, it was found that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a >30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fate determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate (Roubinet, 2021).

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. This study shows that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex (de Torres-Jurado, 2022).

A precise regulation of ASCD is critical in development, tissue homeostasis, and tumorigenesis. Extrinsic signals from specialized microenvironments, the niches, importantly contribute to that regulation by promoting the stem cell fate in the daughter cell that receives those signals, whereas the other daughter cell enters a differentiation program. Intriguingly, the ASCD of some stem cells, including the Drosophila CNS stem cells called NBs, the subject of this study, seems to depend exclusively on intrinsic regulatory mechanisms. Then, are those stem cells completely independent of their surrounding environment? Also, how general is the requirement of niches and the signals secreted from them for maintaining the stem cell fate in an ASCD (de Torres-Jurado, 2022)?

This study shows that the ASCD of Drosophila NBs, a traditional paradigm for studying intrinsic ASCD regulatory mechanisms, do also depend on extrinsic cues secreted by a glial niche, which are in close contact with those neural stem cell lineages in the larval brain. However, these extrinsic signals are not required to maintain the stem cell fate. They ultimately impact on the regulation of intrinsic factors to induce differentiation in one daughter cell, repressing the self-renewal 'basal state' in this cell. Different studies have shown the possibility of growing in culture isolated larval NBs, which are able to form crescents and divide asymmetrically without any additional extrinsic signal. However, most of these experiments were performed using central brain type I NB lineage (NBI) NBs, which do not require Fra and Robo1 signaling for their correct development, or included type II NB lineage (NBII,) but only particular markers were analyzed (i.e., Baz and Pon). This is relevant as, for example, it was observed that the localization of some ASCD regulators were not affected without extrinsic signals (i.e., Insc, Mira, and Brat). In the experiments in culture, some of the latter regulators are frequently used. It is also key to properly quantify the cases of crescent formation at metaphase, as the phenotypes are never fully penetrant and can be even totally rescued at telophase. For example, in the system used in this study, aPKC showed about 50% localization failures at metaphase, implying that there are NBIIs that show aPKC crescents. Nevertheless, finding that glia-secreted cues are specifically required in NBII lineages was indeed very intriguing. NBII lineages are larger than NBI, as they undergo an additional proliferation phase through INPs and hence are more prone to induce tumor-like overgrowth when ASCD fails. Thus, additional levels of regulation might have evolved in these lineages to ensure the correct division of the NB and INPs to avoid overgrowths. This issue will be further examined in the future (de Torres-Jurado, 2022).

This work has unveiled a novel function for the axon guidance cues Netrins and their Fra/DCC-like receptor in regulating self-renewal versus differentiation in neural stem and progenitor cells of larval NBII lineages. The cortex glial niche that surrounds those lineages secretes Netrins, which modulate Robo1 signaling threshold levels through Fra and Abl kinase in stem and progenitor cells. Whereas Robo1 signaling is activated by its ligand Slit, also secreted by the glial niche, Abl kinase represses this signaling, and these balanced Robo1 signaling levels appear to be critical for the cell-fate commitment of the daughter cell prone to differentiate. The cortex glia dynamically undergoes remodeling through larval stages; only at late third instar larval stages (L3) the glia chamber enwrapping each NB lineage forms completely. This study already observed the presence of the secreted ligands Slit and NetA in the cortex glia at L2, suggesting that these cues are being secreted from the glia since the reactivation of dormant NBs at early L2 (de Torres-Jurado, 2022).

Slit-Robo signaling regulates progenitor cell proliferation in the mammalian CNS and promotes the terminal asymmetric division of a differentiation-committed cell in the Drosophila CNS. Likewise, in mammary stem cells, Robo1 favors their asymmetric mode of cell division. Slit-Robo signaling is also required in other stem or progenitor cells to regulate their lineage specification, identity, or their adhesion/anchoring to the niche. No role for Netrin-Fra/DCC signaling has been previously described in all those contexts (de Torres-Jurado, 2022).

Ultimately, a transcriptional control has been pointed out as the most common way of action by which Robo signaling regulate the above-mentioned cellular processes. This study shows a novel, transcription-independent mechanism by which Robo1 signaling regulates ASCD. Robo1 signaling would be required to activate the small GTPases Rac1 and Cdc42 by repressing its inhibitor RhoGAP93B as well as by recruiting the Dock-Pak complex, which, through Pak, can also bind activated forms of both Rac1 and Cdc42 Rac1 and Cdc42 downregulation directly impacted on the intrinsic machinery that modulate ASCD in neural stem and progenitor cells. Specifically, compromising those small GTPases led to defects in the localization of the ASCD regulators, Par-6, aPKC, Cno, and Numb, and the concomitant formation of ectopic NBs (eNBs) within brain neural lineages, a phenotype that recapitulates that of Slit-Robo1 signaling impairment. The overexpression of robo1 caused similar defects than the loss of robo1. It was also a similar phenotype than for the loss of fra, the downregulation of Abl, or the expression of a kinase dead form of Abl (unable to repress Robo1). Hence, based on all those experiments, a working model proposes that Netrin-Fra signaling would be modulating, through Abl kinase, the Robo1 signaling threshold levels necessary to regulate in turn the correct activity of the small GTPases Rac1 and Cdc42. In fact, and according to this, the expression of Rac1V12 within NBII lineages caused the formation of eNBs and led to defects in the localization of aPKC in NB and progenitor cells, a similar phenotype than that observed after overexpressing robo1 in these NBII lineages. It would be interesting to determine whether this novel function of Netrin-Fra/DCC signaling regulating ASCD is also conserved in vertebrates, and whether Robo/Rac1-Cdc42 signaling threshold levels in the above-mentioned contexts are also critical for and dependent on Netrin-DCC signaling, as was have found in Drosophila (de Torres-Jurado, 2022).

Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage

During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. this study tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. Unique features were found that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination (Ranjan, 2022).

The last-born daughter cell contributes to division orientation of Drosophila larval neuroblasts

Controlling the orientation of cell division is important in the context of cell fate choices and tissue morphogenesis. However, the mechanisms providing the required positional information remain incompletely understood. This study used stem cells of the Drosophila larval brain that stably maintain their axis of polarity and division between cell cycles to identify cues that orient cell division. Using live cell imaging of cultured brains, laser ablation and genetics, this study reveals that division axis maintenance relies on their last-born daughter cell. It is proposed that, in addition to known intrinsic cues, stem cells in the developing fly brain are polarized by an extrinsic signal. It was further found that division axis maintenance allows neuroblasts to maximize their contact area with glial cells known to provide protective and proliferative signals to neuroblasts (Loyer, 2018).

Deciphering the signals that provide positional information is a central issue in understanding how cell divisions are oriented. This study addressed this question in the highly proliferative NBs in the Drosophila larval brain, which maintain their division axis from one cell cycle to the next in part by using an apical microtubule network as a spatial cue to specify their apico-basal polarity axis and consequently the orientation of mitosis. Attempts were made to understand why NBs only partially fail to maintain their division axis upon loss of this intrinsic polarizing cues, and it was found out that the last-born daughter cell of NBs participates to their division axis maintenance. These results also shed light on some aspects of the physiological importance of division axis maintenance in larval NBs, which has remained elusive. Control of NB division orientation may provide a means to maximize NB/cortex glia surface area to allow optimum protection against environmental stresses by the cortex glia (Loyer, 2018).

Under normal conditions, about 80% of the surface of NBs is in direct contact with a cortex glia and NBs with partially defective division axis maintenance display reduced contact with cortex glia. This most likely directly results from NBs producing progeny between themselves and the cortex glia when the last-born daughter cell derived cue that positions normally the apico-basal polarity axis is damaged. This seems to be important for the protective function of these glial cells on NB proliferation under stress conditions. Indeed, NBs with reduced surface contact to cortex glia appear to be less well protected by glial cells, as was observed a significant increase of sensitivity to oxidative stress using an established assay. However, despite this reduction being statistically significant, only a 9% reduction was measured in NB/cortex glia contact area. On a normal diet, addition of the oxidant tert-butyl hydroperoxide (tbh) results in a 14% drop in NB proliferation when the formation of lipid droplets mediating this protection is prevented. It is therefore surprising that in these experiments reducing the NB/cortex glia contact area by only ~9% in Cindr-depleted NB is already accompanied by a similar drop in proliferation upon tbh treatment. Therefore, although this decrease may directly result from interfering with the protection provided by cortex glia, other unrelated functions of Cindr in protecting NBs against the effect of tbh cannot be ruled out (Loyer, 2018).

It was initially hypothesized that the last-born GMC could act as an additional, extrinsic cue maintaining NB division orientation. A number of observations are consistent with this possibility: it was observed that, upon (perhaps artefactual) last-born GMC movements, NBs realign their division axis toward this GMC; ablation of the last-born GMC and depletion of proteins specifically observed at the last-born GMC/NB interface affect division axis maintenance by misorienting the apico-basal polarity of NBs. It cannot be excluded that the entire NB and any intrinsic spatial cue that it carries simply rotate upon migration or ablation of the last-born GMC or depletion of proteins specifically observed at the last-born GMC/NB interface. Thus the last-born GMC may participate in division axis maintenance by preventing NB rotation. This function could be mediated by specific adhesive contacts at the interface with the NB, plausible given the numerous specific characteristics that were observed at that interface. In particular, the midbody carried by this interface, although not likely to act itself as a stable physical link given its possible ability to migrate within the fluid mosaic of the plasma membrane and the fact that its internalization does not affect division orientation maintenance, may be able to organize specific adhesive contacts at the NB/last-born GMC interface (Loyer, 2018).

An alternative hypothesis is that the last-born GMC provides a cue that more directly functions in specifying the orientation of the apico-basal polarity axis by polarizing Baz, which functions upstream of NB division orientation control. Consistently, despite affecting division orientation maintenance, neither GMC ablation nor RNAi of Cindr disrupt alignment of the mitotic spindle with the polarity axis. In this case, the molecular mechanism through which a positional information provided by the last-born GMC is transduced to the NB polarization machinery remains to be determined. Although bearing similarities with division axis maintenance in budding yeasts, relying on a Septin-rich cytokinesis remnant, the midbody of NBs is unlikely to directly control polarization as midbody internalization does not affect division axis maintenance. Instead, it is proposed that the midbody may organize various other specific components of the last-born GMC/NB interface that in turn may directly control NB polarization. This could be the case of cell-cell contacts organized by the midbody, consistent with the involvement of an adhesion molecule such as Roughest, whose mammalian orthologue physically interacts with Septins, and the fact that GMC ablation, although not directly targeting the interface, affects division axis maintenance. Another promising candidate potentially controlling NB polarity are the plasma membrane tubules probably organized by the midbody, given their physical origin (the midbody) and the timing (immediately after cytokinesis) of their appearance. Interestingly, a physical interaction was observed between Septins and the mammalian orthologue of Cindr, found enriched at the tubules and involved in division axis maintenance. Tubules function might be linked to the integrity of the last-born GMC/NB interface, which itself probably depends on the integrity of the last-born GMC. While these tubules do not disappear upon GMC ablation, it would be of particular interest to monitor whether tubules morphology, dynamics or the enrichment of Flare and Cindr are affected by ablation of the last-born daughter cell (Loyer, 2018).

Interestingly, proteins that were found to be involved in division axis maintenance were described to interact with polarity complexes in other contexts: Septins genetically interact with Baz during Drosophila embryogenesis, and the mammalian orthologues of Roughest regulate podocyte polarity by physically interacting with Par-3. However, both Septins and Roughest localize to the basal pole of NBs, whereas Baz polarizes apically. Therefore, how could a cue received at the basal pole direct polarization of Baz, at the opposite apical pole of the NB? In the C. elegans zygote, the sperm entry point acts as a cue inducing an actomyosin flow establishing Par complex polarity at the opposite end of the cell. Septins, Cindr, Roughest and Flare can be linked in one way or another to the regulation of actomyosin, and at least the maintenance of Baz localization in mitotic NBs is also actin-dependent. Intracellular long-range control of polarization has been further observed in eight-cell stage mouse blastomeres, where cell-cell contacts induce apical polarization at the opposite end of the cell. A promising lead for future work is the possible involvement of actomyosin-dependent mechanical forces in such long-range control of polarity in NBs. Indeed, tensions participate in polarization in the C. elegans zygote, were proposed to mediate polarization of eight-cell stage mouse blastomeres and maintain polarity in migrating neutrophils (Loyer, 2018).

Asymmetric recruitment and actin-dependent cortical flows drive the neuroblast polarity cycle

During the asymmetric divisions of Drosophila neuroblasts, the Par polarity complex cycles between the cytoplasm and an apical cortical domain that restricts differentiation factors to the basal cortex. This study used rapid imaging of the full cell volume to uncover the dynamic steps that underlie transitions between neuroblast polarity states. Initially, the Par proteins aPKC and Bazooka form discrete foci at the apical cortex. Foci grow into patches that together comprise a discontinuous, unorganized structure. Coordinated cortical flows that begin near metaphase and are dependent on the actin cytoskeleton rapidly transform the patches into a highly organized apical cap. At anaphase onset, the cap disassembles as the cortical flow reverses direction toward the emerging cleavage furrow. Following division, cortical patches dissipate into the cytoplasm allowing the neuroblast polarity cycle to begin again. This work demonstrates how neuroblasts use asymmetric recruitment and cortical flows to dynamically polarize during asymmetric division cycles (Oon, 2019).

The dynamics that accompany transitions between unpolarized and polarized states of Drosophila neuroblasts were examined using rapid imaging throughout the full volume of the cell. These data reveal that canonical neuroblast polarity, with the Par complex's catalytic component aPKC tightly localized around the apical pole at metaphase, results from a multistep process. Initially, asymmetric recruitment to the apical cortex leads to a discontinuous structure composed of apical cortical patches. Coordinated cortical flows that begin late in prophase lead to coalescence of the patches into an apical cap. Also, a remarkably dynamic depolarization step following metaphase polarity was discovered in which the apical cap is broken up into cortical patches that spread to the cleavage furrow and ultimately dissipate back into the cytoplasm (see The Neuroblast Polarity Cycle). This study examined the role of the actin cytoskeleton in the steps that make up the neuroblast polarity cycle and found that it is critical for several different aspects of polarization and depolarization (Oon, 2019).

In principle, cortical polarity could result from directional cortical flow of initially symmetric cortical molecules, or from asymmetric cortical targeting directly from the cytoplasm. In the early worm embryo, aPKC is initially symmetrically localized to evenly distributed cortical foci. The cortical cue provided by sperm entry induces anterior directed flows that deplete aPKC foci from the posterior cortex and concentrate it in the anterior hemisphere (Rose, 2014). In contrast to the early worm embryo, neuroblasts begin their polarization cycle with cytoplasmic aPKC such that asymmetry in the cortical recruitment process could be sufficient for polarization. It was observed that neuroblast polarity begins with asymmetric recruitment but that this process alone leads to a discontinuous polarized structure in the apical hemisphere. Coordinated cortical flows toward the apical pole that begin near metaphase and resemble the polarization of the early worm embryo, transform this unorganized structure into the tightly focused metaphase polarity state. Thus, neuroblast polarity results not from a single process, but from the stepwise activity of two very different cellular processes: asymmetric targeting and actin- dependent cortical flow (Oon, 2019).

Given that neuroblasts undergo repeated asymmetric divisions, the neuroblast polarity cycle also includes a depolarization step to regenerate cytoplasmic aPKC, the initial state in the cycle. Rather than directly returning to the cytoplasm from the apical cap,a dramatic cap disassembly step was observed that appears similar to the assembly step but in reverse: the cap breaks up into aPKC patches that move toward the basal rather than apical pole. It is speculates that cap disassembly may play an especially important role in segregating fate determinants by extending aPKC fully along the cortex to the cleavage furrow, but not beyond. Cortical spreading of aPKC could provide a mechanism for ensuring basal fate determinants such as Miranda and Numb are completely excluded from the cortex that becomes part of the self-renewed neuroblast following cytokinesis. Is cap disassembly an active process? As it is initiated precisely when the dramatic morphologic changes in anaphase occur, it may be that this step utilizes a passive mechanism, in which disassembly is driven by the mechanical stresses that the cortex undergoes during this step of the cell cycle (Oon, 2019).

The cycle that was identified in this study represents a new framework for understanding the mechanisms that regulate neuroblast polarity. This framework is being utilized to examine the role the actin cytoskeleton plays in the polarity cycle. While the actin cytoskeleton has been known to be required for metaphase polarity for some time with normally apical proteins such as Inscuteable becoming fully cortical at metaphase when actin filaments are depolymerized, its precise role has been unclear. This study found that the fully cortical depolarized state can result from a polarized intermediate: in interphase treated neuroblasts aPKC is asymmetrically recruited during prophase but rapidly spreads onto the basal cortex. Thus, at least for aPKC, the actin cytoskeleton is not required for polarized cortical recruitment, but is instead necessary for retention at the apical cortex. Treatment of neuroblasts with LatA at various stages of the cell cycle also revealed that the coalescence of aPKC and Baz patches into a metaphase apical cap and cap disassembly both require an intact actin cytoskeleton. It is suspected that the analysis of other perturbations in terms of the neuroblast polarity cycle, such as mutants of previously described polarity genes, will lead to new insight into the mechanisms by which animal cells become polarized (Oon, 2019).

Phases of cortical actomyosin dynamics coupled to the neuroblast polarity cycle

The Par complex dynamically polarizes to the apical cortex of asymmetrically dividing Drosophila neuroblasts where it directs fate determinant segregation. Previously it was shown that apically directed cortical movements that polarize the Par complex require F-actin. This paper report the discovery of cortical actomyosin dynamics that begin in interphase when the Par complex is cytoplasmic but ultimately become tightly coupled to cortical Par dynamics. Interphase cortical actomyosin dynamics are unoriented and pulsatile but rapidly become sustained and apically-directed in early mitosis when the Par protein aPKC accumulates on the cortex. Apical actomyosin flows drive the coalescence of aPKC into an apical cap that is depolarized in anaphase when the flow reverses direction. Together with the previously characterized role of anaphase flows in specifying daughter cell size asymmetry, the results indicate that multiple phases of cortical actomyosin dynamics regulate asymmetric cell division (Oon, 2021).

The results reveal previously unrecognized phases of cortical actomyosin dynamics during neuroblast asymmetric division, several of which coincide with the neuroblast's cortical polarity cycle. During interphase, transient cortical patches of actomyosin undergo highly dynamic movements in which they rapidly traverse the cell cortex, predominantly along the cell's equator, before dissipating and beginning a new cycle. Shortly after mitotic entry the movements become more continuous and aligned with the polarity axis (orthogonal to the equatorial interphase pulses). The transition to apically directed cortical actin movements occurs shortly before the establishment of apical Par polarity, when discrete cortical patches of aPKC undergo coordinated movements toward the apical pole to form an apical cap. Importantly, cortical actin dynamics are required for aPKC to coalesce into an apical cap. Apically directed actin dynamics continue beyond metaphase when apical aPKC cap assembly is completed, suggesting that actomyosin dynamics may also be involved in cap maintenance. A role for actomyosin in aPKC cap assembly and maintenance is supported by the lack of coalescence when the actin cytoskeleton is completely depolymerized, or when actin dynamics are inhibited but the cytoskeleton is left intact. The cycle of cortical actomyosin dynamics is completed when the movement abruptly changes direction at anaphase leading to the cleavage furrow-directed flows that have been previously characterized. While this study examined the relationship between actomyosin dynamics and cortical protein polarity, it is noted that a neuroblast membrane polarity cycle was recently discovered and found to require the actin cytoskeleton. The mechanical phases of the membrane polarity cycle may be related to the phases of cortical actomyosin dynamics reported in this study (Oon, 2021).

While cortical actomyosin dynamics had not been reported during neuroblast polarization, myosin II pulses have been observed in delaminating neuroblasts from the Drosophila embryonic neuroectoderm. The actomyosin dynamics reported in this study may be related to those that occur during delamination and provide a framework for understanding how actomyosin participates in neuroblast apical polarity. First, apically directed movements of actomyosin are consistent with the requirement for F-actin in the coalescence of discrete aPKC patches into an apical cap. How might cortical actomyosin dynamics induce aPKC coalescence and maintenance? In the worm zygote, pulsatile contractions generate bulk cortical flows (i.e. advection) that lead to non-specific transport of cortically localized components. Whether the cortical motions of polarity proteins that occur during the neuroblast polarity cycle are also driven by advection will require further study (Oon, 2021).

The more rapid depolarization of aPKC in Lat- compared to CytoD-treated neuroblasts, is also consistent with a potentially passive role for the actin cytoskeleton in polarity maintenance. Complete loss of the cortical actin cytoskeleton (LatA) leads to more rapid entry of aPKC into the basal neuroblast membrane compared to when cortical actin dynamics is inhibited but the structure maintained (CytoD). The difference could arise simply from an increase in cortical diffusion constant when the cortical actin mesh is removed. In this case, the actin cytoskeleton would participate in Par polarity via at least two mechanisms: by generating non-diffusive movements of polarity proteins through actomyosin-generated cortical flows, and by maintaining the polarized state by slowing the rate of diffusion (Oon, 2021).

Scribble and Discs-large direct initial assembly and positioning of adherens junctions during the establishment of apical-basal polarity

Apical-basal polarity is a fundamental property of animal tissues. Drosophila embryos provide an outstanding model for defining mechanisms that initiate and maintain polarity. Polarity is initiated during cellularization, when cell-cell adherens junctions are positioned at the future boundary of apical and basolateral domains. Polarity maintenance then involves complementary and antagonistic interplay between apical and basal polarity complexes. The Scribble/Dlg module is well-known for promoting basolateral identity during polarity maintenance. This study reports a surprising role for Scribble/Dlg in polarity initiation, placing it near the top of the network-positioning adherens junctions. Scribble and Dlg are enriched in nascent adherens junctions, are essential for adherens junction positioning and supermolecular assembly, and also play a role in basal junction assembly. The hypotheses were tested for the underlying mechanisms, exploring potential effects on protein trafficking, cytoskeletal polarity or Par-1 localization/function. The data suggest that the Scribble/Dlg module plays multiple roles in polarity initiation. Different domains of Scribble contribute to these distinct roles. Together, these data reveal novel roles for Scribble/Dlg as master scaffolds regulating assembly of distinct junctional complexes at different times and places (Bonello, 2019).

Identifying the earliest symmetry-breaking events that initially position AJs, thereby setting the boundary between apical and basolateral domains, is a key aspect of understanding how polarity is established. This study reports that Scrib/Dlg, best known for roles as basolateral determinants during polarity maintenance, play a separate and surprising role in organizing AJs during polarity establishment, positioning them at the top of the polarity network (Bonello, 2019).

Scrib and Dlg are multidomain proteins with many partners, allowing them to serve diverse biological functions, from synaptogenesis to oriented cell division. The data reveal they play distinct roles during polarity establishment and polarity maintenance, likely engaging very different sets of binding partners. This is supported by the evolving localization pattern of Scrib/Dlg on the plasma membrane, with sequential co-localization with and roles in positioning AJ versus SJ proteins, suggesting the capacity to engage with and position distinct junctional and polarity proteins. These analyses also begin to dissect the underlying molecular basis. Scrib's PDZ domains are important for the precision of initial polarity establishment but are redundant with other mechanisms for polarity maintenance after gastrulation, though they regulate SJ positioning (Bonello, 2019).

AJs play a key role at the boundary between apical and basolateral domains, and building a functional junction is a multistep process. This includes assembling the core cadherin-catenin complex, positioning it, and supermolecular assembly. Assembly of the core complex appears to occur coincident with synthesis, and thus small puncta are already present before cellularization. As cellularization proceeds, these are captured at the apicolateral interface in a process requiring Baz, Cno, and an intact actin cytoskeleton, where they coalesce into SAJs, with ~1500 AJ complexes and 200 Baz proteins. Cadherin-catenin complexes form independently of either Baz or Cno, but AJ positioning and full supermolecular assembly depend on both. This study found that Scrib/Dlg are also key for AJ apicolateral retention and supermolecular assembly, although Arm and Cno remain associated in misplaced puncta, and thus core AJ complexes remain intact. Further, a second junctional complex that arise during polarity establishment, the BJs, also require Scrib/Dlg for its supermolecular organization. Unlike AJs, BJ organization is not dependent on other polarity determinants including Cno, Rap1 or Par-1. It will be of interest to examine if Scrib/Dlg act via known regulators of cadherin clustering, including intrinsic (e.g., cis- and trans-interactions of cadherins) and extrinsic (e.g., local actin regulation, endocytosis) factors (Bonello, 2019).

The ultimate goal is to define molecular mechanisms underlying polarity establishment. The new data place Scrib/Dlg in a critical position near the top of the network, but also suggest they act via multiple effectors. Perhaps the strongest evidence for multiple roles with distinct effectors comes from analysis of scrib4. Supermolecular organization of both SAJs and BJ must involve interactions with specific partners via the PDZ domains- one speculative possibility is that these include core AJ proteins, as βcatenin can coIP with Scribble and interact with PDZ domains 1 and 4. Testing this idea will be an important future direction. This initial role may also involve modulating Par-1. During cellularization, Scrib/Dlg and Par-1 localize in 'inverse gradients': Scrib and Dlg enriched at the SAJ level, and Par-1 with higher cortical intensity basolaterally. Scrib/Dlg play a role in effective membrane recruitment of Par-1 at this stage, and effects of par-1-RNAi on SAJ protein localization during cellularization are largely similar to those of scrib-RNAi. However, regulating Par-1 is not the only mechanism by which Scrib/Dlg act, as AJs are rescued during gastrulation after par-1-RNAi (Bonello, 2019).

Scrib then plays a second PDZ-independent role as gastrulation begins, ensuring focusing of cadherin-catenin complexes and Baz into apical belt AJs. This requires the N-terminal LRRs but not the PDZs. Positioning Baz at this stage involves at least two inputs which are redundant with one another, one via Par-1 and one via an apical transport mechanism. One speculative possibility is that Scrib/Dlg also regulate protein trafficking, a role they have in other contexts. However, disrupting Scrib/Dlg function has very different consequences than disrupting Rab5-dependent trafficking, suggesting they do not act via Rab5. aPKC also provides important cues at this stage-perhaps Scrib/Dlg regulate aPKC localization or function. It will be important to further explore the nature of this second role (Bonello, 2019).

The initial goal more than a decade ago was to define roles of AJs in polarity establishment. However, it rapidly became apparent AJs are not at the top of the hierarchy. Cno, Rap1 and Baz act upstream of AJ positioning and supermolecular assembly. The new data moves understanding another step upward in the network, revealing a key role for Scrib/Dlg in regulating AJ positioning and assembly. However, they also reveal that the process is not a simple linear pathway, and raise new questions. Loss of Scrib or Dlg almost completely disrupts AJs during cellularization. However, effects on Baz and Cno are less complete-supermolecular assembly is affected, but they are retained in the apical half of the membrane. This suggests other cues are involved. The ultimate polarizing cue during syncytial development is the oocyte membrane, which then directs cytoskeletal polarization. Cytoskeletal cues regulate Cno localization. While the data rule out a role for Scrib/Dlg in establishing basic cytoskeletal polarity, they do not rule out roles, for example, in localizing a special 'type' of actin cytoskeleton in the apical domain. Retention of Cno at the membrane after Scrib/Dlg knockdown suggests that minimally basal Rap1 activity remains intact. Changes to early Par-1, and to a lesser extent Baz, cortical localization with loss of Scrib/Dlg, also raise the possibility that lipid-based regulation is impaired. At this time, it is not known what cues regulate Scrib/Dlg apical enrichment but AJs do not appear to direct this, nor are they essential for polarizing Cno or Baz. Continued characterization of the full protein network and molecular mechanisms governing polarity establishment will keep the field busy for years to come (Bonello, 2019).

Mutations in ANKLE2, a ZIKA virus target, disrupt an asymmetric cell division pathway in Drosophila neuroblasts to cause microcephaly

The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. This study shows that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase Ballchen-VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. It is suggested that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division (Link, 2019).

Proper development of the human brain requires an exquisitely coordinated series of steps and is disrupted in disorders associated with congenital microcephaly. Congenital microcephaly in humans is characterized by reduced brain size (using occipital frontal circumference [OFC] as a surrogate measure) more than two standard deviations below the mean (Z score < -2) at birth. It is associated with neurodevelopmental disorders such as developmental delay and intellectual disability and can be caused by external exposures to toxins, in utero infections, or gene mutations. Pathogenic gene variants for microcephaly have been identified through targeted genetic testing, genomic copy number studies, and exome sequencing (ES), identifying 18 primary microcephaly loci. Many syndromes significantly overlap with classic microcephaly phenotypes, and together, these disorders can be caused by defects in a wide variety of biological processes, including centriole biogenesis, DNA replication, DNA repair, cell cycle and cytokinesis, genome stability, and multiple cell signaling pathways. In flies, microcephalic phenotypes are referred to when the third instar brain lobes are reduced in size or when adult flies exhibit small heads relative to the their body size. As in humans, microcephaly in flies can be a result of mutations that affect cell division and centrosome biology as demonstrated with mutations in WDR62 and ASPM or ASP and neuroblast (NB) proliferation (Link, 2019).

A forward, mosaic screen for neurodevelopmental and neurodegenerative phenotypes associated with lethal mutations on the X chromosome in Drosophila identified 165 loci, many with corresponding human genetic disease trait phenotypes. Among them, a mutation in Ankryin repeat and LEM domain containing 2 (Ankle2) causes loss of peripheral nervous system (PNS) organs in adult mutant clones and severely reduced brain size in hemizygous third instar larvae. To identify patients with pathogenic variants in ANKLE2, the exome database of the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) was surveyed; compound heterozygous mutations were identified in ANKLE2 in two siblings. Both infants exhibited severe microcephaly, and the surviving patient displayed cognitive and neurological deficits alongside extensive intellectual and developmental disabilities. Mutations in Ankle2 led to cell loss of NBs and affected NB division in the developing third instar larval brain. Remarkably, expression of the wild-type human ANKLE2 in flies rescued the observed mutant phenotypes. This study explored the molecular pathways and proteins that are affected by Ankle2 loss (Link, 2019).

ANKLE2 belongs to a family of proteins containing LEM (LAP2, Emerin, and MAN1) domains that typically associate with the inner nuclear membrane. Conventional LEM proteins have been shown to interact with barrier to autointegration factor (BAF), which binds to both DNA and the nuclear lamina to organize nuclear and chromatin structure. However, the LEM domain in Drosophila and C. elegans Ankle2 is not obviously conserved. Studies in C. elegans indicate that a homolog of ANKLE2 regulates nuclear envelope morphology and functions in mitosis to promote reassembly of the nuclear envelope upon mitotic exit. During this process, ANKLE2 modulates the activities of Vaccina-Related Kinase 1 (VRK1) and protein phosphatase 2A (PP2A). However, all experiments in worms were performed at the embryonic two-cell stage and no other phenotypes were reported except early lethality. While mutations in ANKLE2 have been associated with severe microcephaly, human VRK1 pathogenic variant alleles can cause a neurological disease trait consisting of complex motor and sensory axonal neuropathy and microcephaly (Link, 2019).

Mutations in both Ankle2 and the fly homolog of VRK1, ballchen, cause a loss of NBs in 3rd instar larval brains in Drosophila. NBs divide asymmetrically and are often used as a model to investigate stem cell biology and asymmetric cell division. Most NBs in the larval central brain give rise to another NB and a smaller ganglion mother cell (GMC), which then divides once again to produce neurons or glia. Proper NB maintenance and regulation is essential for precise development of the adult nervous system, and misregulation of NB number or function can lead to defects in brain size (Link, 2019).

Congenital Zika virus infection in humans during pregnancy has been associated with severe microcephaly that can be as dramatic as certain genetic forms of microcephaly including phenotypes associated with biallelic mutations in MCPH16/ANKLE2. Recently, it has been showed that a Zika virus protein, NS4A, physically interacts with ANKLE2 in human cells. Expression of NS4A in larval brains causes microcephaly, induces apoptosis, and reduces proliferation. Importantly, expression of human ANKLE2 in flies that express NS4A suppresses the associated phenotypes, demonstrating that NS4A interacts with the ANKLE2 protein and inhibits its function . Interestingly, Zika virus crosses the blood brain barrier and targets radial glial cells, the neural progenitors in the vertebrate cortex (Link, 2019).

This study shows that Ankle2 is localized to the endoplasmic reticulum and nuclear envelope, similar to NS4A, and genetically interacts with ball-VRK1 to regulate brain size in flies. An allelic series at the ANKLE2 and VRK1 loci shows that perturbation of this pathway results in neurological disease including microcephaly. The data indicate that the Ankle2-Ball (VRK1) pathway is required for proper localization of asymmetric proteins and spindle alignment during NB cell division by affecting two proteins, atypical protein kinase C (aPKC) and L(2)gl, which play critical roles in the asymmetric segregation of cell fate determinants. In addition, NS4A expression in NBs mimics phenotypes seen in Ankle2 mutants, and NS4A induced microcephaly is suppressed by removing a single copy of ball or l(2)gl. Human genomics variant data and disease trait correlations extend this asymmetric cell division pathway from proteins identified in flies and reveal insights into neurological disease. In summary, NS4A hijacks the Ankle2-Ball (VRK1) pathway, which regulates progenitor stem cell asymmetric division during brain development and defines a human microcephaly pathway (Link, 2019).

This study reports six additional patients with microcephaly that carry mutations in ANKLE2 and shows that three variants identified in probands cause a loss of ANKLE2 function when tested in flies, providing compelling evidence that its loss causes reduced brain size in flies and severe microcephaly in humans. Ankle2 is a dosage-sensitive locus whose product is inhibited by the Zika virus protein NS4A. Ankle2, similar to NS4A, is localized to the ER and it targets the nuclear envelope during mitosis. Loss of Ankle2 affects the nuclear envelope and ER distribution and results in a redistribution of Ball or VRK1, a kinase that is normally localized to the nucleus except when the nuclear envelope breaks down during mitosis. Loss of Ankle2 disrupts the localization of NB apical-basal polarity determinants such as aPKC, Par-6, Baz, and Mir, and aPKC phosphorylation is reduced by Ankle2 mutations. Importantly, loss of one copy of ball or l(2)gl suppresses the reduced brain volume associated with a partial loss of Ankle2, suggesting that much of the biological function of Ankle2 is modulated by aPKC and L(2)gl. Finally, the negative influence of NS4A on the activity of ANKLE2 can also be suppressed by removal of one copy of ball or l(2)gl, suggesting the following pathway: NS4A -| ANKLE2 -| Ball-VRK1 -> L(2)gl-LLGL1 -| aPKC. This pathway, regulated by ANKLE2, plays an important role in NB stem cell divisions in flies and microcephaly and potentially other neurological disease phenotypes in humans (Link, 2019).

Interestingly, the above pathway links environmental cues with several genetic causes of sporadic and autosomal recessive microcephaly in humans; moreover, it implicates this pathway in microcephaly accompanying congenital infection. As one example of the latter, the Zika virus has been shown to cross the infant blood brain barrier and has been identified in radial glial cells as well as intermediate progenitor cells and neurons. It is proposed that NS4A affects the function of Ankle2 leading to the release of Ball-VRK1 from the nucleus. It is speculated that this in turn affects the phosphorylation of aPKC and L(2)gl directly by masking phosphorylation sites or indirectly by promoting the activity of one or more phosphatases. Loss of VRK1 has been shown to cause microcephaly and some variant alleles are also associated with pontocerebellar hypoplasia (PCH) in humans, consistent with the loss of ball in flies that causes a severe reduction in brain size. Note that ANKLE2, VRK1, LLGL1, and aPKC as well as other components of the apical complex such as PARD3 are all present in radial glial cells during cortical development. These data suggest that ANKLE2 and its partners such as LLGL1 and asymmetric determinants are important proteins during neural cell proliferation and that the proper levels and relative amounts of these proteins determine how many neurons will eventually be formed in vertebrates. These data also indicate that variant alleles at either ANKLE2 or VRK1 are responsible for some causes of embryonic lethality and severe congenital microcephaly (Link, 2019).

LLGL1 has recently been shown to play an important role in radial glia in mice during neurogenesis, and its loss in clones increases the number of divisions. In addition, aPKCζ or λ localizes at the apical membrane of proliferating neural stem cells in chicken embryos during division and has been shown to provide an instructive signal for apical assembly of adherens junctions. Mouse knockouts of aPKCλ and aPKCι are embryonic lethal; however, aPKCζ knockouts are viable, perhaps suggesting redundant functions within the aPKC family. These proteins have not been linked to microcephaly in mice, but conditional removal of an apical complex protein Pals1 in cortical progenitors resulted in complete cortex loss. Finally, Numb is asymmetrically localized by the Par complex protein in Drosophila, segregated to the daughter cell during asymmetric cell division, and essential for daughter cells to adopt distinct fates. In mice, Numb localization is also asymmetric and null mutations exhibit embryonic lethality, neural tube closure defects, and premature neuron development. These data indicate that asymmetric division may be important for vertebrate neuronal development, but microcephaly is not a phenotype that typically associates with loss of the mice homologs of asymmetric-localized determinants identified in Drosophila. However, the observations reported in this study indicate that the ANKLE2-PAR complex pathway is evolutionarily conserved from flies to humans, although the precise mechanisms remain to be determined as different cells may use this pathway in different contexts (Link, 2019).

In order to determine whether predicted deleterious biallelic variants in PAR-complex-encoding genes or their paralogs associated with a neurologic disease trait, The BHCMG database was searched for mutations associated with neurological disease. Homozygous predicted deleterious missense variants in were found PARD3B (c.1222G>A, p.G408S) in a patient that has microcephaly and compound heterozygous mutations in PARD3B (c.1654G>A, p.A552T) that are associated with other neurological defects. The human ortholog of L(2)gl, LLGL1, is deleted in Smith-Magenis syndrome (SMS), and 86%-89% of the SMS patients have brachycephaly. These observations extend the mutational load beyond ANKLE2 and VRK1 and suggest an association between congenital disease and variants within the PAR complex, potentially by a compound inheritance gene dosage model (Link, 2019).

The Aurora A (AurA) kinase has been shown to phosphorylate the Par complex as well as L(2)gl and regulates cortical polarity and spindle orientation in NBs. The aberrant localization of Ball-VRK1 in Ankle2 mutants may lead to gain-of-function phenotypes that are highly dosage sensitive, as they can be repressed by removing a single copy of Ball-VRK1 in Ankle2A. Mislocalized Ball-VRK1 may mask or interfere with the function of AurA in NB asymmetric division as they share similar kinase substrate consensus sequences. Future studies are needed to assess Ball-VRK1 redundancy or interference with AurA function (Link, 2019).

Another possible evolutionarily parallel with implications in multicellular organismal development is the genetic interaction between the C. elegans homolog of VRK1 and an ANKLE2-like protein at the two-cell stage. Whereas VRK1 in both Drosophila and humans is localized to the nucleus, except during mitosis when the nuclear envelope is broken down, the worm VRK1 protein is localized to the nuclear envelope. The worm ANKLE2-like protein, Lem-4L, also interacts with the phosphatase PP2A, and the fly PP2A regulates NB asymmetric division by interacting with aPKC and excluding it from the basal cortex. PP2A also antagonizes the phosphorylation of Baz by PAR-1 to control apical-basal polarity in dividing embryonic NBs and regulates Baz localization in other cells such as neurons. This raises the possibility that the Ankle2 pathway also acts with PP2A in NB asymmetric division (Link, 2019).

This study identified a pathway that plays a significant role in NB asymmetric division. By combining functional studies in Drosophila together with human subject data, this study has linked several microcephaly-associated genes and congenital infection to a single genetic pathway. These studies allowed the highlighting of conserved functions of the ANKLE2 pathway and provide mechanistic insight into how a Zika infection might affect asymmetric division. This ANKLE2-VRK1 gene dosage-sensitive pathway can be perturbed by genetic variants that disturb biological homeostasis resulting in neurological disease traits or by environmental insults such as Zika virus impinging on neurodevelopment. Hence, lessons learned from the study of rare diseases can provide insights into more common diseases and potential gene- environment interactions (Link, 2019).

aPKC regulates apical constriction to prevent tissue rupture in the Drosophila follicular epithelium

Apical-basal polarity is an essential epithelial trait controlled by the evolutionarily conserved PAR-aPKC polarity network. Dysregulation of polarity proteins disrupts tissue organization during development and in disease, but the underlying mechanisms are unclear due to the broad implications of polarity loss. This study uncovered how Drosophila aPKC maintains epithelial architecture by directly observing tissue disorganization after fast optogenetic inactivation in living adult flies and ovaries cultured ex vivo. Fast aPKC perturbation in the proliferative follicular epithelium produces large epithelial gaps that result from increased apical constriction, rather than loss of apical-basal polarity. Accordingly, it is possible to modulate the incidence of epithelial gaps by increasing and decreasing actomyosin-driven contractility. The origin of these large epithelial gaps were traced to tissue rupture next to dividing cells. Live imaging shows that aPKC perturbation induces apical constriction in non-mitotic cells within minutes, producing pulling forces that ultimately detach dividing and neighboring cells. It was further demonstrated that epithelial rupture requires a global increase of apical constriction, as it is prevented by the presence of non-constricting cells. Conversely, a global induction of apical tension through light-induced recruitment of RhoGEF2 to the apical side is sufficient to produce tissue rupture. Hence, this work reveals that the roles of aPKC in polarity and actomyosin regulation are separable and provides the first in vivo evidence that excessive tissue stress can break the epithelial barrier during proliferation (Osswald, 2002).

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. This study demonstrates that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development (Gass, 2022).

Abnormal larval neuromuscular junction morphology and physiology in Drosophila prickle isoform mutants with known axonal transport defects and adult seizure behavior

Previous studies have demonstrated the striking mutational effects of the Drosophila planar cell polarity gene prickle (pk) on larval motor axon microtubule-mediated vesicular transport and on adult epileptic behavior associated with neuronal circuit hyperexcitability. Mutant alleles of the prickle-prickle (pk(pk)) and prickle-spiny-legs (pk(sple)) isoforms (hereafter referred to as pk and sple alleles, respectively) exhibit differential phenotypes. While both pk and sple affect larval motor axon transport, only sple confers motor circuit and behavior hyperexcitability. However, mutations in the two isoforms apparently counteract to ameliorate adult motor circuit and behavioral hyperexcitability in heteroallelic pk/pk/pk(sple) flies. The consequences of altered axonal transport was further investigated in the development and function of the larval neuromuscular junction (NMJ). Robust dominant phenotypes were uncovered in both pk and sple alleles, including synaptic terminal overgrowth (as revealed by anti-HRP and -Dlg immunostaining) and poor vesicle release synchronicity (as indicated by synaptic bouton focal recording). However, recessive alteration of synaptic transmission was observed only in pk/pk larvae, i.e. increased excitatory junctional potential (EJP) amplitude in pk/pk but not in pk/+ or sple/sple. Interestingly, for motor terminal excitability sustained by presynaptic Ca(2+) channels, both pk and sple exerted strong effects to produce prolonged depolarization. Notably, only sple acted dominantly whereas pk/+ appeared normal, but was able to suppress the sple phenotypes, i.e. pk/sple appeared normal. Thesd observations contrast the differential roles of the pk and sple isoforms and highlight their distinct, variable phenotypic expression in the various structural and functional aspects of the larval NMJ (Ueda, 2022).

Polarized SCAR and the Arp2/3 complex regulate apical cortical remodeling in asymmetrically dividing neuroblasts

Although the formin-nucleated actomyosin cortex has been shown to drive the changes in cell shape that accompany animal cell division in both symmetric and asymmetric cell divisions, the mitotic role of cortical Arp2/3-nucleated actin networks remain unclear. In this study, using asymmetrically dividing Drosophila neural stem cells as a model system, a pool of membrane protrusions was identified that form at the apical cortex of neuroblasts as they enter mitosis. Strikingly, these apically localized protrusions are enriched in SCAR, and depend on SCAR and Arp2/3 complexes for their formation. Because compromising SCAR or the Arp2/3 complex delays the apical clearance of Myosin II at the onset of anaphase and induces cortical instability at cytokinesis, these data point to a role for an apical branched actin filament network in fine-tuning the actomyosin cortex to enable the precise control of cell shape changes during an asymmetric cell division (Cazzagon, 2023).

Girdin is a component of the lateral polarity protein network restricting cell dissemination

Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. This study shows that Drosophila Girdin (girders of actin filaments) and its human ortholog GIRDIN or GIV (Galpha-interacting vesicle associated protein) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, cell dissemination was observed from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, this study discovered a core mechanism contributing to epithelial cell polarization from flies to humans. These data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination (Biehler, 2020).

The ability of epithelia to form physical barriers is provided by specialized cell-cell junctions, including the zonula adherens (ZA). The latter is a belt-like adherens junction composed primarily of the transmembrane homotypic receptor E-cadherin, which is linked indirectly to circumferential F-actin bundles through adaptor proteins such as β-catenin and α-catenin. In Drosophila embryonic epithelia, the protein Girdin stabilizes the ZA by reinforcing the association of the cadherin-catenin complex with the actin cytoskeleton (Ha, 2015). This function in cell-cell adhesion is preserved in mammals, and supports collective cell migration (Wang, 2018; Wang, 2015). Fly and human Girdin also contribute to the coordinated movement of epithelial cells through the organization of supracellular actin cables (Biehler, 2020).

In addition to creating barriers, epithelial tissues generate vectorial transport and spatially oriented secretion. The unidirectional nature of these functions requires the polarization of epithelial cells along the apical-basal axis. In Drosophila, the scaffold protein Bazooka (Baz) is crucial to the early steps of epithelial cell polarization, and for proper assembly of the ZA. Baz recruits atypical Protein Kinase C (aPKC) together with its regulator Partitioning defective protein 6 (Par-6) to the apical membrane. The small GTPase Cdc42 contributes to the activation of aPKC and p21-activated kinase (Pak1), thereby acting as a key regulator of cell polarity. Baz also contributes to apical positioning of the Crumbs (Crb) complex, which is composed mainly of Crb, Stardust (Sdt), and PALS1-associated Tight Junction protein (Patj). Once properly localized, the aPKC-Par-6 and Crb complexes promote the apical exclusion of Baz, which is then restricted to the ZA. The apical exclusion of Baz is essential to the positioning of the ZA along the apical-basal axis, and for full aPKC activation (Biehler, 2020).

The function of aPKC is evolutionarily preserved, and human atypical PKCι (PKClambda in other mammals) and PKCζ PKCzeta)contribute to epithelial cell polarization. aPKC maintains the identity of the apical domain through phospho-dependent exclusion of lateral polarity proteins such as Yurt (Yrt) and Lethal (2) giant larvae (Lgl). In return, these proteins antagonize the Crb- and aPKC-containing apical machinery to prevent the spread of apical characteristics to the lateral domain. In combination with the function of Baz, these feedback mechanisms provide a fine-tuning of aPKC activity in addition to specifying its subcellular localization. This is crucial, as both over- and under-activation of aPKC is deleterious to epithelial polarity in fly and mammalian cells, and ectopic activation of aPKC can lead to cell transformation (Biehler, 2020).

Cell culture work has established that mammalian GIRDIN interacts physically with PAR3 -the ortholog of Baz-and PKCλ. Depletion of GIRDIN in Madin-Darby Canine Kidney (MDCK) epithelial cells delays the formation of tight junctions in Ca2+ switch experiments. GIRDIN is also an effector of AMP-activated protein kinase (AMPK) in the maintenance of tight junction integrity under energetic stress. Moreover, mammalian GIRDIN is required for the formation of epithelial cell cysts with a single lumen, supporting a role for this protein in epithelial morphogenesis as reported in flies. As cyst morphogenesis is linked to epithelial cell polarity, these studies suggest that GIRDIN is involved in establishing the apical-basal axis. However, further studies are required to clarify the role of GIRDIN in apical-basal polarity per se, as other cellular processes could explain the phenotype associated with altered GIRDIN expression. For instance, spindle orientation defects impair the formation of epithelial cysts. Of note, PAR3, aPKC, and AMPK are all required for proper spindle positioning in dividing epithelial cells. The molecular mechanisms sustaining the putative role of GIRDIN in epithelial cell polarity also need to be better deciphered. This study investigated the role of fly and human Girdin proteins in the regulation of epithelial cell polarity, and showed that these proteins are part of the lateral polarity protein network. One crucial function of Girdin proteins is to repress aPKC function. It was also discovered that loss of Girdin proteins promotes overgrowth of cell cysts, and cell dissemination from these multicellular structures. Consistent with these data, it was found that low GIRDIN expression correlates with poor overall survival in subtypes of breast and lung cancers (Biehler, 2020).

Using classical genetics in flies, this study has shown that mutation in Girdin exacerbates the polarity defects in zygotic lgl or yrt mutant embryos and concludes that Girdin is part of the lateral polarity network. It was also found that Girdin opposes the function of aPKC, which plays a crucial role in the establishment and maintenance of the apical domain by antagonizing lateral proteins such as Lgl and Yrt. Thus a model is proposed in which Girdin supports the activity of Yrt and Lgl by restricting the activity of aPKC. This work demonstrates that the role of Girdin in restricting aPKC activity is evolutionarily conserved. This function confers on human GIRDIN the ability to maintain apical-basal polarity in Caco-2 cells, and to support epithelial cyst morphogenesis. These results are in line with previous studies suggesting a role for GIRDIN in polarity and cystogenesis in MDCK and MCF10A epithelial cells. It was shown that PKCλ enhances GIRDIN expression in MDCK cells. Moreover, knockdown of aPKC or GIRDIN gives a similar phenotype characterized by defects in tight junction integrity and cyst formation. It was thus proposed that GIRDIN is an effector of PKCλ. Although cell-type-specific mechanisms may exist, the current data suggest that this hypothesis needs to be revisited in favor of a model in which the induction of GIRDIN expression by PKClambda in MDCK cells initiates a negative feedback loop instead of cooperation between these proteins. The fact that both overactivation of aPKC or inhibition of its activity is deleterious to epithelial cell polarity and cyst morphogenesis may underlie the conflicting interpretations of the data in the literature. GIRDIN is also known to modulate heterotrimeric G protein signaling-a role that seems to contribute to the formation of normal cysts by MDCK cells (Sasaki, 2015). In addition, it was demonstrated recently that GIRDIN acts as an effector of AMP-activated protein kinase (AMPK) under energetic stress to maintain tight junction function (Aznar, 2016). Of note, these two functions are not shared by fly Girdin (Ghosh, 2017; Garcia-Marcos, 2009; Ghosh, 2017), and were thus acquired by GIRDIN during evolution to fulfill specialized functions. In contrast, the discovery in this study of the Girdin-dependent inhibition of aPKC reveals a core mechanism contributing to epithelial cell polarization from flies to humans (Biehler, 2020).

GIRDIN is considered to be an interesting target in cancer due to its role in cell motility, and high levels of GIRDIN have been reported to correlate with a poor prognosis in some human cancers. Notwithstanding that GIRDIN may favor tumor cell migration, the current study indicates that inhibition of GIRDIN function in the context of cancer would be a double-edged sword for many reasons. Indeed, this study showed that knockdown of GIRDIN exacerbates the impact of aPKC overexpression, and leads to overgrowth and lumen filling of Caco-2 cell cysts. Of note, overexpression of aPKC can lead to cell transformation, and was associated with a poor outcome in several epithelial cancers. This study thus establishes that inhibiting GIRDIN in patients showing increased aPKC expression levels could worsen their prognosis. According to the data, abolishing GIRDIN function in tumor cells with decreased levels of the human Lgl protein LLGL1, as reported in many cancers, could also support the progression of the disease by altering the polarity phenotype. Cell detachment and dissemination was observed from GIRDIN knockdown cysts, thus showing that GIRDIN is required for the cohesion of multicellular epithelial structures. Of note, cells, either individually or as clusters, detaching from cysts are alive and some of them remain viable. This is analogous to what was reported in Girdin mutant Drosophila embryos in which cell cysts detach from the ectoderm and survive outside of it. Other phenotypes in Girdin mutant embryos are consistent with a role for Girdin in epithelial tissue cohesion, including rupture of the ventral midline and fragmentation of the dorsal trunk of the trachea. Mechanistically, Girdin strengthens cell-cell adhesion by promoting the association of core adherens junction components with the actin cytoskeleton. A recent study established that this molecular function is evolutionarily conserved, and that GIRDIN favors the association of β-CATENIN with F-ACTIN. Since knockdown of GIRDIN results in cell dispersion from Caco-2 cell cysts, and since weakening of E-CADHERIN-mediated cell-cell adhesion contributes to cancer cell dissemination and metastasis, it is plausible that reduced GIRDIN expression contribute to the formation of secondary tumors and cancer progression. This may explain why this study found that low mRNA expression levels of GIRDIN correlates with decreased survival in more aggressive breast cancer subtypes and lung adenocarcinoma. Future studies using xenograft in mice, and investigating the expression of GIRDIN protein in cancer patients will help validating whether GIRDIN can repress the progression of certain types of epithelial cancers (Biehler, 2020).

In conclusion, using a sophisticated experimental scheme combining in vivo approaches in D. melanogaster with 3D culture of human cells, this study defined a conserved core mechanism of epithelial cell polarity regulation. Specifically, Girdin was shown to repress the activity of aPKC to support the function of Lgl and Yrt, and ensure stability of the lateral domain. This is of broad interest in cell biology, as proper epithelial cell polarization is crucial for the morphogenesis and physiology of most organs. In addition, the maintenance of a polarized epithelial architecture is crucial to prevent various pathological conditions such as cancer progression. Importantly, this study showed that normal GIRDIN function potentially impairs the progression of epithelial cancers by preserving cell polarity whilst restricting cell growth and cell dissemination. Thus, these results place a caveat on the idea that GIRDIN could be an interesting target to limit cancer cell migration, and indicate that inhibition of GIRDIN in the context of cancer could be precarious. Potential drugs targeting GIRDIN would thus be usable only in the context of precision medicine where a careful analysis of aPKC, LLGL1, and E-CAD expression, as well as the polarity status of tumor cells would be analyzed prior to treatment. Inhibition of GIRDIN in patients carrying tumors with altered expression of these proteins would likely worsen the prognosis (Biehler, 2020).

Distinct activities of Scrib module proteins organize epithelial polarity

A polarized architecture is central to both epithelial structure and function. In many cells, polarity involves mutual antagonism between the Par complex and the Scribble (Scrib) module. While molecular mechanisms underlying Par-mediated apical determination are well-understood, how Scrib module proteins specify the basolateral domain remains unknown. This study demonstrates dependent and independent activities of Scrib, Discs-large (Dlg), and Lethal giant larvae (Lgl) using the Drosophila follicle epithelium . The data support a linear hierarchy for localization, but rule out previously proposed protein-protein interactions as essential for polarization. Cortical recruitment of Scrib does not require palmitoylation or polar phospholipid binding but instead an independent cortically stabilizing activity of Dlg. Scrib and Dlg do not directly antagonize atypical protein kinase C (aPKC), but may instead restrict aPKC localization by enabling the aPKC-inhibiting activity of Lgl. Importantly, while Scrib, Dlg, and Lgl are each required, all three together are not sufficient to antagonize the Par complex. These data demonstrate previously unappreciated diversity of function within the Scrib module and begin to define the elusive molecular functions of Scrib and Dlg (Khoury, 2020).

Despite being central regulators of cell polarity in numerous tissues from nematodes to mammals, the mechanisms of Scrib module activity have remained obscure. The current work highlights previously unappreciated specificity in these activities, and begins to define the molecular functions of Scrib, Dlg, and Lgl. The data focus on the Drosophila follicle epithelium, as well as in some cases Drosophila embryos, but it is important to note that tissue contexts can differ in polarity programs: For example, in the adult Drosophila midgut epithelium, where Scrib module proteins are dispensable for epithelial organization. The failure to detect phenotypic enhancement in double-mutant follicle cells, compared to single mutants, which together with the complete penetrance of single-mutant phenotypes suggest full codependence of function rather than functional overlap. Moreover, Scrib module mutants could not be bypassed in any combination by overexpression of other genes in the module, consistent with unique roles for each protein. Thus, while Scrib, Dlg, and Lgl act in a common 'basolateral polarity' pathway, they each contribute distinct functions to give rise to the basolateral domain (Khoury, 2020).

Cell polarity is particularly evident at the plasma membrane, and most polarity regulators act at the cell cortex. Therefore, a key question in the field has concerned the mechanisms that allow cortical localization of the Scrib module and Par complex proteins, which exhibit no classic membrane-association domains. A simple linear hierarchy was found for cortical localization in the follicle that places Dlg most upstream, and contrasts with that recently described in the adult midgut, where Scrib appears to be most upstream. This work highlights the requirement of Dlg for Scrib localization, and provides insight into the mechanism, in part by ruling out previous models. One model involves a direct physical interaction, mediated by the Scrib PDZ domains and Dlg GUK domain. However, in vivo analyses show that follicle cells mutant for alleles lacking either of these domains have normal polarity; these results are supported by data from imaginal discs. In contrast, this study showa that the SH3 domain is critical for Scrib cortical localization as well as polarity. The Dlg SH3 and GUK domains engage in an intramolecular 'autoinhibitory' interaction that negatively regulates binding of partners, such as Gukh and CASK. The dispensability of the GUK domain provides evidence against an essential role for this mode of regulation in epithelial polarity, and highlights the value of investigating the GUK-independent function of the Dlg SH3 (Khoury, 2020).

A second mechanism of Scrib cortical association was also excluded. Mammalian Scrib is S-palmitoylated and this modification is required for both cortical localization and function. As Drosophila Scrib was also recently shown to be palmitoylated, an appealing model would involve Dlg regulating this posttranslational modification. However, no changes to Scrib palmitoylation were detected in a dlg mutant, and chemically or genetically inhibiting Drosophila palmitoyltransferases also had no effect on Scrib localization, although the possibility that Scrib palmitoylation may be part of a multipart localization mechanism cannot be excluded. Surprisingly, palmitoylated Scrib is incapable of reaching the cortex in dlg mutants. While a constitutively myristoylated Scrib can bypass this requirement for localization, it is nevertheless insufficient to support polarity in the absence of Dlg. These results indicate that Dlg regulates additional basolateral activities beyond localizing Scrib (Khoury, 2020).

Lgl's role as an aPKC inhibitor is well-characterized, but how Scrib and Dlg influence this antagonism is not understood. This study shows that Scrib and Dlg maintain cortical Lgl by regulating its phosphorylation by aPKC, rather than by direct physical recruitment to the membrane. A contemporaneous study by Ventura (2020) supports this finding, further showing that the major factor in Lgl cortical stabilization is PIP2. The current data also suggest that the basolateral-promoting activities of Scrib and Dlg are not via direct inhibition of aPKC kinase activity or intrinsic antagonism of aPKC localization. Instead, they are consistent with models in which Scrib and Dlg regulate the three specific aPKC-targeted residues in Lgl. Previous work has demonstrated that these phosphorylated serines (656, 660, 664) are neither functionally nor kinetically equivalent, and a recent model proposes that S664 is required for basolateral polarization by mediating a phosphorylation-dependent interaction with the Dlg GUK domain. Beyond the dispensability of the GUK domain, the enhanced ability of LglAAS to inhibit aPKC and its ability to do so largely independently of Scrib and Dlg, argues against this model. Moreover, only LglAAS among the phospho-mutants can dominantly affect aPKC activity, while WT Lgl can do the same only if Scrib and Dlg are present. Together, these results suggest that S656 is the critical inhibitory residue whose phosphorylation must be limited to enable Lgl's activity (Khoury, 2020).

The mechanism by which Lgl^{S656A,LglS660A(AAS)} (LglAAS) can suppress even constitutively active aPKCΔN remains unclear. aPKC substrates can act as competitive inhibitors; either an increased substrate affinity for aPKC or reduced ability to be inhibited by virtue of having fewer phosphorylation sites could make LglAAS a more effective inhibitor than WT Lgl. Supporting this idea, it was previously shown that S664, the only residue still available in LglAAS, is phosphorylated with higher kinetic preference than S656 or S660. It is also possible that some LglAAS phenotypes may be due to aPKC-independent effects resulting from reduced phosphorylation on S656 and S660. Nevertheless, a model is proposed in which Scrib and Dlg 'protect' Lgl by limiting phosphorylation of S656, thus tipping the inhibitory balance to allow Lgl to inhibit aPKC and establish the basolateral domain (Khoury, 2020).

What mechanism could underlie Scrib and Dlg protection of Lgl? One mechanism could involve generating a high phospholipid charge density at the basolateral membrane, which has been shown to desensitize Lgl to aPKC phosphorylation in vitro. However, the current data do not find evidence for regulation of phosphoinositides by Scrib and Dlg. A second possibility is that Scrib and Dlg could scaffold an additional factor, such as protein phosphatase 1, which counteracts aPKC phosphorylation of Lgl. Alternative mechanisms include those suggested by recent work on PAR-1 and PAR-2 in Caenorhabditis elegans zygotes, a circuit with several parallels to the Scrib module. In this system, PAR-2 protects PAR-1 at the cortex by shielding it from aPKC phosphorylation through physical interaction-dependent and -independent mechanisms. By analogy, binding with Scrib or Dlg could allosterically regulate Lgl to prevent phosphorylation, although this study has ruled out the Lgl-Dlg interaction documented in the literature. Scrib or Dlg might also act as a 'decoy substrate' for aPKC, as PAR-2 does in PAR-1 protection. Indeed, Scrib is phosphorylated on at least 13 residues in Drosophila embryos, although the functional relevance of this is not yet known (Khoury, 2020).

Overall, this work highlights the multifaceted nature of Scrib module function. The failure to bypass Scrib module mutants by transgenic supply of any single or double combination of other module components, including several that were constitutively membrane-tethered, suggests that every member contributes a specific activity to polarity. Nevertheless, even the simultaneous ectopic localization of all three Scrib module proteins was insufficient to disrupt the apical domain. This insufficiency in basolateral specification may reflect an inability of apical Scrib and Dlg to protect Lgl from aPKC phosphorylation, perhaps due to the distinct molecular composition of the apical and basolateral domains. This supports the idea that in addition to intrinsic activity via Lgl, the Scrib module must recruit or activate additional, as yet unidentified effectors in basolateral polarity establishment. The independent as well as cooperative activities of the Scrib module delineated in this study demonstrate previously unappreciated complexity in the determination of basolateral polarity and set the stage for future mechanistic studies of Scrib module function (Khoury, 2020).

Par complex cluster formation mediated by phase separation

The evolutionarily conserved Par3/Par6/aPKC complex regulates the polarity establishment of diverse cell types and distinct polarity-driven functions. However, how the Par complex is concentrated beneath the membrane to initiate cell polarization remains unclear. This study shows that the Par complex exhibits cell cycle-dependent condensation in Drosophila neuroblasts, driven by liquid-liquid phase separation. The open conformation of Par3 undergoes autonomous phase separation likely due to its NTD-mediated oligomerization. Par6, via C-terminal tail binding to Par3 PDZ3, can be enriched to Par3 condensates and in return dramatically promote Par3 phase separation. aPKC can also be concentrated to the Par3N/Par6 condensates as a client. Interestingly, activated aPKC can disperse the Par3/Par6 condensates via phosphorylation of Par3. Perturbations of Par3/Par6 phase separation impair the establishment of apical-basal polarity during neuroblast asymmetric divisions and lead to defective lineage development. It is proposed that phase separation may be a common mechanism for localized cortical condensation of cell polarity complexes (Li, 2020).

How the conserved Par (Par3/Par6/aPKC) complex is selectively recruited and concentrated on membranes for polarity establishment remains unclear. In this study, different from previously reported crescent localization patterning, the endogenous Par complex is revealed to exhibit cell cycle-dependent discrete puncta formation on the apical cortex in Drosophila NBs. The condensed Par puncta emerge from prophase, further condensate and enlarge as a clustered puncta structure in metaphase, then subsequently disassemble into scattered small puncta from anaphase. The cell cycle-dependent clustering of Par proteins in Drosophila NBs were also observed by two recent studies. In vitro biochemical data together with heterologous cell-based studies showed that the Par3/Par6 complex can undergo liquid-liquid phase separation (LLPS) at very low protein concentrations, and mutations of Par3 or Par6 that impair LLPS were found to alter asymmetric cell division (ACD) in Drosophila NBs. It has been recently shown that the basal condensation of Numb in dividing NBs is also regulated by LLPS of the Numb/Partner of numb complex (Shan, 2018). Thus, LLPS may be a common mechanism for the local condensation of apical and basal polarity determining protein complexes (Li, 2020).

It is important to note that the Par proteins, each at their endogenous level, can form clustered puncta via LLPS on the cortex. Though the measured endogenous Baz level in Drosophila NBs was too low to induce its LLPS in the cytoplasm, two-dimensional membrane attachment was expected to locally enrich the protein and lead to its LLPS. In return, LLPS-mediated Par complex condensates formation acts as an effective way for cells to further concentrate limited amount of Par proteins to specific cell cortices for polarity establishment. It is proposed that apical Baz/Par3 localization is a balanced result of apical anchoring and LLPS-mediated local condensation (via multivalent protein-protein interaction, self-association, protein-membrane interaction, etc.). Thus, for the knock-in mutant Baz ΔNTD, partially impaired LLPS ability due to its defective oligomerization led to its less condensed localization and significant cytoplasmic diffusion. However, the situation was different for the overexpressed Baz NTDmu (driven by UAS/GAL4) in the rescue assay, which is ectopically localized. As LLPS is very sensitive to concentrations of biological components, an overexpression of Par proteins especially Baz/Par3, the core driving factor of LLPS, may cause artificial promotion of the Par complex condensation via LLPS. Whereas the apical anchoring capacity of NBs seems to have a limitation. In UAS/GAL4-based rescue assay, the overexpressed Baz WT phase condensates may just have reached the threshold of apical anchoring capacity, whereas the LLPS deficient, overexpressed Baz NTDmu broke the balance, and led to its cortical and cytoplasmic diffusion. If the expression level goes higher, even Baz WT can not be afforded apically. Consistent with this notion, high Flag-Baz expression in a WT background (driven by insc-gal4), has a dominant-negative effect and leads to ectopic localization of endogenous Par complex throughout the cortex, and consequently disrupts localization of basal proteins. Similarly, ectopic Baz localization was observed when exogenous Baz is forcedly expressed in embryonic NBs. It was recently shown that overexpression of Par3-induced cell polarity in apolar S2 cells by forming concentrated Par-dots that further fused into amorphous Par-islands. According to a study of protein LLPS on lipid membrane bilayers, protein clusters gradually grew and fused into larger ones with irregular shapes, and finally coalesced into a mesh-like network. Thus, the amorphous structure of Par-islands in S2 cells may arise from the overexpression and overaccumulation of Par3 in the membrane region. Therefore, caution should be taken in interpreting the overexpression phenotypes of Par3 (Li, 2020).

Another key finding in this study is that aPKC can be recruited and concentrated in Par3/Par6 condensates as an inactive client. Such condensed phase droplets could be an efficient mechanism for local condensation of aPKC. Spatiotemporal activation of aPKC (e.g., by Cdc42) and consequent phosphorylation on Par3 CR3 leads to disassembly of the Par complex condensates. Another cell cycle regulator that might play a role in Par LLPS regulation is Plk1, which inhibits the oligomerization of Par3 by phosphorylating NTD in C. elegans. A critical but currently unknown point is how the autoinhibition of Par3 is relieved, as the open conformation of Par3 is critical for the Par complex condensate formation. Nonetheless, it is likely that multilayered regulatory mechanisms can act concertedly to control the spatiotemporal assembly and disassembly of the Par complex phase separation, and hence the cell polarity regulations (Li, 2020).

It is increasingly recognized that LLPS is a common strategy for cells to form membrane-less compartments by selectively recruiting and condensing proteins/RNAs/lipids. In a broader sense, the local condensation of other master polarity complexes, such as the conserved Lgl/Dlg/Scribble complex in the apical-basal polarity, and the Prickle/Vangl and Frizzled/Disheveled/Diego complexes in the planar cell polarity, may adopt a similar LLPS-driven mechanism to establish cell polarity in different tissues. Like the Par complex proteins, all these complexes share several common features: (1) these proteins contain multiple domains, which mutually interact with each other or self-associate in vitro to form complex platform, which further recruits other binding partners to assemble into higher order protein interaction network; (2) these complexes are found to form condensed patches or puncta attached to the inner surface of plasma membranes in vivo; and (3) proteins within these condensed patches or puncta are highly dynamic and rapidly exchange with corresponding proteins in the cytoplasm. The multivalent interaction-induced LLPS theory can perfectly explain above phenomena, allowing the stable existence of large concentration gradients of the proteins within the local protein condensates and those in the cytoplasm, and at the same time, keeping the proteins in the condensed phase highly dynamic. Such dynamic association may be essential for the fast assembly/disassembly of these polarity complexes in responding to extrinsic/intrinsic cues/signals to rearrange the cell polarity. It is postulated that LLPS of polarity protein complexes induced by multivalent interactions is a general mechanism for the cell polarization (Li, 2020).

Moesin is involved in polarity maintenance and cortical remodelling during asymmetric cell division

An intact actomyosin network is essential for anchoring polarity proteins to the cell cortex and maintaining cell size asymmetry during asymmetric cell division of Drosophila neuroblasts. However, the mechanisms that control changes in actomyosin dynamics during asymmetric cell division remain unclear. This study finds that the actin-binding protein, Moesin, is essential for neuroblast proliferation and mitotic progression in the developing brain. During metaphase, phosphorylated Moesin (p-Moesin) is enriched at the apical cortex and loss of Moesin leads to defects in apical polarity maintenance and cortical stability. This asymmetric distribution of p-Moesin is determined by components of the apical polarity complex and Slik kinase. During later stages of mitosis, p-Moesin localization shifts more basally, contributing to asymmetric cortical extension and myosin basal furrow positioning. These findings reveal Moesin as a novel apical polarity protein that drives cortical remodelling of dividing neuroblasts, which is essential for polarity maintenance and initial establishment of cell size asymmetry (Abeysundara, 2017).

Studies investigating ERM function have highlighted the importance of the ERM proteins in regulating the mechanical properties of the cell cortex. This study provides new insight into the role of Moesin in organizing the cortex of cells that establish intrinsic polarity and undergo asymmetric cell division (ACD) in vivo. When Moesin was knocked down in Insc-expressing cells, the larval CNS was reduced in size due to a decrease in the proportion of dividing NBs throughout larval development. Expressing Moe^dsRNA using the Insc-GAL4 driver affected overall larval development and resulted in larval lethality. However, viable progeny were obtained when Moesin levels were reduced using other NB-GAL4 drivers, asense-GAL4 and worniu-GAL4. When upstream activation sequence-GFP (UAS-GFP) was expressed using the different NB GAL4 drivers, GFP mRNA expression was ~5 fold greater using Insc-GAL4 compared with asense- or worniu-GAL4. Thus, the differences in viability are likely due to the increased strength of Insc-GAL4. Recent studies that identified the Hippo pathway as an essential regulator of NB quiescence also used Insc-GAL4 in their analyses. As it cannot be excluded that the reduced proportion of mitotic NBs may partially be due to impaired cell cycle reentry or an overall delay in larval development, the analysis focussed on the mitotic NBs that had exited quiescence. It was confirmed that defects in mitotic progression and polarity maintenance were observed at both early and late stages of the Moesin knockdown and in the late hypomorphic mutants, demonstrating a functional requirement of Moesin within the larval NBs (Abeysundara, 2017).

Proper regulation and function of the ERM proteins are required during cell division in both flies and mammals . In Drosophila S2 cells, the increased and uniform distribution of p-Moesin at the metaphase cortex enhanced cortical rigidity and cell rounding, proposed to be essential for stable spindle positioning. Drosophila Moesin was also shown to bind and stabilize microtubules at the cortex of cultured cells. Thus, an asymmetric ERM distribution during metaphase would be predicted to influence spindle position and orientation accordingly. In human colorectal Caco2 cells, polarized ezrin locally stabilized actin, providing a physical platform for astral microtubule-mediated centrosome positioning during interphase. HeLa cells cultured on L-shaped micropatterns also displayed restricted ERM activation at the cell cortex adjacent to the adhesive substrate, which was essential for LGN/NuMA polarization and guiding spindle orientation. In Drosophila wing imaginal epithelial cells, p-Moesin was enriched at the basal cortex of mitotic cells and the loss of Moesin led to defects in planar spindle orientation and recruitment of the pericentriolar material marker, Centrosomin. Thus, a role for Moesin in guiding spindle orientation and centrosome behaviour has been well documented. In Drosophila NBs, this study found that p-Moesin was apically enriched at the metaphase cortex, although the mitotic spindle has been reported to be symmetric and centrally located during metaphase. Thus, apical p-Moesin is likely not involved in generating spindle asymmetry during metaphase. The possibility of its involvement in preparing for the establishment of an asymmetric spindle during anaphase cannot be excluded. Furthermore, the loss of Moesin affected spindle orientation in only a small proportion of NBs, and the localization of the Drosophila LGN orthologue, Pins, was largely unaffected in Moesin knockdown NBs during metaphase. Thus, Moesin does not appear to play a prominent role in regulating spindle orientation in NBs. However, Moesin may affect the localization or activity of interacting partners downstream of Pins such as Mud or the heterotrimeric G protein subunit Gαi. Alternatively, the loss of both Moesin and Pins may cause more severe defects in spindle orientation and cell size asymmetry. Thus, future studies examining the loss of both Moesin and Pins may reveal a role for Moesin in maintaining centrosome positioning and spindle orientation in NBs (Abeysundara, 2017).

This study found that overall NB cell size was reduced in the Moesin knockdown. The reduced size of interphase NBs during early larval stages (48 h ALH) suggests that Moesin may be involved in NB enlargement prior to NB exit from quiescence. NB reactivation also appeared impaired in the ventral nerve cords of Moesin knockdown larvae. Previous studies have implicated Insulin/PI3K signaling in NB growth and reactivation during early larval stages. Further examination of these signaling pathways in the Moesin knockdown NBs are required to determine the mechanisms underlying its potential role in NB enlargement and reactivation. Of the NBs that had exited quiescence, a large proportion of mitotic defective NBs were observed during early and late larval stages. These NBs were not round and may reflect the importance of Moesin in cell rounding during early mitosis, as previously shown in Drosophila cell culture. Alternatively, the mitotic defective NBs may represent a population of NBs that have failed to undergo cell division. As the loss of Moesin also resulted in a reduced proportion of mitotic NBs undergoing each stage of mitosis, it is proposed that Moesin is essential for cell shape changes and mitotic progression during ACD (Abeysundara, 2017).

ERM proteins localize to the apical cortex of a wide variety of polarized cells and are essential for maintaining the apical identity and surface properties of epithelial tissues across multiple organisms. By binding directly to filamentous actin and linking membrane-associated proteins to the underlying actin cytoskeleton, the ERM proteins localize to numerous actin-rich structures. Thus, it is possible that the apical p-Moesin represents areas rich in actin filaments at the NB cortex. Although the actin cytoskeleton is important for cortical tethering of polarity complexes in NBs, filamentous actin does not display an asymmetric distribution. Thus, apical p-Moesin may correlate with enhanced cortical stability at the apical cortex necessary for polarity maintenance and integrity (Abeysundara, 2017).

Confirming a role for p-Moesin in stabilizing cortical actin, it was found that Bazooka and aPKC crescents were not observed in a proportion of Moe^dsRNA NBs undergoing prophase and actin appeared discontinuous at the cell cortex. As Bazooka and aPKC polarity is established by prophase, prior to the polar enrichment of p-Moesin, it is concluded that Moesin is involved in polarity maintenance rather than establishment. Similarly, in the Mus musculus and Caenorhabditis elegans intestinal epithelium, ERM proteins are involved in apical membrane assembly and integrity but do not appear to be required for polarity establishment. During metaphase, a proportion of Moe^dsRNA NBs lacked both Par-6 and aPKC polar crescents. However, the majority of Moe^dsRNA NBs displayed Bazooka and Pins polar crescents at the metaphase cortex. In the absence of Par-6 and aPKC, apical domains consisting of Bazooka, Inscuteable, Pins, and Discs large are still able to form. Thus, Moesin may be specifically maintaining Par-6/aPKC polarity during metaphase but have little effect on other apical polarity proteins such as Bazooka and Pins. Furthermore, the aPKC polar domain was disorganized, and cortical blebbing was observed in the Moe^G0323 mutant NBs. Thus, Moesin regulates the integrity and maintenance of the apical domain, likely through affecting cortical stability during ACD (Abeysundara, 2017).

The complex spatiotemporal regulation of Moesin activity during mitosis has been demonstrated in symmetrically dividing S2 cells and requires the coordinated activities of PP1-87B phosphatase, Slik kinase, and regulators of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) levels at the cell cortex. This study showed that Slik was uniformly distributed at the NB cell cortex. As Slik is regulated by phosphorylation, it is possible that the phosphorylated Slik is asymmetrically distributed in mitotic NBs. Furthermore, Slik was found to be essential for NB proliferation and polarity maintenance, likely through regulating Moesin phosphorylation at the NB cortex. The loss of Flapwing and PP1-87B phosphatases did not alter the apical enrichment of p-Moesin in metaphase NBs. Future studies examining other phosphatases and regulators of PI(4,5)P2 levels at the NB cortex are essential for further understanding Moesin regulation during ACD (Abeysundara, 2017).

In addition to Slik kinase, this study found that known apical polarity proteins (Cdc42, Par-6, aPKC, Lgl, and Pins) are important for the proper apical enrichment of p-Moesin during metaphase. As Moesin is also important for maintenance of the apical domain, these findings support a mutually dependent interaction among the apical polarity proteins that has been extensively reported in NBs. Components of the apical polarity complexes also mediate spindle asymmetry and asymmetric cortical extension during anaphase, leading to the generation of unequal-sized daughter cells. Similarly, this study found that Moesin was important for initial positioning of an asymmetric basal furrow during anaphase (Abeysundara, 2017).

In Drosophila NBs, a cortical polarity-induced pathway, consisting of Pins and the heterotrimeric G-proteins, is essential for apical cortical extension and formation of a Myosin-induced basal furrow, independent of the mitotic spindle. This study found that the relative fluorescent intensity (FI) of p-Moesin was reduced at the apical cortex during anaphase when compared with metaphase NBs. Furthermore, the loss of Moesin resulted in the absence of p-Myosin at the basal cortex, affecting basal furrow positioning during anaphase. In Drosophila S2 cells, reduced p-Moesin at the cell poles was shown to lead to cortical relaxation and membrane elongation. Thus, it is proposed that p-Moesin regulation at the apical cortex is important for asymmetric cortical extension and furrow positioning during early anaphase, likely along with Pins and the heterotrimeric G-proteins. However, Moesin also appeared to influence Myosin-mediated cortical contractility during metaphase as well. This study showed that with the loss of Moesin, p-Myosin and Rok-GFP displayed a nonuniform distribution at the metaphase cortex, revealing unstable actomyosin dynamics and a delay in anaphase onset. Although no observable differences were found in cortical Rho1 localization at the metaphase cortex, future studies using alternative biosensor approaches may allow for more precise visualization and analysis of Rho1 signaling. In addition, further investigation of the mechanical properties of cultured NBs will provide great insight into how Moesin function influences the mitotic cortex in the absence of physical constraint or external cues. While this work was under review, another group showed that Rok and Protein Kinase N are involved in the precise spatiotemporal regulation of Myosin flow during the establishment of physical asymmetry. Given the current findings, it will be interesting to further examine how Moesin precisely regulates Myosin dynamics, along with the other components of the polarity-induced cleavage furrow positioning pathway (Abeysundara, 2017).

Mad dephosphorylation at the nuclear pore is essential for asymmetric stem cell division

Stem cells divide asymmetrically to generate a stem cell and a differentiating daughter cell. Yet, it remains poorly understood how a stem cell and a differentiating daughter cell can receive distinct levels of niche signal and thus acquire different cell fates (self-renewal versus differentiation), despite being adjacent to each other and thus seemingly exposed to similar levels of niche signaling. In the Drosophila ovary, germline stem cells (GSCs) are maintained by short range bone morphogenetic protein (BMP) signaling; the BMP ligands activate a receptor that phosphorylates the downstream molecule mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) accumulates in the GSC nucleus and activates the stem cell transcription program. This study demonstrates that pMad is highly concentrated in the nucleus of the GSC, while it quickly decreases in the nucleus of the differentiating daughter cell, the precystoblast (preCB), before the completion of cytokinesis. A known Mad phosphatase, Dullard (Dd), is required for the asymmetric partitioning of pMad. Mathematical modeling recapitulates the high sensitivity of the ratio of pMad levels to the Mad phosphatase activity and explains how the asymmetry arises in a shared cytoplasm. Together, these studies reveal a mechanism for breaking the symmetry of daughter cells during asymmetric stem cell division (Sardi, 2021).

Cell cycle expression of polarity genes features Rb targeting of Vang

Specification of cellular polarity is vital to normal tissue development and function. Pioneering studies in Drosophila and C. elegans have elucidated the composition and dynamics of protein complexes critical for establishment of cell polarity, which is manifest in processes such as cell migration and asymmetric cell division. Conserved throughout metazoans, planar cell polarity (PCP) genes are implicated in disease, including neural tube closure defects associated with mutations in VANGL1/2. PCP protein regulation is well studied; however, relatively little is known about transcriptional regulation of these genes. Earlier study revealed an unexpected role for the fly Rbf1 retinoblastoma corepressor protein, a regulator of cell cycle genes, in transcriptional regulation of polarity genes. This study analyzes the physiological relevance of the role of E2F/Rbf proteins in the transcription of the key core polarity gene Vang. Targeted mutations to the E2F site within the Vang promoter disrupts binding of E2F/Rbf proteins in vivo, leading to polarity defects in wing hairs. E2F regulation of Vang is supported by the requirement for this motif in a reporter gene. Interestingly, the promoter is repressed by overexpression of E2F1, a transcription factor generally identified as an activator. Consistent with the regulation of this polarity gene by E2F and Rbf factors, expression of Vang and other polarity genes is found to peak in G2/M phase in cells of the embryo and wing imaginal disc, suggesting that cell cycle signals may play a role in regulation of these genes. These findings suggest that the E2F/Rbf complex mechanistically links cell proliferation and polarity (Payankaulam, 2021).

Pilot RNAi Screen in Drosophila Neural Stem Cell Lineages to Identify Novel Tumor Suppressor Genes Involved in Asymmetric Cell Division

A connection between compromised asymmetric cell division (ACD) and tumorigenesis was proven some years ago using Drosophila larval brain neural stem cells, called neuroblasts (NBs), as a model system. Since then, it has been learned that compromised ACD does not always promote tumorigenesis, as ACD is an extremely well-regulated process in which redundancy substantially overcomes potential ACD failures. Considering this, a pilot RNAi screen was performed in Drosophila larval brain NB lineages using Ras(V)(12) scribble (scrib) mutant clones as a sensitized genetic background, in which ACD is affected but does not cause tumoral growth. First, as a proof of concept, this study has tested known ACD regulators in this sensitized background, such as lethal (2) giant larvae and warts. Although the downregulation of these ACD modulators in NB clones does not induce tumorigenesis, their downregulation along with Ras(V)(12) scrib does cause tumor-like overgrowth. Based on these results, 79 RNAi lines randomly screened detecting 15 potential novel ACD regulators/tumor suppressor genes. It is concluded that Ras(V)(12) scrib is a good sensitized genetic background in which to identify tumor suppressor genes involved in NB ACD, whose function could otherwise be masked by the high redundancy of the ACD process (Manzanero-Ortiz, 2021).

Cell polarity opposes Jak/STAT-mediated Escargot activation that drives intratumor heterogeneity in a Drosophila tumor model

In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, the expression of Snail family transcription factor Escargot (Esg) was detected and further validated. A similar relationship was ascertained between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, these results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity (Chatterjee, 2023).

Drosophila Phosphatase of Regenerating Liver Is Critical for Photoreceptor Cell Polarity and Survival during Retinal Development

Establishing apicobasal polarity, involving intricate interactions among polarity regulators, is key for epithelial cell function. Though phosphatase of regenerating liver (PRL) proteins are implicated in diverse biological processes, including cancer, their developmental role remains unclear. This study explored the role of Drosophila PRL (dPRL) in photoreceptor cell development. dPRL, requiring a C-terminal prenylation motif, is highly enriched in the apical membrane of developing photoreceptor cells. Moreover, dPRL knockdown during retinal development results in adult Drosophila retinal degeneration, caused by hid-induced apoptosis. dPRL depletion also mislocalizes cell adhesion and polarity proteins like Armadillo, Crumbs, and DaPKC and relocates the basolateral protein, alpha subunit of Na(+)/K(+)-ATPase, to the presumed apical membrane. Importantly, this polarity disruption is not secondary to apoptosis, as suppressing hid expression does not rescue the polarity defect in dPRL-depleted photoreceptor cells. These findings underscore dPRL's crucial role in photoreceptor cell polarity and emphasize PRL's importance in establishing epithelial polarity and maintaining cell survival during retinal development, offering new insights into PRL's role in normal epithelium (Chen, 2023).

Polarized branched Actin modulates cortical mechanics to produce unequal-size daughters during asymmetric division

The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. This study reports that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. This was demonstrated by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. It is thereby possible to engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. These findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size (Daeden, 2023).

Centromere proteins are asymmetrically distributed between newly divided germline stem and daughter cells and maintain a balanced niche in Drosophila males

Stem cells can undergo asymmetric cell division (ACD) giving rise to one new stem cell and one differentiating daughter cell. In Drosophila germline stem cells (GSCs), the centromeric histone CENP-A (CID in flies) is asymmetrically distributed between sister chromatids such that chromosomes that end up in the GSC harbor more CID at centromeres. A model of "mitotic drive" has been proposed in GSCs such that stronger and earlier centromere and kinetochore interactions with microtubules bias sister chromatid segregation. This study shows that in Drosophila males, centromere proteins CID, CAL1, and CENP-C are asymmetrically distributed in newly divided GSCs and daughter cells in S phase. Overexpression of CID (either with or without CAL1) or CENP-C depletion disrupts CID asymmetry, with an increased pool of GSCs relative to daughter cells detectable in the niche. This result suggests a shift toward GSC self-renewal rather than differentiation, important for maintaining tissue homeostasis. Overexpression of CAL1 does not disrupt asymmetry, but instead drives germ cell proliferation in the niche. These results in male GSCs are comparable to female GSCs, indicating that despite differences in signaling, organization, and niche composition, the effects of centromere proteins on GSC maintenance are conserved between the sexes (Kochendoerfer, 2023).

Apical-basal polarity precisely determines intestinal stem cell number by regulating Prospero threshold

Apical-basal polarity and cell-fate determinants are crucial for the cell fate and control of stem cell numbers. However, their interplay leading to a precise stem cell number remains unclear. Drosophila pupal intestinal stem cells (pISCs) asymmetrically divide, generating one apical ISC progenitor and one basal Prospero (Pros)(+) enteroendocrine mother cell (EMC), followed by symmetric divisions of each daughter before adulthood, providing an ideal system to investigate the outcomes of polarity loss. Using lineage tracing and ex vivo live imaging, this study identified an interlocked polarity regulation network precisely determining ISC number: Bazooka inhibits Pros accumulation by activating Notch signaling to maintain stem cell fate in pISC apical daughters. A threshold of Pros promotes differentiation to EMCs and avoids ISC-like cell fate, and over-threshold of Pros inhibits miranda expression to ensure symmetric divisions in pISC basal daughters. This work suggests that a polarity-dependent threshold of a differentiation factor precisely controls stem cell number (Wu, 2023).

Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling

The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. This study demonstrates that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics. This study show sthat while the apical polarity network, in part via atypical protein kinase C (aPKC), tethers Kibra at the junctional cortex to silence its activity, medial actomyosin flows promote Kibra-mediated Hippo complex formation at the medial cortex, thereby activating the Hippo pathway. This study provides a mechanistic understanding of the relationship between the Hippo pathway, polarity, and actomyosin cytoskeleton, and it offers novel insights into how fundamental features of epithelial tissue architecture can serve as inputs into signaling cascades that control tissue growth, patterning, and morphogenesis (Tokamov, 2023).

Diamond controls epithelial polarity through the dynactin-dynein complex

Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells. However, it remains not fully understood how they are targeted to the apical domain. Using Drosophila ovarian follicular epithelia as a model, it was show that epithelial polarity is lost and Crb protein is absent in the apical domain in follicular cells (FCs) in the absence of Diamond (Dind). Interestingly, Dind is found to associate with different components of the dynactin-dynein complex through co-IP-MS analysis. Dind stabilizes dynactin and depletion of dynactin results in almost identical defects as those observed in dind-defective FCs. Finally, both Dind and dynactin are also required for the apical localization of crb transcripts in FCs. Thus these data illustrate that Dind functions through dynactin/dynein-mediated transport of both Crb protein and its transcripts to the apical domain to control epithelial apico-basal (A/B) polarity (Zhao, 2023).

Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex

Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In Drosophila neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). This study identified Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (Hts(HEAD)) to bind a short Mud coiled-coil domain (Mud(CC)) that is adjacent to the Pins-binding domain (Mud(PBD)). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. In vitro analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, this study found that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via Mud(CC) oligomerization) and protein disorder (via an intrinsically disordered region in Hts; Hts(IDR)) in phase separation of the Hts/Mud/Pins complex. This study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output (Parra, 2023).

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Zygotically transcribed genes

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