Epithelial folding is typically driven by localized actomyosin contractility. However, it remains unclear how epithelia deform when myosin levels are low and uniform. In the Drosophila gastrula, dorsal fold formation occurs despite a lack of localized myosin changes, while the fold-initiating cells reduce cell height following basal shifts of polarity via an unknown mechanism. This study shows that cell shortening depends on an apical microtubule network organized by the CAMSAP protein Patronin. Prior to gastrulation, microtubule forces generated by the minus-end motor dynein scaffold the apical cell cortex into a dome-like shape, while the severing enzyme Katanin facilitates network remodelling to ensure tissue-wide cell size homeostasis. During fold initiation, Patronin redistributes following basal polarity shifts in the initiating cells, apparently weakening the scaffolding forces to allow dome descent. The homeostatic network that ensures size/shape homogeneity is thus repurposed for cell shortening, linking epithelial polarity to folding via a microtubule-based mechanical mechanism (Takeda, 2018).
Epithelial folding is a fundamental morphogenetic process in which two-dimensional (2D) epithelial sheets deform into 3D, folded structures. Epithelial folds form throughout animal development, giving rise to internal tissue structures and functional organ units. Folding of an epithelial monolayer can be induced by local forces generated at the site of initiation, or resulting from global stresses that buckle the tissue by exploiting intrinsic mechanical inhomogeneities. Both types of folding have been predominantly associated with modulation of myosin-dependent contractility. However, for epithelial folding events that lack overt myosin changes, it remains unclear what active mechanism underlies tissue deformation (Takeda, 2018).
Dorsal fold formation during Drosophila gastrulation has emerged as a crucial model for alternative folding mechanisms since myosin levels are low and uniform across the tissue. Following the completion of cellularization that forms the first embryonic epithelial layer, folding begins as two stripes of initiating cells straddling across the dorsal surface at stereotypical locations become shorter than their neighbours, leading to the formation of anterior and posterior dorsal folds eventually. Prior to cell shortening, Par-1, the MARK family kinase that specifies the basal-lateral membrane, retracts its apical margin and becomes downregulated, resulting in basal repositioning of adherens junctions in the initiating cells. Following such basal polarity shifts, the apices of initiating cells shrink and descend below the embryonic surface. How basal polarity shifts result in shrinkage and descent of the apices to reduce cell height was not known. As Par-1 has recently been shown to control the localization of the microtubule minus-end protector Patronin, this study investigated the possibility that Patronin-dependent modulation of the microtubule network underlies dorsal fold initiation (Takeda, 2018).
This study has identified a non-centrosomal microtubule network that is anchored and crosslinked to the apical cortex to exert pushing forces, thereby scaffolding the spherical apical shape of the epithelial cells in the Drosophila embryo prior to gastrulation. The data suggest that this microtubule network possesses an intrinsic, feedback-dependent remodelling mechanism that helps dampen the mechanical noises arising within the tissue, thus ensuring size homogeneity, echoing previous reports on microtubule-dependent cell size homeostasis. As gastrulation commences, coupling to the basal polarity shifts repurposes this homeostatic network for cell shortening (Takeda, 2018).
The microtubule forces exerted by the apical network must exist prior to, and in the opposite direction to, the movement of dome descent, thus contrasting with myosin-dependent apical constriction whose amplitude and directionality positively correlate with cell shape changes. It is proposed that dome descent in the dorsal fold system may be induced by residual stresses that compress inward and initially counterbalance the microtubule-dependent outward pushing forces, but become dominant as the basal redistribution of Patronin weakens the outward forces. This model is mechanically similar to the shape control of red blood cells where the microtubule-based marginal band pushes the cortex to counterbalance cortical tension generated by the Spectrin/actin-based membrane skeleton. It is possible that Spectrin and/or additional microtubule/actin dual linkers, such as the spectraplakin protein Shot, underlie the hypothetical counterbalancing stresses, and it is worth noting that recent work implicated links between Spectrin/Shot and Patronin/CAMSAP. Thus, in the context of the current model, Par-1 downregulation initially causes a transient expansion of the apical volume as a result of basal shifts of junctions. The expanded apical dome probably becomes mechanically imbalanced due to basal redistribution of the Patronin-anchored microtubule network, such that the pre-existing cortical compression stresses shrink the apex to drive dome descent. When such an imbalance occurs in a localized manner, a mechanical weak point could be produced to allow for initiation of folding. Conversely, global perturbation via stiffening (overexpression of Patronin) or softening (knockdown of Dynein) of the cortices probably causes a uniform deviation of such a balanced state, which instead blocks folding (Takeda, 2018).
The Patronin/CAMSAP proteins have emerged as key regulators of the microtubule minus ends, particularly for the non-centrosomal microtubules. During early cellularization prior to its transitioning to the apical cortex, however, Patronin is localized to the centrosome and appears to be required for the anchorage of centrioles and nucleus. Similar phenotypes have also been reported for the mammalian intestinal epithelia. These data could suggest an additional function on the centrosomal microtubules, which may also account for Patronin's involvement in the apical translocation of Bazooka. Intriguingly, junctional positioning in the patronin bazooka double RNAi embryos exhibits both a wide basal spread and an apical shift, respective features observed in the patronin and bazooka single knockdowns. Thus, Patronin may be required for junctional positioning in a Bazooka-independent manner. Moreover, the double RNAi embryos do not fold, even though the junctions are differentially positioned. It may be possible that the tissue-level mechanical coupling via junctions becomes defective due to the loss of Bazooka, resulting in ineffective transmission of tensile or compressive forces necessary for folding (Takeda, 2018).
The data implicate a cell shape control function for the Patronin/CAMSAP proteins in the epithelial system. The apical microtubule network that was observed resembles a similar structure previously described in the mammalian epithelial cells and could be a common feature for some epithelial systems. Highly pliable epithelial cells owing to low actomyosin-based rigidity such as those found on the dorsal side of the Drosophila gastrula may depend on the microtubule-based forces to define their shape, contrasting with epithelial contexts where cortical actomyosin forces dominate and cell shapes themselves dictate the spatial arrangement of microtubule filaments, but not vice versa. Evidence has emerged in certain contexts wherein actomyosin and microtubule networks are both involved in cell shape changes for the initiation of folding. It will be interesting to see whether context-specific interplay between these two mechanical systems underlies the formation of distinct morphological features (Takeda, 2018).
Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, persistent minus end growth is observed, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, it was hypothesized that minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, dendrite development and regeneration were analyzed. Minus ends extended into growing dendrites in the presence of Patronin. In sum, these data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules (Feng, 2019).
Microtubule plus ends exhibit rapid growth and shrinkage phases, a behavior termed dynamic instability. This behavior occurs in vitro with pure αβtubulin dimers and in vivo, where it can be regulated by plus end binding proteins and microtubule age. Minus ends also exhibit dynamic instability in vitro, although their growth rate is two to three times slower than plus ends. In vivo, however, minus ends are generally thought not to contribute to microtubule dynamics or exploration of cellular space (Feng, 2019).
In cells, the γtubulin ring complex acts as a template and nucleator to allow new microtubules to form. As long as it remains attached at the nucleating or minus end, it acts as a cap to block addition of subunits and prevent depolymerization. Free minus ends can then be generated by severing proteins. Two well-established fates for free minus ends are depolymerization and stabilization. In plants, cortical microtubules that are freed from nucleation sites depolymerize at the minus end while growing at the plus end to generate treadmilling behavior. In animal cells, depolymerization at the minus end is an important aspect of mitotic spindle dynamics, and minus end recognition by ASPM (abnormal spindle-like microcephaly associated) is important to help control depolymerization. In interphase, most free minus ends seem to be rapidly stabilized by calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin proteins (Feng, 2019).
Identification of CAMSAP/Patronin proteins was a major breakthrough in understanding the cellular stability of minus ends. They were the first proteins identified that could directly recognize free minus ends and are likely responsible for their resistance to depolymerization in interphase. The repertoire of minus end-binding proteins was recently expanded by the addition of ASPM, which can also recognize minus ends and controls their depolymerization in the spindle. It is not known whether this family of proteins also functions in post-mitotic cells like neurons (Feng, 2019).
Invertebrates have one CAMSAP/Patronin family member, termed Patronin, while vertebrates generally have three, CAMSAP1-3, with some extras where genome duplications have occurred. For example, in zebrafish, there are two each of CAMSAP1 and 2 and one CAMSAP3. Drosophila melanogaster Patronin stabilizes minus ends against depolymerization by kinesin-13 in cultured cells and in vitro. Mammalian CAMSAPs also protect minus ends from depolymerization by kinesin-13s in vitro and in cells. Overall stabilization of minus ends by CAMSAPs and Patronin is agreed upon as a critical function without which the microtubule network in cultured Drosophila cells and mammalian cells including neurons is dramatically destabilized (Feng, 2019).
The relationship between CAMSAPs/Patronin and minus end growth is more complex. In vitro CAMSAP2 and 3, as well as two domains of Patronin, suppress addition of tubulin subunits to the minus end in a concentration-dependent manner. In contrast, CAMSAP1 tracks minus ends as they grow without altering the rate of subunit addition. In cells, CAMSAP1 also tracks growing minus ends, but reduction of CAMSAP1 does not result in any change in microtubule behavior. CAMSAP2 has been described as suppressing minus end growth and also as promoting addition of short stretches of microtubule to the minus end. Although these two models for CAMSAP2 sound incompatible, they are actually not so different. Minus ends grow slowly in the presence of CAMSAP2, and this allows short stretches of CAMSAP2 to become stably associated with the microtubule. The stretches are on average 1 µ in control cells and 2 µ when katanin is depleted, and so, although they are derived from tubulin subunit addition, this does not result in much net growth at the minus end. In primary neuron cultures, stretches >10 µ of CAMSAP2 along microtubules have been observed, but growth has only been tracked for stretches of about a micron, so it is not clear how the longer stretches arise. Thus, it is still ambiguous whether extended growth at the minus end occurs in cells and, if so, how it contributes to global microtubule organization (Feng, 2019).
CAMSAP/Patronin proteins are particularly important in neurons where most, if not all, microtubules are noncentrosomal. In cultured hippocampal neurons, reduction of CAMSAP2, the major family member in this cell type, destabilizes microtubules and reduces dendrite complexity. Caenorhabditis elegans Patronin is required for maintenance of normal neuronal morphology, neuronal microtubule stability, and axon regeneration. Beyond stabilizing microtubules, it is not clear whether Patronin regulates specific aspects of microtubule organization in neurons. In epithelial cells, CAMSAP3 is responsible not only for stability of microtubules but also their polarized arrangement with minus ends concentrated at the apical surface. Neuronal microtubules are even more dramatically polarized than epithelial ones, with uniform plus-end-out polarity in axons and mixed or minus-end-out polarity in dendrites. It is therefore hypothesized that CAMSAP/Patronin proteins might function to control not only microtubule stability in neurons, but also their polarity (Feng, 2019).
Drosophila dendrites are strikingly polarized with >90% minus-end-out microtubules. It is conceptually straightforward to imagine plus-end-out processes in which fast-growing microtubule plus ends allow microtubules to populate an extending structure, while more complex models are thought to be required for population of processes with minus-end-out microtubules. In dendrites, local nucleation can generate new minus ends, and outgrowth of minus ends has not been considered as an alternative way to get minus-end-out microtubules into dendrites. However, in Drosophila neurons, nucleation sites are concentrated at dendrite branch points, so how the terminal dendrite beyond the branch point is populated with minus-end-out microtubules remains a conundrum (Feng, 2019).
Using live imaging of microtubule dynamics with tagged end-binding (EB) proteins in Drosophila and zebrafish neurons, this study identified a population of slow-growing microtubule ends that move in the opposite direction to fast-growing plus ends. In Drosophila dendrites, these slow-moving structures are labeled with Patronin, confirming that they are growing microtubule minus ends. This study demonstrates that sustained growth of minus ends requires Patronin, and in turn is important for getting minus-end-out microtubules into distal regions of mature dendrites, as well as into developing and regenerating dendrites (Feng, 2019).
In contrast to the traditional view that microtubule plus end growth is exclusively responsible for extending microtubules, this study found that in neurons, minus ends undergo extensive growth, with some individual runs lasting ≥10 min. Surprisingly, Patronin facilitates the persistent slow growth of minus ends, allowing them to elongate microns in single growth runs. Moreover, this minus end growth has critical functions in organizing the neuronal microtubule cytoskeleton, including populating growing dendrites and distal regions of mature dendrites with minus-end-out microtubules (Feng, 2019).
Neuronal DLK/JNK stress signaling is a common response to reduced Patronin in C. elegans and Drosophila. As this signaling is responsible for some of the phenotypes observed in both systems, it will be important to determine whether any of the phenotypes that have been described in mammalian neurons with reduced CAMSAP2 are downstream of DLK or JNK. Changes in microtubule dynamics are a key output of neuronal stress from expression of proteins that cause neurodegeneration in flies and mammals, so some of the phenotypes attributed to direct function of CAMSAP2 could be secondary to stress. The current data suggest that in Drosophila, Patronin directly regulates minus end extension and microtubule polarity, but that changes in microtubule dynamics may be secondary to stress signaling (Feng, 2019).
Persistent minus end growth facilitated by Patronin could be an evolutionary innovation in animals like Drosophila that have mostly minus-end-out dendrites. However, this study was able to visualize growing minus ends that behaved similarly to the ones in Drosophila dendrites in plus-end-out zebrafish sensory endings. If this phenomenon is widespread across evolutionarily distant taxa, then why has it not previously been reported? EB proteins have been used to visualize microtubule plus end growth in neurons since 2003, but slow-moving dots have not been noted before in neurons. One reason for this is likely improvements in sensitivity and signal-to-noise detection in microscopes used for live imaging. However, upon close inspection, there seem to be occasional minus ends in published EB data; for example, slow dim EB structures are present in kymographs from Drosophila and mammalian neurons. In the future, it will be important to investigate behavior of microtubule minus ends in neurons from other species in detail to determine whether minus ends grow for long periods and over substantial distances (Feng, 2019).
It is particularly interesting to compare minus end growth across species because of the expansion of the Patronin/CAMSAP family in vertebrates. In zebrafish, clear minus end growth is observed, but it is not yet known which of the five CAMSAPs (two each of CAMSAP1 and 2 and one CAMSAP3) in fish might be present at these growing minus ends. In contrast to CAMSAP2 and 3, which suppress minus end growth, CAMSAP1 tracks minus ends as they grow, so it is a good candidate. However, knockdown of CAMSAP1 has not been associated with changes in microtubule behavior in immortalized cell cultures and was not detected in neurons in the hippocampus. In contrast, CAMSAP2 knockdown has dramatic effects on microtubule stability in immortalized cells and neurons, so it is an open question what family member might be present on the growing minus ends tracked in zebrafish sensory neurons and how different types of CAMSAPs might cooperate or compete with one another (Feng, 2019).
The finding that Drosophila Patronin facilitates, rather than reduces, minus end growth in neurons is surprising in light of its previous characterization. Rather than tracking growing minus ends like CAMSAP1, it is thought to dampen minus end growth like CAMSAP2 and 3. One possible explanation for this discrepancy between Patronin behavior in vitro and in neurons is that in specific cell types or contexts, its behavior at minus ends could be regulated. In vitro, the coiled coil domain of Patronin tracks dynamic minus ends, and when the neighboring CKK domain is added, the behavior switches to growth suppression. In vivo, perhaps the effect of the CKK domain is modulated to uncover the minus end growth tracking ability of the coiled coil domain (Feng, 2019).
The neuronal phenotypes from reduction of Patronin are consistent with a role in minus end growth, and it is proposed that extended periods of minus end growth allow minus-end-out microtubules to grow into dendrites. Consistent with this idea, Patronin reduction resulted in fewer minus-end-out microtubules in distal regions of mature dendrites as well as in developing and regenerating dendrites. However, other mechanisms likely work in parallel with minus end growth to populate dendrites with microtubules (Feng, 2019).
Sliding of minus-end-out microtubules along plus-end-out ones by kinesin-6 motors was the mechanism first proposed to populate dendrites with minus-end-out microtubules in mixed polarity mammalian neurons, and the function of kinesin-6 has been confirmed more recently with newer methods. In Drosophila, these motors could play a role in dendritic microtubule organization early in development when microtubules in dendrites are mixed, but it is harder to imagine how they might function in mature minus-end-out dendrites. Analysis of Pavarotti, one of the Drosophila kinesin-6 motors, confirms that it is unlikely to play a major role in polarity of mature dendrites, and actually reduces sliding of microtubules by kinesin-1 into developing axons (Feng, 2019).
Local nucleation of microtubules in dendrites can also help populate dendrites with microtubules and, in contrast to sliding, has been shown to be important in Drosophila and C. elegans. In mammalian neurons, noncentrosomal microtubule nucleation also predominates and is known to be critical for axon growth and microtubule organization, though the localization and role of nucleation sites in dendrites has not been detailed so far. During C. elegans development, microtubule nucleation operates in parallel to minus end regulation by Patronin, and both types of minus end regulation may cooccur broadly in different cell types and species. In different contexts, nucleation or Patronin may vary in relative importance. For example, during initial dendrite outgrowth, no strong morphological defects were observed with Patronin knockdown, but during regeneration, dendrite growth was strongly perturbed. While differences in RNAi knockdown could account for the phenotypic differences, it is also possible that nucleation can compensate more completely for Patronin reduction in development compared with regeneration (Feng, 2019).
Overall, this study has demonstrated that, rather than existing as static structures, microtubule minus ends can grow in Drosophila and zebrafish neurons. Minus end growth is slow, but persistent, and up to 10 µ can be added in a single episode of growth. While both EB proteins and Patronin can bind to minus ends, they do so independently, and Patronin is critical for extended stretches of minus end growth. Without Patronin-mediated minus end growth, minus-end-out microtubules were reduced in dendrite tips and developing dendrites. Moreover, extra Patronin was sufficient to convert dendrite tips from mixed polarity to minus-end-out. Dendrite regeneration was particularly sensitive to Patronin reduction. It is concluded that minus end microtubule growth is likely to be broadly important in helping minus-end-out microtubules reach cellular regions that are distal to nucleation sites (Feng, 2019).
Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, this study resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, these results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed (Sun, 2021).
The structural reconstruction reveals that microtubules in the outer segment are highly organized and stable. They form two structurally separate arrays (see Molecular organization of microtubules in the outer segment). In particular, the array in the mechanoreceptive organelle (MO) contains mostly short microtubules, which facilitate the structural optimization of the MO and provide anchoring sites to a large number of mechanosensory molecules (e.g., NompC), thereby serving key sensory functions. Furthermore, the microtubule arrays in the tubular body (TB) and MO are differentially regulated, suggesting dedicated patterning and regulatory mechanisms in the outer segment of fly external mechanoreceptors (Sun, 2021).
The present study provides two implications in understanding how the microtubule arrays in fly sensory cilia are formed. First, a large quantity of microtubules is required to form dense arrays in the outer segment. Kat-60L1 and Patronin together amplify the mass of microtubules. In addition, the presence of a small number of microtubules in one piggyBAC insertion mutant/null mutant (c01236/BE6) implies that there is a kat-60L1–independent mechanism (denoted as X1) to assemble microtubules. These microtubules may serve as the initial substrates of kat-60L1 to create more templates. Second, the nanoscopic cytoskeleton in the MO determines the number and spatial arrangement of NompC force-sensitive channels that can be accommodated in the MO. Kat-60L1 coordinates the number and length of microtubules to build the cytoskeleton in the MO, which likely facilitates the structural–mechanical design of the MO, for example, providing adequate anchoring sites for NompC or withstanding resting tensions for optimal mechanosensitivity. An ensuing issue is how short microtubules in the MO can be stabilized. The lower susceptibility of the MO microtubules to the reduction in the level of Patronin suggests the presence of additional stabilizing mechanisms (Sun, 2021).
The results suggest that kat-60L1 has two functions in fly ES cilia. First, Kat-60L1 increases the mass of microtubules in the outer segment. This agrees with the proposed function of microtubule-severing enzymes in amplifying the local mass of microtubules. Second, kat-60L1 is required to generate short microtubules. It has been shown that microtubule-severing enzymes sculpt cellular microtubule networks; for example, the noncentrosomal microtubule arrays in plant cells and the meiotic spindle. Previous studies have also implicated the potential functions of microtubule-severing enzymes in neurons. However, due to the limited optical resolution, visualizing dense microtubule networks (e.g., in neurons) where the severing activity is potentially important remains challenging. ET could be useful in such cases. In the present study, structural reconstruction provides direct evidence to demonstrate the severing activity of Kat-60L1 in fly ciliated sensory neurons, thereby providing new insights into the neuronal and ciliary functions of microtubule-severing enzymes (Sun, 2021).
A potential caveat to the above conclusion is that the loss of kat-60L1 also changes the homeostatic microtubule dynamics in the inner segment, so it is formally possible that the structural and functional defects in the outer segment might be a secondary effect. However, this is not thought to be the case based on three reasons. First, in kat-60L1i, no significant microtubule accumulation was observed in the inner segment, but the cytoskeleton in the outer segment was also disrupted, suggesting that microtubules in the outer segment are more susceptible to the level of kat-60L1, and that the defects in the outer segment are a primary consequence of losing kat-60L1. Second, more microtubule polymerizations, suggested by the observation of more frequent EB1 comets, are observed in the inner segment in c01236/BE6, suggesting that the level of free tubulin dimers is fairly normal. Because tubulin is transported or diffuses into cilium as dimers, it is argued that tubulin influx into the cilia, which matters for microtubule dynamics in the outer segment, is not down-regulated in c01236/BE6. Third, no major defects in the morphology of dendritic inner segments were observed in c01236/BE6, suggesting that the functional defects can be mostly attributed to the defects in the outer segment (Sun, 2021).
Previous in vitro studies proposed that the cellular effects of microtubule-severing enzymes depend on the ratio between the amount of severing enzymes and the local microtubule dynamics. This study shows that in the outer segment, where microtubules are stable and the signal of Kat-60L1 is weak, Kat-60-L1 acts as a constructor, while in the inner segment, where microtubules are more dynamic and Kat-60L1 shows an enriched signal, Kat-60-L1 tends to act as a destructor. These results provide an in vivo example that supports the predictions from the previously proposed model (Sun, 2021).
The function of katanin family members has been implicated in ciliogenesis, especially in the assembly of central pair microtubules in motile cilia. Fly mechanosensory cilia contain no central pair microtubules but numerous noncentrosomal microtubules. The disruption of the cytoskeleton in the absence of Kat-60L1 in fly mechanosensory cilia resembles the loss of central pair microtubules in the katanin mutants of other species. This implies a common component in the assembly mechanism of noncentrosomal microtubules inside cilia, which, however, remains a hypothesis waiting for future studies (Sun, 2021).
Spatially organized macromolecular complexes are essential for cell and tissue function, but the mechanisms that organize micron-scale structures within cells are not well understood. Microtubule-based structures such as mitotic spindles scale with cell size, but less is known about the scaling of actin structures within cells. Actin-rich denticle precursors cover the ventral surface of the Drosophila embryo and larva and provide templates for cuticular structures involved in larval locomotion. Using quantitative imaging and statistical modeling, denticle number and spacing were demonstrated to scale with cell size over a wide range of cell lengths in embryos and larvae. Denticle number and spacing are reduced under space-limited conditions, and both features robustly scale over a ten-fold increase in cell length during larval growth. The relationship between cell length and denticle spacing can be recapitulated by specific mathematical equations in embryos and larvae, and accurate denticle spacing requires an intact microtubule network and the microtubule minus-end-binding protein, Patronin. These results identify a novel mechanism of microtubule-dependent actin scaling that maintains precise patterns of actin organization during tissue growth (Spencer, 2017).
Noncentrosomal microtubules play an important role in polarizing differentiated cells, but little is known about how these microtubules are organized. This study identified the spectraplakin, Short stop (Shot), as the cortical anchor for noncentrosomal microtubule organizing centers (ncMTOCs) in the Drosophila oocyte. Shot interacts with the cortex through its actin-binding domain and recruits the microtubule minus-end-binding protein, Patronin, to form cortical ncMTOCs. Shot/Patronin foci do not co-localize with gamma-tubulin, suggesting that they do not nucleate new microtubules. Instead, they capture and stabilize existing microtubule minus ends, which then template new microtubule growth. Shot/Patronin foci are excluded from the oocyte posterior by the Par-1 polarity kinase to generate the polarized microtubule network that localizes axis determinants. Both proteins also accumulate apically in epithelial cells, where they are required for the formation of apical-basal microtubule arrays. Thus, Shot/Patronin ncMTOCs may provide a general mechanism for organizing noncentrosomal microtubules in differentiated cells (Nashchekin, 2016).
The recent discovery of the Patronin family of MT minus-end-binding proteins, consisting of Patronin in Drosophila, CAMSAP1, 2, and 3 in mammals, and PTRN-1 in worms, has begun to reveal how the minus ends of noncentrosomal MTs are organized and maintained (Akhmanova, 2015; Baines, 2009; Goodwin, 2010: Marcette, 2014; Meng, 2008; Richardson, 2014). The Patronins recognize and stabilize free MT minus ends by protecting them from depolymerization (Goodwin, 2010; Hendershott, 2014; Jiang, 2014). Patronins appear to play a particularly important role in organizing MTs in differentiated cells. CAMSAP3 localizes to the apical domain in epithelial cells, where it is required for the formation of the apical-basal array of MTs (Tanaka, 2012; Toya, 2016, Zheng, 2013). CAMSAP2 stabilizes neuronal MTs in axon and dendrites, and its knockdown leads to defects in axon specification and dendritic branch formation (Yau, 2014). Similarly, Caenorhabditis elegans PTRN-1 is required for normal neurite morphology and axon regeneration (Chuang, 2014; Marcette, 2014; Richardson, 2014). The function of Drosophila Patronin has only been examined in cultured S2 cells, where its depletion leads to a decrease in MT number and an increase in free moving MTs (Goodwin, 2010; Nashchekin, 2016 and references therein).
The polarized arrangement of the MTs in the Drosophila oocyte depends on the posterior crescent of the Par-1 kinase, which excludes MT minus ends from the posterior cortex (Doerflinger, 2010; Parton, 2011). This study shows that Par-1 acts by preventing the association of Shot with the posterior actin cortex, thereby restricting the formation of noncentrosomal MTOCs to the anterior and lateral cortex. Computer modeling has shown that this asymmetric localization of MT minus ends is sufficient to explain the formation of the weakly polarized MT network that directs the transport of oskar mRNA to the posterior pole. Thus, the regulation of the interaction of Shot with the cortex by Par-1 transmits cortical PAR polarity into the polarization of the MT cytoskeleton that localizes the axis determinants (Nashchekin, 2016).
The mechanism by which Par-1 excludes Shot is unknown. The interaction of Shot with the cortex depends on its N-terminal calponin homology domains, which bind to F-actin. Thus, Par-1 could phosphorylate Shot to inhibit its binding to the cortex. If this is the case, Par-1 would have to modify the activity or accessibility of the N-terminal ABD of Shot, as this domain recapitulates the posterior exclusion and cortical recruitment of the full-length protein. Phosphorylation of the ABD by Par-1 was not detected in vitro, however, and it seems more likely that Par-1 acts by modifying the cortex to block the binding of Shot (Nashchekin, 2016).
Shot and its vertebrate ortholog, MACF1, have previously been shown to interact with the MT plus-end tracking protein EB1 through their C-terminal SxIP motifs and with the MT lattice through their Gas2 and C-terminal domains (Nashchekin, 2016).
The current results indicate that in addition to binding to MT plus ends and to the MT lattice, Shot also interacts with MT minus ends through its association with the Patronin/Katanin complex. The exact nature of the interaction between Shot and the Patronin complex is unclear, but Shot was found to interact with Katanin 60 in a high-throughput yeast two-hybrid screen. Thus, one possibility is that Katanin acts as a link between Shot and Patronin. Since Shot is exclusively cortical in the oocyte, the protein does not appear to bind to MT plus ends or along the body of MTs in this system. It will therefore be interesting to investigate whether the different modes of MT binding by Shot are mutually exclusive and how this is regulated (Nashchekin, 2016).
Several models have been proposed to explain the formation of noncentrosomal MTs. Upon centrosome inactivation in postmitotic Drosophila tracheal cells and C. elegans intestinal cells, γ-TuRC complexes and other pericentriolar material (PCM) components are released from the centrosome and transported toward the apical membrane, where they nucleate MT. Whole MTs released from the centrosome can also be delivered and anchored to the apical domain or cell junctions by Ninein. Alternatively, new MTs can grow from MT ends generated by severing enzymes, a mechanism that is thought to be important in plant cells and neurons (Nashchekin, 2016).
This study presents evidence that this latter mechanism is responsible for the formation of the MT array that directs Drosophila axis formation. Firstly, Shot/Patronin ncMTOCs contain stable minus ends even after treatment with the MT-depolymerizing drug, colcemid, as shown by the persistent recruitment of Tau-GFP and EB1-GFP to these foci. This is consistent with the ability of Patronin and CAMPSAPs to capture and stabilize minus ends of single MTs in vitro and in cells. Secondly, MTs start to grow out in all directions from the Shot/Patronin foci immediately after colcemid inactivation. Indeed all visible growing MTs emanate from Patronin foci, indicating that they are the principal source of MTs in the oocyte. Thirdly, the foci contain no detectable γ-tubulin and do not co-localize with PCM proteins. This is consistent with observations in Caco-2 cells, which showed that CAMSAP2 and CAMSAP3 do not co-localize with γ-tubulin and in the C. elegans epidermis, where PTRN-1 and γ-tubulin function in parallel pathways to assemble circumferential MTs (Nashchekin, 2016).
Taken together, these results suggest a model in which the Shot/Patronin foci act as ncMTOCs by capturing and stabilizing MT minus-end stumps that then act as templates for new MT growth. One attractive feature of this model is that it uncouples MT organization from MT nucleation in both space and time. The Shot/Patronin complex bypasses the need to continually nucleate new MTs by preventing existing microtubules from completely depolymerizing. Thus, once a cell has nucleated sufficient MTs, it can maintain and reorganize its MT cytoskeleton by stabilizing MT minus-end stumps in appropriate locations and using these, rather than the γ-tubulin ring complex, to provide the seeds from which new MTs grow. The number of MTs can even increase in the absence of new MT nucleation if MT-severing proteins chop up existing MTs to produce new minus ends that can then be captured and stabilized. The presence of the severing protein, Katanin, in the Shot/Patronin foci is intriguing in this context, as it raises the possibility that it severs existing MTs to provide a local source of minus ends for Patronin to capture (Nashchekin, 2016).
Shot and Patronin also co-localize at the apical cortex of the epithelial follicle cells, where they are required for apical-basal MT organization. This consistent with the recent observation that CAMSAP3 is required for the recruitment of MT minus ends to the apical cortex of mammalian intestinal epithelial cells (Toya, 2016). Thus, this function of Patronin has been evolutionarily conserved. Furthermore, the similarities between roles of Shot and Patronin in the oocyte and the follicle cells suggest that the complex may provide a general mechanism for organizing noncentrosomal MTs. The relationship between Shot and Patronin is different in the follicle cells compared with the oocyte, however, as Shot is not required for the apical recruitment of Patronin. Nevertheless, loss of either protein produces a very similar loss of apical MT and a reduction in overall MT density. Although it cannot be ruled out that they act in parallel pathways, this observation suggests that they collaborate to anchor MTs to the apical cortex. The combination of Patronin binding to the MT minus ends and Shot binding to the MT lattice may therefore provide a robust anchor to retain MTs at the apical cortex (Nashchekin, 2016).
In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. This study examines the hierarchy of events during cytoskeletal polarisation in Drosophila and human epithelia. Core apical-basal polarity determinants polarise the Spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1/2/3 in humans) and Shortstop (Shot; MACF1/BPAG1 in humans) to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV actin motor to deliver the key microvillar determinant Cadherin99C to the apical membrane to organise the biogenesis of actin microvilli (Khanal, 2016).
These results reveal a mechanism linking determinants of cell polarity with stepwise polarisation of the spectrin cytoskeleton, microtubule cytoskeleton and biogenesis of actin microvilli through apical trafficking of Cad99C. The results suggest that polarisation of the apical spectrin βH-Spectrin is dependent on polarity determinants, likely through interactions with the FERM domain proteins and the apical polarity determinant Crb. The spectraplakin Shot is highly similar to βH-Spectrin, and is able to bind to and colocalise with it at the apical domain of epithelial cells, suggesting that the two proteins might have a similar function. βH-Spectrin is linked to microtubules through Patronin, whereas Shot can directly bind microtubules. Consequently, redundancy is anticipated between βH-Spectrin and Shot, or between Patronin and Shot. Accordingly, this study found that mutation of βH-spectrin only had a mild phenotype, whereas mutation of α-spectrin simultaneously disrupted both pairs of proteins in parallel and caused a drastic phenotype, completely disrupting the apical trafficking of Cad99C and microvillar biogenesis. More importantly, double mutants for shot and βH-spectrin had a more severe effect on microtubule and Cad99C localisation than either alone, therefore demonstrating that the two proteins act in a redundant fashion (Khanal, 2016).
Downstream of the spectrin cytoskeleton, Patronin and Shot are required in parallel to drive apical-basal polarisation of microtubules, which is then responsible for orienting the apical transport of Cad99C, within Rab11 endosomes, by the Dynein motor protein. Eliminating microtubules from cells by overexpressing Katanin60 results in loss of Nuf-Dynein-based apical Rab11 endosome transport and failure to efficiently deliver Cad99C to the apical membrane. The effect on Cad99C polarisation is not an indirect effect of loss of polarity due to impaired Rab11 and Dynein function in localising the apical polarity determinant Crumbs to the apical membrane because, firstly, polarity is maintained in cells expressing Rab11 or Dynein RNAi, as indicated by the normal localisation of aPKC and, secondly, loss of Crb does not strongly affect cell polarity in the follicle cell epithelium owing to redundancy with Bazooka. The results indicate that even under conditions with severe depletion of microtubules, the overall shape of the follicle cell epithelium is relatively normal, indicating that polarised microtubules are required to influence formation of apical microvilli, rather than for other functions of the actin cytoskeleton in epithelial cells. Similarly, no strong effects are seen on cell shape upon loss of either Patronin or Shot (or both), raising questions over the claimed requirement for Patronin homologs and microtubules in formation or maintenance of adherens junctions epithelial cells in culture (Khanal, 2016).
The final step in delivery of Cad99C to the apical membrane also requires actin-based transport through the action of Rip11-MyoV complex. Compromising normal MyoV function in Drosophila follicle cells by expressing a dominant-negative version of the protein, results in loss of Rab11 polarisation from the apical membrane and its abnormal accumulation in the sub-apical region. This phenotype in Drosophila shows similarities with the human microvillus inclusion disease, where mutations in the Myo5b gene also cause loss of Rab11 endosomes from the apical membrane (Khanal, 2016).
In summary, these results reveal how the spectrin cytoskeleton acts to polarise microtubules in epithelial cells, and how polarised microtubules then direct trafficking of Rab11 endosomes carrying Cad99C to the apical membrane. This process relies on a hierarchy of events, and disruption at any stage can lead to failure in delivering Cad99C to the apical membrane, resulting in defective biogenesis of microvilli. These findings are directly relevant to human diseases such as Usher's Syndrome Type 1 and microvillus inclusion disease, helping to outline the molecular and cellular basis for these conditions (Khanal, 2016).
During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division (Coumailleau, 2009; Loubéry, 2014). Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. This study shows that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. This mechanism was demonstrated by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. These data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division (Derivery, 2015).
Klp98A was first identified as the kinesin mediating Sara endosome motility during sensory organ precursor (SOP) division. Klp98A is the Drosophila homologue of mammalian KIF16B, an early endosomal kinesin containing a phosphatidylinositol 3-phosphate-binding PX domain. Indeed, Klp98A localizes to Sara-positive early endosomes (Derivery, 2015).
During SOP division, Klp98A-GFP-positive Sara endosomes segregate to the pIIa daughter, but not the pII (Coumailleau, 2009; Loubéry, 2014). Sara endosomes were monitored by following Delta 20 min after internalization (iDelta20) through an improved antibody internalization assay. iDelta20 parallels Sara endosome dynamics in the controls and mutants studied here (in vivo and primary cultures. Like KIF16B, purified Klp98A binds specifically to phosphatidylinositol 3-phosphate and is a plus-end-directed motor whose velocity is 0.76 ± 0.02 μm s-1 (Derivery, 2015).
To study Klp98A function, deletions within the motor domain (Klp98AΔ6, Klp98AΔ7 and Klp98AΔ8, 6, 7 and 8-base-pair deletions, respectively) and a clean coding sequence deletion (Klp98AΔ47). Except Klp98AΔ6, all are protein nulls. In Klp98A-, Sara endosomes move diffusively. Therefore, Klp98A mediates Sara endosome motility (Derivery, 2015).
In wild-type cells, Sara endosomes move on microtubules to the Pavarotti-positive central spindle and, late in cytokinesis, to pIIa. Spindle microtubule plus-ends are oriented towards the equator, explaining central spindle endosomal targeting by a plus-end motor. Indeed, Sara endosome central spindle targeting fails in Klp98A- mutants. Importantly, in Klp98A- mutants and upon RNAi-mediated Klp98A knockdown, endosomes are symmetrically dispatched (Derivery, 2015).
Klp98A-mediated motility contributes to cell fate assignation through asymmetric Notch signalling, but this activity is redundantly covered by Neuralized and Numb (Fürthauer, 2009). Indeed, Klp98A-;pnr > neurRNAidouble mutants show a synergistic fate assignation phenotype: the notum is largely void of bristles. Conversely, Klp98A;Numb double mutants strongly suppress the diagnostic Numb- multiple socket phenotype. Therefore, having established the role of Klp98A motility in Notch signalling, this study focused on the mechanisms orchestrating asymmetric motility (Derivery, 2015).
Central spindle targeting of Sara endosomes precedes asymmetric segregation to pIIa. Focus was therefore placed on Sara endosome motility with respect to the central spindle reference frame. The central spindle is composed of the Pavarotti-positive core (containing antiparallel microtubules) plus the microtubules emanating from it. The Pavarotti core was automatically tracked, defining a 2D cartesian reference frame whose origin is the Pavarotti centroid and whose x axis is the pIIb-pIIa axis. This also defines a Pavarotti width (PW) and length (PL, the length of the microtubule antiparallel array (Derivery, 2015).
Sara endosomes were tracked with respect to this reference frame (with 160 nm accuracy. Automatic tracking and spatio-temporal registration provided a large data set (2,897 traces) from which a spatio-temporal density plot of endosomes at the central spindle was generated. For 500 s, endosomes remain mostly within the Pavarotti region. Remarkably, at the central spindle, motility along the x axis is bidirectional. Motility along the y axis merely follows PW contraction, consistent with motility along central spindle microtubules, parallel to the x axis. Velocities are similar towards pIIa and pIIb and slower than in vitro, possibly due to crowding by microtubule-associated proteins (Derivery, 2015).
Confinement within the Pavarotti region and bidirectional movement are both consistent with a plus-end motor switching direction on antiparallel microtubules. On single microtubules, Klp98A-bound quantum dots always maintain their directionality when resuming after a pause. It was asked whether Klp98A could switch direction in an antiparallel bundle. In an in vitro reconstitution assay, Klp98A-bound quantum dots move bidirectionally within antiparallel MAP65-1-mediated microtubule arrays. 68% tracks change direction after pausing. Therefore, Klp98A supports bidirectional motility in antiparallel array (Derivery, 2015).
Notably, in vivo, bidirectional endosome motility is asymmetric: the residence time in pIIa is 1.8-fold longer than in pIIb. Consistently, the spatio-temporal density plot is asymmetric. Furthermore, tracks overshoot beyond the Pavarotti region more frequently into pIIa (Derivery, 2015).
Eventually, endosomes depart from central spindle microtubules into the cytoplasm and therefore move also on the y axis. The longer pIIa residence time and higher pIIa overshoot frequency make this final departure asymmetric, explaining the biased segregation into pIIa. Therefore, asymmetric endosome motility at the central spindle underlies asymmetric dispatch to pIIa (Derivery, 2015).
It was then asked whether the central spindle itself is asymmetric. Using Pavarotti spatio-temporal registration, an 'average cell' was generated to map the densities of the microtubule markers Jupiter and SiR-tubulin (microtubule markers), Patronin (Goodwin, 2010) (minus-end), and Pavarotti (plus-ends/antiparallel overlap). This 'average cell' reveals a polarity map of the central spindle consistent with electron microscopy reports: plus-ends are in the middle and minus-ends on the outer side. Microtubule densities in general, and Patronin in particular, are ~20% higher on the pIIb side. This asymmetry depends on Par complex activity, and is absent in neighbouring cells dividing symmetrically, but this seems independent of central spindle or endosomal asymmetry (Derivery, 2015).
Microtubule asymmetry builds up during anaphaseB, concomitant with biased endosome motility, while, earlier, the metaphase spindle is symmetrical. During anaphaseB, the central spindle shrinks by microtubule depolymerization through depolymerizing kinesins like Klp10A, among other factors. Depolymerization dynamics are asymmetric: microtubule loss is faster in pIIa. This could be explained by Patronin enrichment in the central spindle pIIb outer side where it binds to minus-ends, counteracting Klp10A-mediated depolymerization (Goodwin, 2010; Wang, 2013; Hendershott, 2014; Jiang, 2014; Derivery, 2015 and references therein).
Indeed, Klp10A/Patronin control asymmetric microtubule depolymerization: their depletion abolishes spindle asymmetry. In Patronin-knockdown cells, both sides exhibit low microtubule densities characteristic of pIIa, consistent with Patronin pIIb enrichment in wild type and its activity against depolymerization. Conversely, upon knockdown of Klp10A, both sides exhibit high microtubule densities resembling pIIb (Derivery, 2015).
The parallelism between central spindle asymmetry and asymmetric endosome motility suggests that spindle asymmetry causes biased motility. Indeed, endosome motility at the central spindle and, therefore, segregation become symmetric in Klp10A- and Patronin-knockdowns, while early central spindle targeting is normal. This uncovers a quantitative correlation between spindle and endosomal asymmetry (Derivery, 2015).
Together, a plus-end motor and microtubule plus-ends facing the centre explain why a higher pIIb microtubule density (~20% enrichment) targets endosomes to pIIa (~80% pIIa, that is, >300% enrichment). In other words, endosomes move away from higher microtubule densities in pIIb (Derivery, 2015).
Based on a theoretical model of plus-end endosomal motility on an antiparallel, asymmetric microtubule overlap, the steady-state endosome distribution is Where PpIIa, PpIIb, the probabilities for an endosome to be on either side of the antiparallel overlap; ρa, ρb, microtubule densities in pIIa/pIIb, respectively; kon, koff, microtubule association/dissociation constants of the motor, respectively; v, the endosome motor-driven velocity; D, the diffusion coefficient of endosomes detached from microtubules; and l, the antiparallel overlap length (Derivery, 2015).
To generate this inverted spindle, 'nanobody assay' was established based on GFP-binding-peptide (GBP)-Pon, a nanobody fused to the Pon localization domain. GBP-Pon traps GFP-Patronin away from the spindle at the pIIb cortex thereby reducing, specifically in pIIb, Patronin-dependent protection against central spindle depolymerization. This inverts spindle asymmetry, which consequently inverts endosomal asymmetry. SiR-Tubulin and endogenous acetyl-tubulin stainings confirmed this spindle inversion (Derivery, 2015).
Interestingly, this assay generates a phenotypic series of different levels of spindle reversal and their corresponding endosomal reversals. These data fall on the theoretical curve obtained with independently measured parameters. Therefore these results uncover the quantitative dependence of asymmetric endosome targeting on spindle asymmetry (Derivery, 2015).
This study has identified Klp10A/Patronin as the machinery generating spindle asymmetry, which is read out by Klp98A to achieve asymmetric targeting of signalling endosomes. Asymmetric endosomal targeting contributes in turn to asymmetric cell fate assignation, confirming previous reports in flies and fish. These data thus uncover a mechanism by which intracellular cargoes in general, and signalling endosomes in particular, can be targeted to one of the daughter cells during asymmetric division (Derivery, 2015).
How could then other cargoes segregate symmetrically, if the spindle is asymmetric? Asymmetric targeting would only be efficient if kon, koff and v are optimized to amplify the mild asymmetry of the spindle, otherwise concealed by noise sources in the cell. More generally, plus- and minus-end motors are present simultaneously in the same vesicle and thereby may counteract each other to achieve symmetrical dispatch (a sort of 'tug of war'). Therefore, the precise landscape of microtubule polarity trails combined with the right cocktail of motors in vesicles provides the plasticity required to generate the plethora of molecular spatial patterns observed in polarized cells (Derivery, 2015).
The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at the plus- or minus-ends of the polymer, is essential for MTs to carry out their biological functions. A variety of proteins act on MT ends to regulate their dynamics, including a recently described family of MT minus-end binding proteins called calmodulin-regulated spectrin-associated protein (CAMSAP)/Patronin/Nezha. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minus-ends against depolymerization in vitro and in vivo. This study shows that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13-induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. By using truncation analysis, this study found that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, in both cases, a highly conserved C-terminal domain and a more variable central domain were found to cooperate to suppress minus-end dynamics in vitro, and both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics (Hendershott, 2014).
Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. This study has identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. It is proposed that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton (Goodwin, 2010).
