BIOLOGICAL OVERVIEW

Asymmetric cell divisions are critically important to generate diverse cell types in the development of multicellular organisms. Polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus are prerequisite for asymmetric divisions. Genetic analysis of Drosophila and C. elegans has led the way in unraveling the origins of cell polarity during embryonic development. The recently cloned Drosophila gene bazooka codes for a protein implicated in the formation of the cell-cell junction called the zonula adherins. This protein is a homolog of C. elegans Par-3 protein, which contributes to cell polarity and spindle alignment in early C. elegans embryos. As these data suggest, there may be a common basis for the establishment of cell polarity throughout the metazoa. What is the origin of apical basal polarity in the epidermis of Drosophila and how does this get converted to a mechanism that faithfully orients the mitotic spindle thus influencing the asymmetric distribution of cytoplasmic determinants during asymmetric cell division? Answering this question provides a key cornerstone to understanding the origins of cellular diversity during development. This review will first describe the role of the Par genes in regulating spindle orientation in C. elegans. Then it will turn to the role of Bazooka in the establishment of the adherins junction. Finally, it will present the evidence that Bazooka plays a role in spindle orientation.

The role of the Par genes in regulating spindle orientation

In the C. elegans embryo, a series of early asymmetric cleavages produce six founder cells with different cleavage patterns and cell fates. Anterior-posterior (AP) polarity is established during the first cell cycle and correlates with a dynamic rearrangement of cytoplasm along the AP axis, which is defined by an extrinsic cue provided by sperm. Microfilaments accumulate at the anterior periphery; central cytoplasm flows toward the posterior pole, and cortical cytoplasm flows in the opposite direction. The first mitotic spindle is placed posteriorly and the first cleavage becomes asymmetric, producing a large cell, termed AB, to form the anterior and a small cell, P1, to form the posterior of the embryo. In concert with the cytoplasmic rearrangement, P granules (germline-specific ribonucleoprotein particles) become localized to the posterior pole. Several other cytoplasmic factors that play roles in cell fate specification are distributed asymmetrically after the first cleavage. For example, SKN-1, which is a putative transcription factor and is required to specify the fate of the EMS blastomere, is present at a higher level in the P1 cell, and MEX-3, a putative RNA-binding protein, is expressed at a higher level in the AB cell (Tabuse, 1998 and references).

The establishment of AP polarity in C. elegans embryos is known to require the activities of the maternally expressed par genes. Interference of normal functions of these genes causes extensive polarity defects in the 1-cell embryo and results in loss of many early asymmetries. Of the six par genes so far identified, par-1, par-2, par-3, par-5 and par-6 (Drosophila homolog: par-6) mutant embryos exhibit a symmetrically placed first cleavage spindle and the equal-sized AB and P1 blastomeres. P granules fail to be distributed exclusively to P blastomeres in par-1, par-3, par-4 (see Drosophila Lkb1), par-5 and par-6 embryos. Both AB and P1 spindles at the second cleavage of par-1, par-2, par-4 and par-5 embryos are aligned transversely, like the wild-type AB spindle, while they are aligned longitudinally in par-3 and par-6 embryos. Three par genes (par-1, par-2 and par-3) have been molecularly characterized. PAR-1 contains an amino-terminal serine/threonine kinase domain and a carboxy-terminal domain with binding activity to non-muscle myosin. PAR-2 is a protein containing a zinc-binding domain of the ring finger class and a myosin-type ATP-binding site. PAR-3 is a novel protein (Etemad-Moghadam, 1995) with three PDZ domains. Consistent with their role in polarity, the PAR proteins are themselves distributed in a polar fashion in the P lineage blastomeres. PAR-1 and PAR-2 localize to the posterior periphery of the 1-cell embryo and the P1 cell and localize to the ventral periphery of P2 and P3 blastomeres, but are absent from the periphery of all other blastomeres. In contrast, PAR-3 is present in a distribution reciprocal to that of PAR-1 and PAR-2; it is localized to the anterior periphery of 1-cell embryos and P1, localizes to the dorsal side of P2 and P3, and is present uniformly at the periphery of all other blastomeres (Tabuse, 1998 and references).

PAR-3 appears to play a central role in polarity establishment. In par-3 1-cell embryos, PAR-1 and PAR-2 are no longer restricted to the posterior pole although they remain peripheral (Etemad-Moghadam, 1995), and the par-3 spindle orientation defect is epistatic to those of the other pars. Proper distribution of PAR-3 itself, however, is dependent on the activities of par-2, par-5 and par-6. Insight into how asymmetric cell divisions are controlled can be gained by identifying the proteins with which the PAR proteins interact. Screens for proteins that can bind to the carboxy-terminus of PAR-1 have identified a non-muscle myosin that was subsequently shown by RNA-mediated depletion of the protein to be required for successful asymmetric divisions and for proper distribution of PAR-1, PAR-2 and PAR-3. Mammalian atypical protein kinase Cs (aPKCs), PKCzeta and PKClambda, associate specifically with a mouse protein similar to C. elegans PAR-3 (Yasushi Izumi. et al., unpublished data, 1998 cited in Tabuse, 1998). Although aPKCs are expressed ubiquitously in animals and have been implicated in mitogenic signal transduction in mammalian cells and the maturation of the Xenopus oocyte, their physiological function is totally unknown. An aPKC of C. elegans, termed PKC-3, is implicated in regulation of asymmetic cell division. PKC-3 mutants die displaying Par-3-like phenotypes, and PKC-3 can bind to PAR-3 in vitro and is co-localized with PAR-3 at the anterior cortex of the 1-cell embryo. Furthermore, the two proteins are mutually dependent for their proper localization: PKC-3 localization is disrupted in par-3 embryos and PAR-3 localization is disrupted in PKC-3-depleted embryos. Other par genes that regulate PAR-3 distribution are also required for PKC-3 localization. Thus, C. elegans PKC-3 plays an indispensable role in establishing embryonic polarity through interaction with PAR-3 (Tabuse, 1998 and references).

The exact functional relationship between PKC-3 and PAR-3 is not clear. Three possibilities are suggested. (1) PAR-3 could be acting to recruit PKC-3 to the cell periphery where it acts as a signaling molecule. (2) PKC-3, like the par-6 product, acts to recruit PAR-3 to the cell periphery or maintain it there throughout the cell cycle or both. It could do this by phosphorylating PAR-3 directly or by modifying the cortical cytoskeleton. The two possibilities are not mutually exclusive; PAR-3 could recruit PKC-3 to the cell periphery where its kinase activity has the dual effect of providing a signal leading to anterior/posterior differences as well as maintaining PAR-3 at the periphery. (3) PKC-3 and PAR-3, perhaps along with the product of the par-6 gene, might act together to form a functional complex whose stability or localization requires the presence of both. In any case, it is likely that the PAR-3/PKC-3 connection is evolutionarily conserved and serves to establish or maintain cell polarity in other species (Tabuse, 1998).

The role of Bazooka in the establishment of the adherins junction

The discussion now turns to Bazooka, a Drosophila PAR-3 homolog, and its role in formation of the circumferential adherens junctions termed zonula adherens (ZA) and maintenance of polarized blastoderm epithelium in Drosophila. Antibodies to Armadillo, an adherins junction component, and to phosphotyrosine (PY) epitopes were used as markers to demonstrate the formation of the ZA during embryogenesis and to analyze defects caused by bazooka mutants and a second mutation termed stardust in ZA formation. The mutations baz and sdt belong to a group in which mutant embryos show severe abnormalities in the differentiation of the larval cuticles, including the genes crumbs (crb) and shotgun (shg). During cellularization, the formation of cell membranes begins with the generation of cleavage furrows extending from the periphery of the embryo in a radial direction, thus establishing the normal boundaries between cells in the ectoderm. New plasma membranes are assembled until a monolayer cell sheet has formed that shares many features with epithelial cell monolayers, including epithelial cell functions and polarized membrane transport. A major redistribution of Arm and PY occurs during cellularization and early gastrulation. At mid-cellularization, staining becomes stronger on the apical aspect than in the more basal part of the lateral domain. By the end of cellularization, Arm and PY staining are strongly reduced at the basal part of the cleavage furrow that separates nuclei of cells in the blastoderm. bazooka and stardust have been identified as X-linked genes required for the formation of the ZA in the embryo. Mutations in either of these genes lead to a zygotic embryonic lethal phenotype, which is characterized by either severe malformation or an absence of the embryonic epidermis (Wieschaus, 1984 and Tepass, 1993).

Hemizygous baz single mutants show normal Arm and NT staining up to stage 10 of embryogenesis. Even at this rather late stage, the ZA is disordered only locally while large regions of the epidermis are still normal. Similarly, sdt single mutant embryos exhibit abnormal ZA morphology only late in development. The effects of the baz;sdt double mutant are much more severe. In postgastrula embryos, adherens junction plaques are strongly reduced and often absent; these plaques are normally present at the apical-lateral junction, indicating the presence of the ZA. The relatively weak zygotic phenotype of baz can be explained by a strong maternal component for the expression of baz. baz null embryos were produced to analyze the ZA phenotype of embryos that lack both maternal and zygotic baz activity. baz null embryos show a early disruption of ZA formation like that seen in baz;sdt zygotic double mutants. In particular, the concentration of Arm in the apical region of cell contact at the beginning of gastrulation is absent in both baz null mutants and the double mutants. In addition to these early alterations in ZA formation, two morphological features of both mutant phenotypes are very similar: (1) when cellularization is complete, cell shapes in the mutant embryos are aberrant; instead of the highly columnar cell shape in the epithelium of wild-type embryos, the cells have irregular outlines and some appear bottle-shaped; (2) the regular hexagonal pattern of Arm staining seen on the surface of wild-type embryos is distorted in the double mutants; the apical cell surfaces appear to vary in size, indicating that the apical domains are expanded in some cells and constricted in others. No ZAs are found in baz null mutant embryos. Germ-line null armadillo mutants exhibit a phenotype similar to baz;sdb mutants, showing that Armadillo is also required for ZA formation. It is suggested that early stages in the assembly of the ZA are critical for the assembly and/or stability of the polarized blastoderm epithelium (Muller, 1996).

Lack of zygotic baz function results in a loss of the coherent epidermal tissue structure. During germ-band expansion, when three post-blastodermal cell divisions take place and neuroblasts delaminate from the neurogenic ectoderm, only slight irregularities in the epithelium can be detected. Widespread defects become obvious from the beginning of germ-band retraction onward: cell shape is modified; cells lose their contacts, and the epidermis adopts a highly irregular appearance (Kuchinke, 1998).

The role of Bazooka in spindle orientation

It is a defect of spindle orientation in zygotic bazooka mutants that links Bazooka to C. elegans PAR-3 and PAR-3's role in establishing the plane of spindle orientation in C. elegans cell division. One manifestation of a polarized phenotype is the orientation of the mitotic spindle. In the developing trunk epidermis of wild-type Drosophila embryos, the mitotic spindle is oriented parallel to the surface, resulting in two cells that remain integrated in the epithelium after cytokinesis. In baz mutant embryos, the mitotic spindle in epidermal cells occasionally adopts an aberrant orientation, leading to an inner and an outer cell after completion of division. This occurrence of the spindle phenotype with low penetrance is consistent with only a mild epithelial phenotype in the epidermis at a stage when all postblastodermal divisions are completed. This suggests that the strong defects observed in the epidermis at later stages of development must be attributed to an additional function of baz that is required late for the maintenance of the epithelial tissue structure (Kuchinke, 1998).

In delaminating neuroblasts of wild-type embryos, one of the centrosomes migrates basally, resulting in a spindle that is oriented perpendicular to the surface. Since the transcription factor Prospero is localized in a basal crescent in the cortical cytoplasm of the neuroblast during methaphase, only the basally located GMC will receive this protein. After cytokinesis, Pros is rapidly translocated into the nucleus of the GMC. In baz mutant embryos, the orientation of the mitotic spindle frequently deviates from the apico-basal axis. During division, Pros remains localized in a cortical crescent, which is, however, not always strictly basally positioned. Following division, Pros is correctly translocated into the nuclei of the smaller GMCs after cytokinesis but, in contrast to wild type, GMCs are often found localized in lateral positions relative to the neuroblasts instead of basal positions (Kuchinke, 1998). Defects in spindle orientation in baz mutants may be contrasted with those in inscuteable, another gene whose protein product is asymetrically localized. In insc mutants, spindle orientation and Pros localization are randomized and no longer coordinated; consequently, many GMCs do not receive Pros protein and, hence, fail to develop properly. This suggests a function of insc in the coordination of additional and/or different aspects of cellular polarity, such as spindle orientation and localization of cytoplasmic determinants (Kuchinke, 1998 and references).

Strikingly, spindle orientation and localization of GMCs are not completely randomized. In most cases, the GMCs can be found lying within a basal quadrant of the neuroblast, whereas hardly any GMCs can be found apical to the neuroblast. The phenotype clearly shows that neuroblasts of baz mutant embryos still develop an apico-basal polarity, which is manifested by the coordinated regulation of spindle orientation and Pros localization. Yet, many neuroblasts have lost the ability to orient their axis of polarity correctly with respect to the axis of the embryo. The fact that many neuroblasts with defective spindle orientation are lying below a phenotypically organized epithelium makes it unlikely that the misorientation of the apico-basal axis of neuroblasts is a consequence of defects in polarity of the overlying epithelium. Therefore, neuroblasts lacking baz still have an intrinsic apico-basal polarity (lost in insc mutants) but they fail to orient their axis of polarity with respect to the axis of the embryo, resulting in a misorientation of the spindle and, hence, a mispositioning of the GMC. It is proposed that Baz is a likely candidate for organizing a multiprotein complex by means of its recruitment of multiple cytoplasmic, cytoskeletal or integral membrane proteins to the apical pole of the cell (Kuchinke, 1998).

Cdc42 acts downstream of Bazooka to regulate neuroblast polarity through Par-6 aPKC

Cdc42 recruits Par-6-aPKC to establish cell polarity from worms to mammals. Although Cdc42 is reported to have no function in Drosophila neuroblasts, a model for cell polarity and asymmetric cell division, this study shows that Cdc42 colocalizes with Par-6-aPKC at the apical cortex in a Bazooka-dependent manner, and is required for Par-6-aPKC localization. Loss of Cdc42 disrupts neuroblast polarity: cdc42 mutant neuroblasts have cytoplasmic Par-6-aPKC, and this phenotype is mimicked by neuroblast-specific expression of a dominant-negative Cdc42 protein or a Par-6 protein that lacks Cdc42-binding ability. Conversely, expression of constitutively active Cdc42 leads to ectopic Par-6-aPKC localization and corresponding cell polarity defects. Bazooka remains apically enriched in cdc42 mutants. Robust Cdc42 localization requires Par-6, indicating the presence of feedback in this pathway. In addition to regulating Par-6-aPKC localization, Cdc42 increases aPKC activity by relieving Par-6 inhibition. It is concluded that Cdc42 regulates aPKC localization and activity downstream of Bazooka, thereby directing neuroblast cell polarity and asymmetric cell division (Atwood, 2007).

Little is currently known about how the Par complex is localized or regulated in Drosophila neuroblasts, despite the importance of this complex for neuroblast polarity, asymmetric cell division and progenitor self-renewal. This study shows that Cdc42 plays an essential role in regulating neuroblast cell polarity and asymmetric cell division. Baz localizes Cdc42 to the apical cortex where it recruits Par-6-aPKC, leading to polarization of cortical kinase activity that is essential for directing neuroblast cell polarity, asymmetric cell division, and sibling cell fate (Atwood, 2007).

Asymmetric aPKC kinase activity is essential for the restriction of components such as Mira and Numb to the basal cortex. The aPKC substrates Lgl and Numb are thought to establish basal polarity either by antagonizing activity of myosin II or by direct displacement from the cortex. This study found that Cdc42 recruits Par-6-aPKC to the apical cortex and that Cdc42 relieves Par-6 inhibition of aPKC kinase activity. In the absence of Cdc42, aPKC is delocalized and has reduced activity, resulting in uniform cortical Mira. Expression of Cdc42-DN leads to cortical overlap of inactive Par-6-aPKC and Mira indicating the importance of Cdc42-dependent activation of aPKC kinase activity. Expression of Cdc42-CA leads to cortical aPKC that displaces Mira from the cortex, presumably because Lgl is phosphorylated at the entire cell cortex. This is similar to what is seen when a membrane-targeted aPKC is expressed (Atwood, 2007).

Baz, Par-6 and aPKC have been considered to be part of a single complex (the Par complex). This study found that, when Cdc42 function is perturbed, Par-6 and aPKC localization is disrupted but Baz is unaffected. Why is Baz unable to recruit Par-6-aPKC in the absence of Cdc42? One explanation is that Cdc42 modulates the Par-6-Baz interaction, although Cdc42 has no direct effect on Par-6-Baz affinity. Alternatively, Baz might only be transiently associated with the Par-6-aPKC complex (e.g. as an enzyme-substrate complex); this is consistent with the observation that Baz does not colocalize with Par-6-aPKC in Drosophila embryonic epithelia and its localization is not dependent on either protein. How does Baz recruit Cdc42 to the apical cortex? Like other Rho GTPases, Cdc42 is lipid modified (prenylated), which is sufficient for cortical localization. Baz is known to bind GDP-exchange factors (GEFs), which may induce accumulation of activated Cdc42 at the apical cortex (Atwood, 2007).

The requirement of Par-6 for robust Cdc42 apical enrichment suggests that positive feedback exists in this pathway, a signaling pathway property that is also found in polarized neutrophils. More work is required to test the role of feedback in neuroblast polarity but one attractive model is that Baz establishes an initial polarity landmark at the apical cortex in response to external cues, which leads to localized Par-6-aPKC activity through Cdc42. Phosphorylation of Baz by aPKC might further increase asymmetric Cdc42 activation, perhaps by increased GEF association, thereby reinforcing cell polarity. Such a mechanism could generate the robust polarity observed in neuroblasts and might explain why expression of dominant Cdc42 mutants late in embryogenesis does not lead to significant defects in polarity (Atwood, 2007).

This study argues that Cdc42 functions downstream of Baz. Cdc42 is required for Baz-Par-6-aPKC localization in C. elegans embryos and mammalian neural progenitors. In C. elegans embryos, RNA interference of cdc42 disrupts Par-6 localization, whereas PAR-3 localization is slightly perturbed. In this case, Cdc42 is required for the maintenance but not establishment of PAR-3-Par-6 asymmetry; however, other proteins have been shown to localize Par complex members independently of Cdc42. Conditional deletion of cdc42 in the mouse brain causes significant Par-3 localization defects, although this may be caused by the loss of adherens junctions. More work will be required in these systems to determine if the pathway that has been proposed is conserved (Atwood, 2007).

This study has identified at least two functions of Cdc42 in neuroblasts: first, to recruit Par-6-aPKC to the apical cortex by direct interaction with its CRIB domain and, second, to promote aPKC activity by relieving Par-6 repression. aPKC activity is required to partition Mira and associated differentiation factors into the basal GMC; this ensures maintenance of the apical neuroblast fate as well as the generation of differentiated neurons. Polarized Cdc42 activity may also have a third independent function in promoting physically asymmetric cell division, because uniform cortical localization of active Cdc42 leads to same-size sibling cells. Loss of active Cdc42 at the cortex by overexpression of Cdc42-DN still results in asymmetric cell division, suggesting that other factors also regulate cell-size asymmetry, such as Lgl and Pins. In conclusion, these data show that Cdc42 is essential for the establishment of neuroblast cell polarity and asymmetric cell division, and defines its role in recruiting and regulating Par-6-aPKC function. These findings now allow Drosophila neuroblasts to be used as a model system for investigating the regulation and function of Cdc42 in cell polarity, asymmetric cell division and neural stem cell self-renewal (Atwood, 2007).

Bazooka inhibits aPKC to limit antagonism of actomyosin networks during amnioserosa apical constriction

Cell shape changes drive tissue morphogenesis during animal development. An important example is the apical cell constriction that initiates tissue internalisation. Apical constriction can occur through a phase of cyclic assembly and disassembly of apicomedial actomyosin networks, followed by stabilisation of these networks. Delayed negative-feedback mechanisms typically underlie cyclic behaviour, but the mechanisms regulating cyclic actomyosin networks remain obscure, as do mechanisms that transform overall network behaviour. This study shows that a known inhibitor of apicomedial actomyosin networks in Drosophila amnioserosa cells, the Par-6-aPKC complex, is recruited to the apicomedial domain by actomyosin networks during dorsal closure of the embryo. This finding establishes an actomyosin-aPKC negative-feedback loop in the system. Additionally, aPKC was found to recruit Bazooka to the apicomedial domain, and phosphorylates Bazooka for a dynamic interaction. Remarkably, stabilising aPKC-Bazooka interactions can inhibit the antagonism of actomyosin by aPKC, suggesting that Bazooka acts as an aPKC inhibitor, and providing a possible mechanism for delaying the actomyosin-aPKC negative-feedback loop. These data also implicate an increasing degree of Par-6-aPKC-Bazooka interactions as dorsal closure progresses, potentially explaining a developmental transition in actomyosin behaviour from cyclic to persistent networks. This later impact of aPKC inhibition is supported by mathematical modelling of the system. Overall, this work illustrates how shifting chemical signals can tune actomyosin network behaviour during development (David, 2013).

These data outline a regulatory circuit for guiding amnioserosa apical constriction. The circuit controls both the localisation and activity of its components. In terms of protein localisation, it was found that amnioserosa actomyosin networks recruit the Par proteins to the apicomedial domain. Although Par protein puncta are not continually dependent on the actomyosin networks, their numbers build over developmental time, apparently owing to the cumulative effect of multiple rounds of actomyosin network assembly. The networks appear to impact aPKC directly, and in turn, aPKC recruits Baz to the apical domain. This recruitment depends on the C-terminal aPKC-binding region of Baz, which aPKC phosphorylates for a dynamic relationship with Baz in the apical domain of amnioserosa cells (David, 2013).

Par-6-aPKC activity inhibits amnioserosa actomyosin networks (David, 2010), and the recruitment of aPKC by the networks implicates a negative-feedback loop. As delayed negative feedback tied to a continual input signal can produce an oscillatory output, the actomyosin-aPKC negative-feedback loop might explain how aPKC regulates actomyosin network assembly-disassembly cycles (David, 2010). However, apical populations of Par-6-aPKC puncta are not fully recruited and fully removed with each actomyosin cycle, suggesting additional mechanisms. Importantly, Par-6-aPKC activity can be tempered by Baz. Thus, aPKC inhibition by Baz might delay the actomyosin-aPKC negative-feedback loop during early DC, promoting the actomyosin assembly-disassembly cycles. As DC proceeds, the additive effects of actomyosin assembly-disassembly cycles could increase apical Par protein levels; additionally, the gradual apical constriction of the cells decreases their apical surface areas and could thus increase apical surface Par protein concentrations. It is proposed that a gradual increase to apicomedial aPKC-Baz interactions inhibits aPKC and thus leads to the stabilisation of actomyosin networks. Simulations indicate that this transition in network behaviour can occur abruptly following incremental reductions to myosin inhibition during earlier DC. It is proposed that Baz acts as a competitive inhibitor to reduce aPKC phosphorylation of cytoskeletal regulators. This idea is consistent with reports of Par-3 inhibiting aPKC in kinase assays in vitro. However, Baz is also known to promote aPKC localisation in the epidermis and amnioserosa. Thus, Baz appears to both promote and inhibit aPKC activity, potentially forming a paradoxical circuit (or incoherent feed-forward loop) in which Baz and aPKC promote each other's recruitment, and in which Baz competitively inhibits aPKC activity. Significantly, Baz has multiple binding sites for the Par-6-aPKC complex [Par-6 binds Baz PDZ1; aPKC binds Baz PDZ2-3; aPKC binds the Baz C-terminal aPKC-binding region], suggesting cooperative binding and that Baz interactions with the Par-6-aPKC complex are stronger than those between the Par-6-aPKC complex and its cytoskeleton targets. Notably, this study found that Baz apical surface levels are ~66% lower than those of Par-6, suggesting that the inhibitory effect of Baz must be dynamic; Baz cannot simply sequester all Par-6-aPKC complexes by outnumbering them. The inhibitory effect must also depend on phosphatases because aPKC interactions with Baz are weakened following phosphorylation (Morais-de-Sá, 2010). Baz/Par-3 is known to be regulated by Protein phosphatase 1 and Protein phosphatase 2A with Protein phosphatase 1 de-phosphorylating the aPKC phosphorylation site of Par-3. Thus, Baz may act as a strong and dynamic inhibitor of Par-6-aPKC to buffer and eventually overcome the actomyosin-aPKC negative-feedback loop (David, 2013).

A crucial unknown is the identity of the cytoskeletal target(s) of aPKC. Cytoskeletal targets of aPKC have been identified but have not been examined during amnioserosa apical constriction. In mammalian cells, Par-6-aPKC can phosphorylate Smurf1, an E3 ubiquitin ligase, in turn leading to RhoA degradation in cellular protrusions (Wang, 2003). During dendritic spine morphogenesis, Par-6-aPKC acts though p190RhoGAP to inhibit RhoA (Zhang, 2008). As well, aPKC phosphorylation of Rho kinase leads to its cortical dissociation in mammalian cell culture (Ishiuchi, 2011), and apparently during salivary gland tubulogenesis in Drosophila (Röper, 2012). Of note, the persistent Par-6-aPKC puncta could actively downregulate actomyosin activity, or prolong the lull between actomyosin activations, or do both. Another question is how actomyosin networks recruit aPKC. The recruitment of Par proteins by actomyosin networks has been documented during Drosophila cellularisation and C. elegans one-cell polarisation, and Baz and aPKC have been shown to co-immunoprecipitate with myosin regulatory light chain from Drosophila egg chambers, but specific linkages have yet to be identified. Defining further components of the actomyosin-aPKC negative-feedback loop will be crucial for understanding its regulation and its effects on actomyosin network dynamics. In particular, despite identifying a potential delay mechanism for the loop, it is unclear how the loop and the delay mechanism could translate into oscillatory network behaviour. Perhaps the cytoskeletal target(s) of aPKC are co-recruited with the assembling networks, which in combination with the buffering effect of Baz, could delay their phosphorylation by aPKC. It is also possible that the clustering of Par protein puncta with each network assembly event could somehow modify the Baz buffering effect (David, 2013).

Another unanswered question is the influence of circumferential anchors for Baz or Par-6-aPKC, as weakening of these anchors could contribute to apicomedial Par protein accumulation over DC. Echinoid (Ed), a transmembrane AJ-associated protein that can directly bind Baz, is normally lost from the amnioserosa during DC. It is hypothesised that this loss might promote the loss of Baz from AJs and its apicomedial accumulation. However, ectopic expression of Ed in the amnioserosa leading to circumferential Ed levels higher than those seen in the epidermis had no apparent effect on apicomedial Baz localisation. Thus, differences in Ed expression alone cannot account for the differential localisation of Par proteins between the amnioserosa and epidermis. It is possible that the effects of actomyosin can overpower ectopic Ed, or that other changes to the apical circumference of amnioserosa cells are involved. More generally, other Par protein interaction partners should be considered. For example, Baz and Stardust also interact and, together with Crumbs and Patj, they form the apical Crumbs complex (Tepass, 2012). Recent results suggest Patj can activate myosin by suppressing myosin light chain phosphatase. Intriguingly, amnioserosa BazS980A apical surface puncta also recruit Patj, suggesting that this pathway might contribute to myosin activity as well (David, 2013).

In summary, the data argue that the differential regulation of amnioserosa actomyosin networks by Baz and Par-6-aPKC can be explained by a single pathway in which Baz inhibits Par-6-aPKC antagonism of the cytoskeletal networks. It was also found that the actomyosin networks recruit aPKC, forming a negative-feedback loop. It is proposed that the inhibition of aPKC by Baz delays the negative feedback at earlier DC for cycling actomyosin networks, and with increased inhibition of aPKC by later DC, the actomyosin networks persist. These findings provide an example of how chemical signalling, and changes to this signalling, can modify the behaviour of actomyosin networks during embryo development (David, 2013).

Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor

In Drosophila epithelial cells, apical exclusion of Bazooka/Par3 defines the position of the Zonula Adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. This study shows that the small GTPase Rap1, its effector AF6/Canoe (Cno) and the Cdc42-effector Pak4/Mushroom bodies tiny (Mbt), converge in regulating epithelial E-Cadherin, and Bazooka retention at the ZA. Furthermore, the results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating their functions during ZA morphogenesis are interlinked. In this context, the Rap1-GEF Dizzy was found to be enriched at the ZA and the results suggest it promotes Rap1 activity during ZA morphogenesis. Altogether, it is proposed the Dizzy, Rap1/Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis (Walther, 2018).

In the pupal photoreceptor, ZA morphogenesis is orchestrated by a conserved protein network that includes Cdc42, Par6, aPKC, Baz, Crb and its binding partner Sdt, and Par1. In turn, AJ material is an essential part of the regulatory network that orchestrates polarity. Previous work has shown that Mbt regulates pupal photoreceptor development by promoting ZA morphogenesis. During this process Mbt contributes in preventing Baz from spreading to the lateral membrane, a regulation that this study found to depend in part on the phosphorylation of Arm by Mbt at S561 and S688. It is proposed that Mbt regulates photoreceptor polarity by promoting the retention of Baz at the developing ZA. Failure in ZA retention leads to Baz spreading to the lateral membrane where it is eliminated through Par1-mediated displacement. In these cells, failure to retain AJ material, including Baz, at the ZA leads to its shortening along the apical basal axis and can impact on the polarization program of the photoreceptor (Walther, 2018).

This study shows that Mbt function is linked to that of Dzy, Rap1 and Cno. First, Cno and Mbt accumulation at the ZA is interdependent, reflecting a tight coupling between the Rap1 and Cno pathway and Mbt. Second, it was found that Cno promotes Baz retention at the ZA, as cnoIR leads to shorter ZAs that can be depleted of Arm and Baz. This phenotype resembles that of mbt mutant cells and is also seen when overexpressing a version of Arm that cannot be phosphorylated by Mbt. These observations prompted a test or the hypothesis that Rap1, Cno and Mbt might function as part of a linear pathway promoting Baz retention at the ZA. In this pathway, it was reasoned that Mbt could mediate Rap1 function through Arm phosphorylation. In testing this hypothesis, it was found that this is not the case. Instead, the observation that expressing a version of Arm that mimics its constitutive phosphorylation by Mbt does not ameliorate the cnoIR phenotype suggests that Rap1, Cno, and Mbt converge in promoting Baz retention at the ZA, and cannot compensate for each other during this process. This conclusion is well supported by the finding that overexpressing cno in mbt mutant cells does not lead to an amelioration of the mbt phenotype. Third, it was found that Mbt influences the distribution of Rap1 along the apical-basal axis of the cell in that Rap1::GFP no longer accumulates preferentially at the ZA. This correlates with a loss of Dzy::GFP at the plasma membrane, raising the possibility that Mbt might regulate Rap1 through Dzy. However, the dzy phenotype is milder than that seen with Rap1 or cno, in that loss of dzy does not lead to cell delamination from the retina. This suggests that, as has been reported in the cellularizing embryo, other GEFs regulate Rap1 during epithelial morphogenesis (Walther, 2018).

An interesting aspect of the cnoIR phenotype is the defects in apical accumulation of aPKC and Crb. These defects are not observed in the dzy mutant or Rap1IR cells, indicating that Cno might function independently of Rap1 during this process. However, it is noted that while Cno was not detected at the ZA of cnoIR cells, it can still be detected in Rap1IR cells. It is therefore hypothesized that residual Cno in Rap1IR cells supports optimum aPKC and Crb accumulation at the apical membrane. In this model, Dzy, Rap1 and Cno function as part of the same pathway, which includes a function in promoting optimum apical accumulation of Crb and aPKC. Baz is required for Par complex assembly and associated aPKC and Crb recruitment at photoreceptor apical membrane. It is hypothesized that the defects in Crb and aPKC that were detect in cnoIR cells are linked to the failure in retaining Baz at the ZA, which leads to its elimination from the lateral membrane by Par1. More work will be required to understand how exactly AJ material and ZA retention of Baz influences apical membrane specification (Walther, 2018).

Rap1 and cno have been shown to regulate apical-basal polarity in the cellularizing embryo. In this model system, Rap1 and Cno regulate the apical localization of Baz and Arm, which precedes the apical recruitment of Crb. In turn, Baz influences the localization of Cno. This work indicates that similar complex regulations are at play in the pupal photoreceptor. However, unlike in the early embryo, AJ material (Arm) is absolutely required for Baz (and Par6-aPKC) accumulation or retention at the cell cortex in the developing pupal photoreceptor. A model is therefore favored whereby Mbt, Rap1 and Cno influence ZA morphogenesis primarily through regulating the interface between E-Cad or Arm, Baz and the F-actin cytoskeleton. In this model, Mbt regulates this interface both through Arm phosphorylation and cofilin-dependent regulation of F-actin, and Cno contributes to this process, at least in part, through its ability to bind to F-actin (Walther, 2018).

To probe Rap1 and Cno function during photoreceptor ZA morphogenesis, the effect of decreasing Rap1 expression on E-Cad stability was assessed. Consistent with the notion that the function of mbt and Rap1 are linked during ZA morphogenesis, it as found that, as it is the case for Mbt , Rap1 is required to stabilize E-Cad::GFP at the photoreceptor ZA. However, the mobile fraction estimated for E-Cad is much higher in Rap1IR cells than in mbt^P1 null cells (evaluated at ~70% for Rap1IR and 45% for mbt^P1). Together with the finding that Mbt accumulation at the ZA is decreased in Rap1IR cells, FRAP data are therefore compatible with Mbt mediating part of the function of Rap1 in promoting E-Cad stability. However, the much larger mobile fraction were estimated in the Rap1IR genotype when compared to mbt^P1 photoreceptors indicates that Rap1 must also regulate E-Cad stability independently of Mbt. The longer time scale for E-Cad::GFP to recover in Rap1IR cells when compared to mbt^P1 mutant cells is compatible with Rap1 functioning, in part, through promoting E-Cad delivery (Walther, 2018).

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).

A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity

Par-3 regulates animal cell polarity by targeting the Par complex proteins Par-6 and atypical protein kinase C (aPKC) to specific cortical sites. Although numerous physical interactions between Par-3 and the Par complex have been identified, this study discovered a novel interaction between Par-3's second PDZ domain and a highly conserved aPKC PDZ-binding motif (PBM) that is required in the context of the full-length, purified Par-6-aPKC complex. This study also found that Par-3 is phosphorylated by the full Par complex and phosphorylation induces dissociation of the Par-3 phosphorylation site from aPKC's kinase domain but does not disrupt the Par-3 PDZ2-aPKC PBM interaction. In asymmetrically dividing Drosophila neuroblasts, the aPKC PBM is required for cortical targeting, consistent with its role in mediating a persistent interaction with Par-3. These results define a physical connection that targets the Par complex to polarized sites on the cell membrane (Holly, 2020).

The catalytic activity of atypical protein kinase C (aPKC) defines mutually exclusive cortical domains in diverse animal cells. Par-6 and aPKC are recruited to specific cellular sites where aPKC phosphorylation polarizes downstream factors by displacing them from the Par cortical domain. For example, in Drosophila neural stem cells or neuroblasts, the Par complex localizes to an apical cortical domain during mitosis where it excludes neuronal differentiation factors. Apical exclusion separates these factors into a distinct cortical domain at the basal cortex, which is segregated into the basal daughter cell following cytokinesis. Par polarized factors such as Miranda and Numb contain sequences that bind the membrane but are also phosphorylation motifs for aPKC. The direct connection of aPKC's catalytic activity to the polarization of downstream factors makes the regulatory pathways that control its cortical targeting critical to animal cell polarity (Holly, 2020).

In many cellular contexts, Par-3 (Bazooka in flies) is essential for recruitment of Par-6 and aPKC to specific cortical sites. Par-3's role in regulating Par complex cortical recruitment is thought to be direct because five physical interactions have been discovered with both Par-6 and aPKC. Four of the interactions involve at least one of Par-3's three PDZ protein-interaction domains: Par-3 PDZ1 binding to the Par-6 PDZ domain, Par-3 PDZ1 and PDZ3 domain interactions with Par-6's PDZ-binding motif (PBM), and an interaction with an undefined region of aPKC that requires both Par-3 PDZ2 and PDZ3. Additionally, because Par-3 is an aPKC substrate, the aPKC kinase domain interacts with Par-3's aPKC phosphorylation motif (APM; also known as CR3). Although protein kinases are typically thought to interact transiently with their substrates, the interaction with the APM has been proposed to mediate complex assembly. Previous investigations used small fragments of the Par complex that did not contain all potential binding motifs, such that it was not possible to assess whether any of the interactions are required for binding in the context of the purified, full-length Par-6-aPKC complex. Furthermore, none of the interactions have been shown to be required for cortical targeting of aPKC in a functional context (Holly, 2020).

Par-3 interactions with the Par-6-aPKC complex were examined by reconstituting full-length Drosophila Par-6 and aPKC. Although it was possible to purify the Par-6-aPKC complex to a high degree, Par-3 is very large (157.4 kDa) and the maltose-binding protein-fused Par-3 (MBP-Par-3; total mass 199.9 kDa) significant amounts of degradation products were obtained in addition to full-length protein. Nevertheless, using this preparation, it was possible to detect an interaction with reconstituted Par complex using a qualitative affinity chromatography (i.e., 'pull-down') assay. Additionally, phosphate transfer to full-length Par-3 (and some smaller fragments with masses consistent with COOH-terminal truncations that contain the APM) were obtained using an antibody specific to the phosphorylated APM. Phosphorylation of Par-3 by aPKC has been controversial. This result contributes to the understanding of the process by demonstrating that aPKC phosphorylates Par-3 in the context of the full-length, purified Par complex in addition to the isolated catalytic domain and APM peptide (Holly, 2020).

Using the system of purified Par complex and MBP fusions of full-length Par-3 and its degradation products, attempts were made to identify Par-3 domains required for interaction with the full Par complex. The Par-6 PBM within the Par complex was tested, as it has been reported to bind both the Par-3 PDZ1 and PDZ3 domains. ATP was included in binding experiments because Par-3 is a substrate in the context of the full Par complex. Using this experimental setup, Par-3 PDZ2 was identified as a required interaction domain for binding to the full Par complex (Holly, 2020).

To determine the mechanism by which Par-3 PDZ2 mediates binding to the Par complex, attempts were made to identify the recognition site on the complex. The Drosophila aPKC COOH-terminal sequence has the characteristics of a 'class 3' PBM and is consistent with the binding specificity of the PDZ2 domain as assessed using a phage-display assay. The aPKC COOH-terminal sequence is also highly conserved among metazoan orthologs, the same evolutionary interval in which Par-3 is found. Tests were performed to see whether the aPKC COOH terminus is required for the interaction with Par-3 by purifying Par complex lacking aPKC's final six residues. The aPKC COOH terminus was shown to be required for Par-3's interaction with the Par complex (Holly, 2020).

To test whether the Par-3 PDZ2 and aPKC COOH terminus are sufficient for binding, the interaction of the isolated motifs was examined. The isolated proteins are sufficient for complex assembly. In general, PDZ-PBM interactions are strongly dependent on the identity of the terminal residue, and it was found that Par-3 PDZ2 failed to bind the aPKC COOH terminus when the final residue was mutated from valine to alanine (aPKC V606A). It is concluded that the aPKC COOH terminus is a bona fide PBM. It was also confirmed that the aPKC PBM and Par-3 PDZ2 interaction is broadly conserved across metazoans by examining orthologs from a chordate (human), a placozoan (Trichoplax), and a cnidarian (Hydra), in addition to the arthropod Drosophila. Binding was observed for each of the orthologous pairs, indicating that the interaction is conserved across diverse metazoan organisms. Together, these results indicate that the Par-3 PDZ2 and aPKC PBM are sufficient for binding and their interaction is conserved across metazoa (Holly, 2020).

To assess the role of the Par-3 PDZ2-aPKC PBM interaction quantitatively, an equilibrium supernatant-depletion assay was implemented. The affinity of the Par-3 PDZ1-APM for the Par complex was measured, as this region could be purified to a level suitable for quantitative measurements. Addition of Par-3 PDZ1-APM depleted Par-6 and aPKC from the supernatant consistent with a Kd of 0.7 μM. To determine the effect of disrupting the Par-3 PDZ2-aPKC PBM interaction on binding affinity, Par-3 PDZ1-APM binding to Par-6-aPKCΔPBM was measured. Sufficient depletion of Par-6 and aPKCΔPBM by PDZ1-APM was not observed to allow fitting to a binding isotherm, indicating the absence of the aPKC PBM substantially decreases the affinity of the Par-3 interaction with the Par complex, consistent with the results of qualitative measurements (Holly, 2020).

The results indicate that the Par-3 PDZ2 and aPKC PBM are required for Par-3's interaction with the Par complex. The requirement for these domains suggests that the Par-3 phosphorylation site (i.e., APM) does not form a persistent interaction with the Par complex. However, this conclusion appears to be in conflict with previous work showing that the Par-3 APM is sufficient for binding to the aPKC kinase domain, both with binding assays and structure determination using X-ray crystallography. Furthermore, a stable APM-kinase interaction forms the basis of a model in which the unphosphorylated Par-3 APM forms a stable, persistent interaction with the aPKC kinase domain that is not phosphorylated until an unknown activating event occurs. The finding that Par-3 is phosphorylated by the full Par complex is inconsistent with this model, but it does not fully resolve whether the Par-3 APM is sufficient for forming a stable, persistent interaction with the Par complex (Holly, 2020).

It was hypothesized that the presence of ATP could influence the binding behavior of the Par-3 APM with the Par complex. A key difference between the current experiments and previous reports is that the current experiments included ATP, whereas previous binding experiments and structural analysis lacked ATP. Without ATP, completion of the protein kinase catalytic cycle is not possible, and interactions that would otherwise form transiently could persist (Holly, 2020).

Tests were performed to see whether the Par-3 APM forms a stable, persistent interaction with the Par complex in the absence of ATP. Vinding between Par-3 and the Par complex was detected after replacing ATP with ADP in a context where the Par-3 PDZ2-aPKC PBM interaction is disrupted. This interaction requires the APM, leading to the conclusion that the Par-3 APM can form a persistent interaction with the Par complex, but only in the absence of ATP. When ATP is present the APM interacts transiently with the Par complex, because it is phosphorylated and subsequently dissociates. Under the same conditions, the Par-3 PDZ2 interaction with the Par complex is not disrupted, however. Although it is possible to form a stalled complex between the Par-3 APM and the Par complex in the absence of ATP, it is proposed that persistent binding of the APM to the kinase domain due to the lack of ATP is unlikely in vivo because ATP concentrations are high under normal cellular conditions (Holly, 2020).

Although numerous interactions have been identified between Par-3 and Par-6-aPKC, none have been demonstrated to be required for cortical targeting of the Par complex. In fact, the interactions of Par-6 with Par-3 have been shown to be dispensable for function. To determine whether the Par-3 PDZ2-aPKC PBM interaction is required for Par complex polarization, the localization of aPKC harboring the V606A PBM point mutation was investigated during neuroblast asymmetric division by expressing aPKC-V606A in larval brain neuroblasts and comparing its localization to that of wild-type aPKC. Consistent with previous observations, wild-type aPKC is polarized to a cortical crescent around the apical pole at metaphase. In contrast, aPKC-V606A remained in the cytoplasm and was not recruited to the cortex, even though the localization of Par-3 was unaffected. The aPKC-V606A protein also failed to be recruited to the apical cortex in neuroblasts lacking endogenous aPKC. It is concluded that the Par-3 PDZ2-aPKC PBM interaction is required for cortical recruitment and polarization of aPKC in neuroblasts (Holly, 2020).

The interaction of Par-3 with the full-length Par complex was investigated, and it was found that Par-3 PDZ2 and a previously unrecognized PBM at the COOH terminus of aPKC are required for complex assembly. The Par-3 phosphorylation site (APM) can also form a persistent interaction with the aPKC kinase domain, but only if phosphorylation is not allowed to occur due to the absence of ATP. Unlike the APM-kinase domain interaction, the Par-3 PDZ2 interaction with the aPKC PBM is not influenced by the presence of ATP, suggesting that additional mechanisms besides APM phosphorylation must exist to dissociate Par-3 from the Par complex, an important component of current polarity models (Holly, 2020).

The identification of the Par-3 PDZ2 domain as a key factor in recruiting the Par complex to the cortex during animal cell polarization is consistent with previous work demonstrating that whereas Par-3 PDZ1 and 3 are dispensable in C. elegans, PDZ2 is required for cortical recruitment of Par-6 and aPKC. It is also consistent with work in both C. elegans and Drosophila showing that the interaction of Par-6 with Par-3 is not required. In Drosophila, the role of PDZ2 is less clear but is known to be required for downstream effects on epithelial structure. It is suggested that the Par-3 PDZ2-aPKC PBM interaction represents an important physical connection for animal cell polarity and that the reconstitution approach used to identify this interaction will likely be useful for understanding how other regulatory molecules, such as Cdc42, control polarity (Holly, 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).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 1464

Structural Domains

Analysis of Baz sequence reveals no obvious hydrophobic regions, suggesting a localization of the protein in the cytoplasm. In the center of the protein, three repeated regions exhibit pronounced similarity to the PDZ motif. The PDZ motif is a globular domain of 80-110 amino-acid residues, which can be present once or several times within a protein and provides an interface for protein-protein interactions. This domain has been identified in a number of intracellular proteins, many of which are associated with the membrane or concentrated at sites of cell-cell contact, such as tight junctions, septate junctions (see Discs large and polychaetoid) or postsynaptic densities. Others, for example the Drosophila protein InaD, have been shown to control the assembly of a multiprotein signaling complex, mediated by specific interactions performed by individual PDZ domains (Kuchinke, 1998 and references).

The Drosophila Bazooka protein exhibits an overall similarity to the PDZ protein Par-3 of C. elegans. Similar to Baz, Par-3 also contains three PDZ domains. Strikingly, the third PDZ domain (PDZ3) of Bazooka is more similar to PDZ3 of Par-3 than to Bazooka PDZ2 or PDZ1. In both proteins, the PDZ1 domains are less similar to the consensus PDZ motif. They lack, for example, the characteristic Gly-Leu-Gly-Phe repeat, which occurs in PDZ2 of Baz and, slightly modified, in PDZ2 of Par-3 and PDZ3 of both proteins. The similarity between the two proteins extends beyond the three PDZ domains and includes two additional regions in the amino and carboxyl termini, which exhibit an amino acid identity of 41% and 39% in a region of 80 and 25 amino acids, respectively. In addition, one mouse and one human expressed sequence tag possess similarity to the amino-terminal region of the Baz protein. Since the remaining sequences of these proteins are not yet known, however, it is uncertain whether these represent homologous proteins (Kuchinke, 1998).

date revised: 25 April 2024

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