InteractiveFly: GeneBrief

mini spindles: Biological Overview | References

BIOLOGICAL OVERVIEW

mini spindles (msps) was identified in a cytological screen for mitotic mutants. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle (Cullen, 1999).

Mini-spindles and D-TACC localize at acentrosomal poles in the female meiotic spindle of Drosophila. Msps and D-TACC are also associated with mitotic centrosomes and interact with one another and are both required for maintaining the bipolarity of acentrosomal spindles. The polar localization of Msps is dependent on D-TACC and Ncd, a kinesin-like microtubule motor. It is proposed that the polar localization of Msps mediated by D-TACC and Ncd may be crucial for the stabilization of meiotic spindle bipolarity (Cullen, 2001).

Molecular cloning revealed that the msps gene encodes a protein which belongs to the dis1-TOG family. This family of proteins is very divergent at the primary sequence level. Conserved residues are limited to four motifs that form part of repeating regions in the NH₂-terminal portion of the molecule. These repeats with their limited conservation could be a structural or functional module in the sense of the tetratrico peptide repeats (TPR), which are found in proteins with various functions. In support of this idea, it was shown that the repeats are dispensable for in vivo function of S. pombe Dis1. This raises the question as to whether these repeats do indeed have a common function and whether the dis1-TOG family shares a conserved function in vivo (Cullen, 1999).

The vertebrate and fly proteins share features that are distinct from lower eukaryotic proteins. They have four repeats in the NH2-terminal region, while the yeast and C. elegans have only two repeats. The Msps protein is highly homologous to human TOGp along its entire length. Their COOH-terminal regions are conserved and share some homology with the C. elegans protein. In contrast, the COOH-terminal regions of the yeast protein has a coiled-coil structure bearing no sequence homology. Drosophila genetics should provide a unique opportunity to study in vivo function of higher eukaryotic members of this MAP family (Cullen, 1999).

Considering the divergence of protein sequence of the family members and structure of the mitotic apparatus among eukaryotes, localization of the dis1-TOG proteins is surprisingly similar among dis1-TOG family from yeasts to human. They concentrate in the vicinity of the centrosome or SPB during the early stages of mitosis, transiently spread along the whole length of the mitotic spindles during mid-mitosis, before localizing back to the vicinity of the centrosome/SPB region in late mitosis (Cullen, 1999).

The cell cycle-dependent interaction between Msps and microtubules or centrosomes could occur as a result of posttranslational modification of Msps protein. Although no evidence is available for such modification of Msps, cell cycle-dependent phosphorylation of the XMAP215 and Dis1 proteins has been observed. In the case of Dis1, cdc2 kinase appears responsible for this phosphorylation. However, the effect of phosphorylation on the interaction of these proteins with microtubules and in vivo function remains to be determined (Cullen, 1999).

Msps protein is abundant in tissues that contain many dividing cells. However, a significant amount of Msps protein was found in nonproliferating tissues, such as the adult head. This is also seen in vertebrates. Both human ch-TOG and Xenopus XMAP215 are highly expressed in adult brains (Charrasse, 1998; Gard, 1987). Since these proteins can regulate microtubule dynamics, it is an attractive possibility that these proteins may also function in post mitotic cells, a question that could in future be addressed in vivo in Drosophila (Cullen, 1999).

The msps mutation affects only a limited aspect of spindle formation. It does not appear to have a strong impact on microtubule nucleation, bipolarity of the spindle, focusing of the poles, or chromosome alignment. Rather the mutant appears defective in holding the mitotic spindle together. Since the Msps protein is localized to centrosomal regions, it is possible that it is involved in the nucleation of microtubules around centrosomes. If centrosomal microtubule nucleation were defective, the effects of chromosomes on stabilizing microtubules would become dominant, resulting in the mini spindles phenotype. Such chromosome driven bipolar spindle formation has been demonstrated in centrosome-free systems. Beads coated with DNA are capable of organizing a bipolar spindle in Xenopus egg extracts and single meiotic chromosomes expelled from the spindle in various mutants can organize a bipolar mini spindle during Drosophila female meiosis. Moreover, mini spindle formation is triggered when chromosomes are detached by micromanipulation from the Drosophila male meiotic spindle which contains centrosomes. However, this model is not consistent with the phenotypes seen in either Drosophila γ-tubulin mutants or abnormal spindle (asp) mutants. The γ-tubulin complex and the Asp protein appear essential for the integrity of microtubule nucleation activity of centrosomes. The Drosophila γ-tubulin mutant shows a variety of defects but no mini spindles phenotype has been reported. Similarly the poles of asp mutant spindles are highly disorganized but the spindles are largely intact and bipolar (Cullen, 1999).

Alternatively, it may be possible that Msps protein is required for anchoring spindle microtubules to centrosomes. It is known that many microtubules are not directly attached to centrosomes, and partial loss of Msps protein may cause a set of spindle microtubules to detach from centrosomes. However, neither this nor the previous model explain the conserved localization of Msps protein along the spindle microtubules during mid-mitosis. Msps protein localizes on the female meiotic spindle at metaphase I. Since the female meiotic spindle at metaphase I lacks centrosomes and its formation is driven by chromosomes, this observation supports the possibility that Msps protein has functions that are independent of centrosomes (Cullen, 1999).

The msps phenotype may be best explained by a failure in the microtubule bundling that holds the mitotic spindle together. Microtubule bundling combined with minus end motors has been proposed as a mechanism to focus the spindle at the polar region. The dynein-dynactin complex in association with NuMA (Nuclear protein that associates with the Mitotic Apparatus), Ncd, and CTK2 are proposed to fulfil such roles. The msps phenotype may suggest that focusing of the polar region requires two steps. In one step, the microtubules that emanate from each chromosome are bundled together, whereas in the second these microtubule bundles are held together. Msps protein may be required mainly for the second step. Although human TOGp and Xenopus XMAP215 have high sequence similarity to Msps protein, no such in vitro activity has been reported for either of the purified proteins. However, it is possible that interaction with other proteins is required for this activity (Cullen, 1999).

The simplest model is that Msps protein is required for formation of long microtubules during mitosis. When cells fail to make long microtubules, the mitotic spindle cannot hold all of its chromosomes and it collapses to form small spindles. This model is supported by the in vitro activity of purified XMAP215 and human TOGp. Purified XMAP215 dramatically increases elongation and shortening velocity and decreases the frequency of the rescue at the plus ends of microtubules while effects on the minus end are much less dramatic. In total, it promotes plus end assembly and turnover, resulting in a population of extremely long but highly dynamic microtubules (Vasquez, 1994). In contrast, it was reported that TOGp increases the elongation rate of both ends equally and appears to inhibit catastrophes. As a result, TOGp promotes microtubule assembly (Charrasse, 1998). Although it is not clear whether these differences reflect experimental approaches, it is evident that both proteins can promote microtubule assembly (Cullen, 1999).

The Drosophila microtubule-associated protein Mini spindles is required for cytoplasmic microtubules in oogenesis

Drosophila mini spindles is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles. msps is also required during oogenesis for the structure and function of cytoplasmic microtubules. Localization of bicoid (bcd) mRNA in the oocyte is a microtubule-mediated event. bcd RNA localization is defective in msps mutants. Defects in cytoplasmic microtubules were identified in both the germ and follicle cells of mutant ovaries, and the expression pattern of msps mRNA and protein in developing egg chambers was determined. The findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions (Moon, 2004; full text of article).

What role might Msps play in bcd mRNA localization? bcd RNA is initially localized in stages 8 and 9 msps^P/msps²⁰⁸ oocytes, but some egg chambers have patchy or dispersed RNA, indicating that localization is less efficient at these stages. Later in oogenesis, bcd RNA localization is completely lost in the msps mutants. This progressive loss of bcd RNA localization mirrors the temporal decline in msps expression that occurs in the mutant egg chambers. A model is favored in which msps is required to regulate the structure of more than one population of cytoplasmic microtubules, including microtubules required in mid-oogenesis for bcd RNA transport and microtubules required later for anchoring bcd RNA at the oocyte anterior. Consistent with this model, defects were obaserved in several populations of microtubules in msps egg chambers. The requirement for msps in the late maintenance of bcd, but not osk mRNA localization, is consistent with studies that show that the maintenance of osk mRNA localization is actin dependent, not microtubule dependent (Moon, 2004).

Biochemical experiments have shown that other XMAP215/TOG family members promote spindle assembly and regulate microtubule dynamics by promoting growth of microtubules at their plus ends. During oogenesis, Msps may affect the dynamics of microtubules that provide the pathway for bcd RNA transport within and between the nurse cells and oocyte; possibly, these microtubules include those observed in this study. The gradient of Msps in the nurse cells is intriguing and may serve to preferentially regulate the plus ends of these microtubules. Alternatively, Msps might affect the minus ends of these and other microtubules required for anchoring bcd RNA in the oocyte. XMAP215/TOG proteins are concentrated at the poles of spindles in dividing cells. It has been proposed that Msps may organize the poles of the female meiotic spindle by capturing microtubules at their minus ends. Msps could act similarly on cytoplasmic microtubules required for bcd RNA localization and stabilize the attachment of their minus ends at the oocyte cortex, where Msps is itself concentrated (Moon, 2004).

Given their localization to the poles and microtubules of spindles in dividing cells, members of the XMAP215 family of proteins might play a role in the association of mRNAs, or even noncoding structural RNAs, with spindles. Recently, mRNA localization to centrosomes has been shown to accompany the asymmetric distribution of patterning mRNAs to daughter cells in Ilyanassa embryos. The factors that mediate this centrosomal attachment are unknown, as is the extent of association of RNAs with spindles in general. MAPs, including the XMAP215/TOG proteins, are candidates for such events and may prove to be valuable tools for identifying spindle-associated RNAs (Moon, 2004).

Mini spindles, the XMAP215 homologue, suppresses pausing of interphase microtubules in Drosophila

Drosophila Mini spindles (Msps) protein belongs to a conserved family of microtubule-associated proteins (MAPs). Intriguingly, this family of MAPs, including Xenopus XMAP215, was reported to have both microtubule stabilising and destabilising activities. While they are shown to regulate various aspects of microtubules, the role in regulating interphase microtubules in animal cells has yet to be established. This study shows that the depletion or mutation of Msps prevents interphase microtubules from extending to the cell periphery and leads to the formation of stable microtubule bundles. The effect is independent of known Msps regulator or effector proteins, kinesin-13/KinI homologues or D-TACC. Real-time analysis revealed that the depletion of Msps results in a dramatic increase of microtubule pausing with little or no growth. This study provides the first direct evidence to support a hypothesis that this family of MAPs acts as an antipausing factor to exhibit both microtubule stabilising and destabilising activities (Brittle, 2005).

The Dis1/TOG proteins are one of the most conserved families of MAPs among eukaryotes. Studies in various systems revealed that this family of MAPs regulates a variety of microtubule functions in the cell (Ohkura, 2001). However, the focus of most studies has been on functions in mitosis, while interphase microtubule regulation has featured less prominently. Crucially, in vivo roles of this family of MAPs in interphase animal cells need to be established. This study revealed that the depletion of Msps results in a dramatic increase in the amount of time spent in a paused state. Strikingly, even the partial depletion of Msps has stronger effects than nearly complete depletion of microtubule end-binding protein EB1, the only protein previously shown to have antipause activity. Previous in vitro experiments revealed activities of the Msps homologues to increase growth and shrinkage rates, and to suppress catastrophe frequency (Gard, 1987; Vasquez, 1994; Tournebize, 2000; Kinoshita, 2001). However, antipause activity has not been reported in other Msps homologues in vivo or in vitro except the budding yeast homologue stu2, the deletion of which modestly increases pausing of cytoplasmic microtubules. It would be interesting to see whether microtubule dynamics are altered by the depletion of TOG in mammalian cells in the light of the current findings (Brittle, 2005).

Dynamic instability is an intrinsic property of microtubules. Microtubules assembled in vitro from purified tubulin either grow or shrink, with infrequent alternation between the two states. In cells, however, microtubule dynamics are strongly influenced by additional proteins, and microtubule plus ends often pause without significant growth or shrinkage. This pausing is more frequent at the cortex, indicating that it is regulated by cellular factors. This regulation is potentially critical for linking microtubule ends to the cell cortex. Despite being a common feature of microtubules in cells, relatively little attention has been paid to the pause state, and consequently only limited information is available on the nature and regulation of this state (Brittle, 2005).

The most enigmatic aspect of this family of MAPs is to have apparently opposing activities, both stabilising and destabilising microtubules (Tournebize, 2000; van Breugel, 2003; Shirasu-Hiza, 2003). In vitro functional analysis of XMAP215 indicates that the protein has the intrinsic ability to promote both the polymerisation and depolymerisation of microtubules (Tournebize, 2000; Shirasu-Hiza, 2003). Shirasu-Hiza (2003) proposed that the two opposing activities of XMAP215 could be reconciled if it functions as an antipause factor. This model is based on the hypothesis that the pause state may be the obligate intermediate between growth and shrinkage. It is also proposed that a pause corresponds to blunt-ended ultrastructures distinct from those of growth (sheet-like) and shrinkage (curled). Destabilising the pause state will promote the transitions from pause to both growth and shrinkage, therefore it can act as a microtubule stabiliser as well as a destabiliser (Brittle, 2005).

The results in Drosophila cells provide the first direct experimental evidence to support the hypothesis proposed by Shirasu-Hiza (2003) that Msps acts as an antipause factor. Consistent with the hypothesis, further analysis shows that Msps promotes transitions from pause to growth and inhibits transitions from growth to pause. In contrast, it was not seen that Msps significantly promoted the transition from pause to shrinkage. However, this may be due to partial depletion of Msps, which allows direct observation of microtubule dynamics. In cells that are well depleted of Msps, microtubules are more resistant to the depolymerising drug colchicine, suggesting that catastrophe or depolymerisation is inhibited (Brittle, 2005).

Msps associates with microtubule plus ends in the cell. Therefore, it could directly destabilise the paused state by altering polymer conformation or it could act indirectly by preventing a pause factor from accessing the polymer. In Xenopus egg extracts both from mitosis and interphase, XMAP215 is shown to stabilise microtubules mainly by antagonising the microtubule depolymerising activity of the kinesin-13/KinI family kinesin, XKCM1 (Tournebize, 2000). In contrast, this study shows that Msps regulation of interphase microtubules in S2 cells is unlikely to be through the kinesin-13 proteins. It was also found that Msps activity and localisation to interphase microtubules are independent of its binding partner D-TACC, suggesting this interphase function is independent of known regulators or effectors. Since knowledge on microtubule pausing is very limited, it is not possible to speculate on the precise mechanism of the antipause activity. However, the current finding represents a significant advance towards the molecular understanding of this third state of microtubule dynamics (Brittle, 2005).

Msps depletion dramatically altered interphase microtubule organisation in Drosophila S2 cells. Microtubules are unable to extend to the periphery of the cell and occasionally form extensive bundles. The importance of Msps for microtubule organisation in developing flies was confirmed by the presence of similar defects in haemocytes from msps mutant larvae. In addition, a very recent report (Moon, 2004) has described defects in microtubule organisation in msps mutant oocytes. Therefore, Msps is likely to be a general regulator of interphase microtubules in Drosophila. Mutations in msps homologues have a dramatic impact on the organisation of interphase microtubules in yeasts, Dictyostelium amoebae and plants (Garcia, 2001; Whittington, 2001; Graf, 2003). In contrast, the depletion of the mammalian homologue, TOG, by RNAi does not alter interphase microtubule organisation (Gergely, 2003; Holmfeldt, 2004), although none of these studies have examined microtubule dynamics (Brittle, 2005).

Extensive pausing can explain the failure of interphase microtubules to extend out to the cell cortex seen in Msps-depleted cells. It is not clear whether the microtubule bundling phenotype is a secondary consequence of extensive pausing or whether Msps somehow acts directly as an antibundling factor. It is also unclear whether microtubule stabilisation caused bundling or vice versa. Interestingly, although microtubule bundling is commonly observed during overexpression of MAPs, Msps is the only protein that induces microtubule bundling when it is depleted (Brittle, 2005).

In conclusion, functional study of Msps demonstrated an essential role in the organisation and dynamics of interphase microtubules in vivo, and uncovered the antipause activity, which provides a crucial insight into this family of MAPs (Brittle, 2005).

Length control of the metaphase spindle by Msps

The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. Using the Drosophila S2 cell line, proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) were either depleted or overexpressed in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, a reduction in spindle size was found after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, a conclusion was reached that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force (Goshima, 2005).

Metaphase spindle length was examined in cells depleted of various proteins by RNAi. One general caveat of RNAi approach is that the targeted proteins are not completely depleted and that the observed phenotypes (especially 'no phenotype') could be still 'hypomorphic' as a result of the residual proteins. Although this is also the case of the current study, significant protein-level reduction was confirmed for most of the genes, and specific spindle/chromosome phenotypes were observed. First, it was found that reduction of Rad21, a protein essential for sister-chromatid cohesion, led to longer spindles, as documented in yeast. After Rad21 RNAi, anti-Cid staining (an inner-kinetochore marker) revealed no paired sister-kinetochore dots, and thin chromosomal masses were scattered, strongly suggesting the precocious separation of sister chromatids. Statistically significant effects also were observed for regulators of MT dynamics. In agreement with previous qualitative descriptions, RNAi of the MT stabilizers EB1, Msps, and Mast caused shortening of metaphase spindle, whereas knockdowns of MT depolymerases (Klp10A and Klp67A) caused expansion. Notably, the outer-kinetochore-enriched regulators of MT plus ends (Klp67A, Mast) affected the length more severely than the global or centrosome-localized MT regulators EB1, Msps, and Klp10A. Klp67A and EB1 RNAi sometimes causes centrosome detachment from the kinetochore microtubules (kMTs), and this detachment could skew the measured centrosome-to-centrosome distance from the actual spindle length. However, the degree of centrosome separation from the kMT for EB1 RNAi is small, only accounting for a 0.2 microm increase to the measurement of spindle length. In the case of Klp67A, the centrosome is detached, but is not always localized along the axis. Indeed, by comparing measurements of centrosome-to-centrosome distance to the distances of the minus ends of the kMT in 32 bipolar spindles, it was found that the centrosome-to-centrosome distance in Klp67A RNAi cells underestimates spindle length by 0.3 μm compared with control cells. Nevertheless, these effects are small and do not affect the conclusions that EB1 and Klp67A RNAi treatments shorten and lengthen spindle length, respectively (Goshima, 2005).

If spindle length is controlled by a balance of MT polymerization and depolymerization, it was reasoned that a phenotype generated by depletion of the MT-depolymerizing protein Klp67A might be rescued by depletion of MT-polymerizing proteins (e.g., Msps or Mast). In accordance with this idea, the simultaneous knockdown of Klp67A and Msps or Mast produced an average length that was intermediate between single Klp67A and single Msps or Mast knockdowns. The important role played by MT-polymerization dynamics in determining spindle length was also discovered recently in Xenopus egg extracts where it was also argued that an unidentified non-MT tensile element, possibly a 'spindle matrix', may constrain spindle length. Although the existence of such an element cannot be excluded, the current results have not uncovered evidence for its existence. For example, shortening of MT length by EB1 or Msps RNAi produced short metaphase spindles without significantly perturbing its shape (Goshima, 2005).

In summary, quantitative analyses indicate that MT dynamics are the major determinants of metaphase spindle length, with MT sliding playing a relatively minor role in determining length. Sister-chromatid cohesion is also critical to restrain the length. The model also provides a possible explanation for the surprising insensitivity of spindle length to sliding forces. This model uses a simple set of force-balance equations combined with a 'coupling assumption' in which increasing motor-driven forces leads to faster rates of depolymerization of MTs. The sliding-depolymerization-coupling model also might explain the difference between the current results showing no change in spindle length with varying Kinesin-5 levels, and those in yeast, where varying the concentration of this motor affects spindle length. Unlike metazoan cells, budding yeast displays no detectable MT depolymerization at spindle poles and thus may lack a means of buffering spindle length in response to pushing effects of microtubule motors. Undoubtedly, aspects of the model will need to be revised, and additional parameters will need to be incorporated (e.g., astral microtubule forces are not considered here). Nevertheless, the model in its present state has already served to predict a 'bistabilty' in spindle morphology in which a threshold amount of Kinesin-5-generated force is needed to prevent spindle collapse. With a combination of RNAi and protein overexpression, this prediction was experimentally verified. The model may make other predictions that could be explored in the future. For example, the model predicts that the degree of central-spindle MT overlap varies in response to Kinesin-5 levels, which could be examined in the future by 3D reconstructions of the spindle by electron or deconvolusion microscopy. As another example, a polymer ratchet mechanism was incorporated to explain sliding-depolymerization coupling. Such a coupling could be achieved by a gradient of the Klp10A MT depolymerase activity at the spindle poles, which would allow stronger sliding forces generated by Klp61F to push MT minus ends farther into the interior region of the pole, where the depolymerase activity might be correspondingly higher. Thus, the current model, although not providing a final answer to how mitotic spindle length is controlled and buffered in response to changing forces, provides a useful framework for additional experimentation and more detailed modeling of this important problem in the future (Goshima, 2005).

Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. Drosophila melanogaster TACC (D-TACC) has been shown to be phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A-dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. It is proposed that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis (Barros, 2005).

The centrosome is the main microtubule (MT) organizing center in animal cells, and it plays an important part in organizing many processes in the cell, including cell polarity, intracellular transport, and cell division. Aurora A protein kinases are centrosomal proteins that are essential for mitosis and have been widely implicated in human cancer. They have several functions in mitosis, and they appear to play a particularly important part in regulating centrosome behavior. They are, for example, required for the dramatic recruitment of pericentriolar material to the centrosome, which occurs as cells enter mitosis. This centrosome 'maturation' is thought to ensure that centrosomes are the dominant sites of MT assembly during mitosis (Barros, 2005 and references therein).

It was recently shown that Aurora A can phosphorylate the transforming acidic coiled coil (TACC) family of centrosomal proteins in vitro. TACC proteins stabilize spindle MTs in flies, humans, worms, and frogs apparently by recruiting the MT-stabilizing protein Minispindles (Msps)/XMAP215/ch-TOG (colonic and hepatic tumor overexpressing gene; hereafter referred to as Msps) to the centrosome. Msps proteins bind directly to MTs and regulate MT dynamics primarily by influencing events at MT plus ends (for review see Cassimeris, 1999; Ohkura, 2001; Kinoshita, 2002). In Xenopus laevis egg extracts (Tournebize, 2000; Kinoshita, 2001), for example, the balance of activity between XMAP215 and the MT-destabilizing protein XKCM1/MCAK (mitotic centromere-associated kinesin) at MT plus ends seems to be the main parameter that determines the overall stability of MTs (Barros, 2005).

These findings present something of a paradox; Msps proteins act mainly on MT plus ends, yet, in vivo, they are most strongly concentrated at centrosomes, where the minus ends of MTs are clustered. To explain this paradox, it has been proposed that TACC proteins recruit Msps to centrosomes to ensure either that Msps is efficiently 'loaded' onto MT plus ends as they grow out from centrosomal nucleation sites or that Msps can stabilize the minus ends of centrosomal MTs after they have been released from their nucleating sites (Lee, 2001). The finding that a GFP-D. melanogaster TACC (D-TACC) fusion protein appears to associate with both the plus and minus ends of MTs in living D. melanogaster embryos is consistent with both possibilities (Barros, 2005).

Ser626 of X. laevis TACC3/maskin has recently been identified as a major site of Aurora A phosphorylation in vitro (Kinoshita, 2005; Pascreau, 2005), and this site is conserved in humans (Ser558) and flies (Ser863). The potential significance of the phosphorylation of this site in D-TACC in regulating MT behavior was investigated in D. melanogaster embryos. The findings suggest that D-TACC-Msps complexes can stabilize MTs in two ways: (1) when not phosphorylated on Ser863, they can stabilize MTs throughout the embryo, presumably through interactions with MT plus ends; (2) when D-TACC is phosphorylated on Ser863, the complexes can stabilize MTs by interactions with MT minus ends. This second mechanism appears to be activated by Aurora A specifically at centrosomes, which perhaps explains why centrosomes are such dominant sites of MT assembly during mitosis (Barros, 2005).

D-TACC phosphorylated on Ser863 (P-D-TACC) is detectable only at centrosomes, whereas nonphosphorylated D-TACC, like most other centrosomal proteins (including γ-tubulin, Aurora A, Msps, CP190, CP60, and centrosomin), has large pools of protein that are present in the cytoplasm of Drosophila embryos. It is concluded that Aurora A stimulates the phosphorylation of D-TACC only at centrosomes and that, once phosphorylated, P-D-TACC is either unable to exchange with the soluble pool of D-TACC or is rapidly dephosphorylated when it leaves the centrosome. Since Ser863 is a conserved site of Aurora A phosphorylation in vitro, it seems likely that Aurora A directly phosphorylates Ser863 in vivo, although the possibility cannot be excluded that Aurora A indirectly stimulates the phosphorylation of Ser863 at centrosomes by activating another kinase. It is unclear why Aurora A stimulates the phosphorylation of D-TACC only at centrosomes, but it is noted that two activators of Aurora A kinase, TPX2 and Ajuba, are themselves concentrated at centrosomes (Barros, 2005).

It has been reported that Aurora A is required to recruit D-TACC to centrosomes. This study found, however, that GFP-S863L still concentrates at centrosomes, although this concentration is somewhat weaker than that seen with GFP-D-TACC, demonstrating that phosphorylation on Ser863 plays some part in recruiting D-TACC to centrosomes but is not absolutely essential. An accompanying paper (Kinoshita, 2005), shows that the X. laevis TACC (X-TACC) protein X-TACC3 is phosphorylated by Aurora A in vitro on three sites that are conserved between frogs and humans, only one of which (Ser863) is conserved in flies. A form of X-TACC3 that was mutated at all three serines localizes to centrosomes very weakly. It is possible, therefore, that there are other, nonconserved, Aurora A phosphorylation sites in D-TACC that have a more important role in recruiting the protein to centrosomes. Importantly, GFP-D-TACC and GFP-S863L interact equally well with Msps in immunoprecipitation experiments, and the localization of Msps to centrosomes appears largely unperturbed in GFP-S863L embryos. Thus, it is concluded that the defects in centrosome/MT behavior that were observe in GFP-S863L embryos are unlikely to arise simply from a failure to recruit D-TACC-Msps complexes to centrosomes (Barros, 2005).

Although GFP-S863L concentrates at centrosomes, it is only partially functional. Whereas spindle MTs are relatively unperturbed in GFP-S863L embryos, astral MTs are dramatically destabilized. In addition, unlike GFP-D-TACC, GFP-S863L appears unable to interact with the minus ends of spindle MTs, suggesting that this interaction requires the Aurora A-dependent phosphorylation of D-TACC. If this were so, one might expect to detect P-D-TACC on the minus ends of spindle MTs. Although this is not usually the case, such a staining can be detected with anti-P-D-TACC antibodies in favorable preparations of fixed embryos. It is suspected, therefore, that P-D-TACC generated at the centrosome can interact with the minus ends of spindle MTs, but this is difficult to visualize in fixed preparations. In addition, it is speculated that P-D-TACC can bind to the minus ends of all centrosomal MTs (not just those in the spindle), but this interaction can be visualized only in the spindle, where large numbers of minus ends are tightly clustered in a region that is slightly separated from the centrosome (Barros, 2005).

Altogether, these observations suggest a model for how Aurora A, D-TACC, and Msps may cooperate to stabilize MTs during mitosis in D. melanogaster embryos. It is proposed that D-TACC-Msps complexes normally stabilize MTs in two ways. First, when D-TACC is not phosphorylated on Ser863, the complexes are present throughout the embryo and can potentially stabilize all MTs through either lateral interactions with MTs or interactions with MT plus ends (see Mechanism 1 of A schematic model of how the D-TACC-Msps complex stabilizes MTs in Drosophila embryos; Barros, 2005). The latter possibility is favored because both D-TACC and Msps appear to concentrate at MT plus ends (Lee, 2001), and Msps family members primarily influence MT dynamics through interactions with plus ends. Since this stabilization is independent of phosphorylation on Ser863, GFP-S863L can fulfill this function, which would explain why the expression of GFP-S863L significantly rescues the viability of d-tacc mutant embryos (from <1% to ~30%). In support of this possibility, it has been shown (Kinoshita, 2005) that nonphosphorylated X-TACC3 can enhance the ability of XMAP215 to stabilize MTs in vitro (Barros, 2005).

The Aurora A-dependent phosphorylation of D-TACC on Ser863 at centrosomes, however, activates a second MT stabilization mechanism that acts exclusively on MTs associated with the centrosome. This mechanism cannot operate in GFP-S863L embryos, and, as a result, astral MTs are dramatically destabilized. The lack of this stabilization mechanism in GFP-S863L embryos, however, appears to have only a limited effect on spindle MTs. It is speculated that this is because there is a chromatin-based acentrosomal pathway of spindle assembly that can compensate for the instability of centrosomal MTs. Such a pathway exists in many cell types and is especially robust in Drosophila. Because centrosomes still nucleate many MTs in GFP-S863L embryos (centrosomal MTs are simply less stable than normal), these centrosomal MTs can interact with the MTs that assembled around the chromatin to form relatively normal spindles. In contrast, astral MTs, which are exclusively nucleated by centrosomes and do not interact with MTs nucleated around the chromosomes, are dramatically destabilized in GFP-S863L embryos. Kinoshita (2005) shows that the Aurora A-dependent phosphorylation of X-TACC3 is also required to stabilize centrosomal (but not spindle) MTs in X. laevis egg extracts, suggesting that this mechanism is conserved at least in frogs and flies (Barros, 2005).

Although it is unclear how the phosphorylation of D-TACC on Ser863 leads to MT stabilization at centrosomes, it is proposes that phosphorylation allows D-TACC to interact with MT minus ends and stabilize them (see Mechanism 2 of A schematic model of how the D-TACC-Msps complex stabilizes MTs in Drosophila embryos; Barros, 2005). This proposal will be controversial, since Msps proteins appear to stabilize MTs mainly through interactions with MT plus ends (Cassimeris, 1999; Ohkura 2001; Kinoshita, 2002). Msps proteins are thought to have such a dramatic effect on MT plus end stability because they specifically counteract the MT destabilizing activity of Kin I kinesins at plus ends. Several Kin I kinesins, however, are also concentrated at centrosomes (for review see Moore, 2004). In D. melanogaster embryos, the Kin I kinesin Klp10A has been reported to destabilize the minus ends of centrosomal MTs (Rogers, 2004). Like D-TACC, Klp10A is concentrated both at centrosomes and on the minus ends of spindle MTs that are clustered close to centrosomes (Rogers, 2004), and this study finds that Klp10A remains clustered at these MT minus ends in GFP-S863L embryos. Perhaps the phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to counteract the destabilizing activity of Klp10A at MT minus ends. If so, then a balance between the activities of Msps/XMAP215 and a Kin I kinesin seems to regulate the stability of MTs at both plus and minus ends (Barros, 2005).

Finally, these findings provide important insight into why centrosomes are the dominant sites of MT assembly during mitosis. As cells enter mitosis, centrioles recruit pericentriolar material in the Aurora A-dependent process of centrosome maturation, which increases the MT nucleating capacity of centrosomes. The results suggest that this increase in nucleating capacity is insufficient on its own to generate large centrosomal arrays of MTs during mitosis; Aurora A must also phosphorylate D-TACC to activate D-TACC-Msps complexes at centrosomes, which can then stabilize these centrosomal MTs. In this new model, Aurora A ensures that centrosomes are the major site of MT assembly during mitosis both by increasing the MT nucleating capacity of centrosomes and by stabilizing centrosomal MTs. Since Aurora A, TACC, and ch-TOG (the human homologue of Msps) have all been implicated in human cancer, it will be interesting to determine whether their common role in stabilizing centrosomal MTs is linked to their roles in oncogenesis (Barros, 2005).

Maelstrom coordinates microtubule organization during Drosophila oogenesis through interaction with components of the MTOC

The establishment of body axes in multicellular organisms requires accurate control of microtubule polarization. Mutations in Drosophila PIWI-interacting RNA (piRNA) pathway genes often disrupt the axes of the oocyte. This results from the activation of the DNA damage checkpoint factor Checkpoint kinase 2 (Chk2) due to transposon derepression. A piRNA pathway gene, maelstrom (mael), is critical for the establishment of oocyte polarity in the developing egg chamber during Drosophila oogenesis. Mael forms complexes with microtubule-organizing center (MTOC) components, including Centrosomin, Mini spindles, and γTubulin. Mael colocalizes with αTubulin and γTubulin to centrosomes in dividing cyst cells and follicle cells. MTOC components mislocalize in mael mutant germarium and egg chambers, leading to centrosome migration defects. During oogenesis, the loss of mael affects oocyte determination and induces egg chamber fusion. Finally, this study shows that the axis specification defects in mael mutants are not suppressed by a mutation in mnk, which encodes a Chk2 homolog. These findings suggest a model in which Mael serves as a platform that nucleates other MTOC components to form a functional MTOC in early oocyte development, which is independent of Chk2 activation and DNA damage signaling (Sato, 2011).

In this study, it was shown that Mael is an MTOC component and that dynamic organization of MTs does not occur in developing mael oocytes, which correlates with mislocalization of other MTOC components. It was also observed that loss of mael affects the number and position of the oocytes in egg chambers and induces fusion of egg chambers. These results indicate that Mael specifically regulates MTOC formation, and thereby plays a key role in coordinating dynamic MT organization during Drosophila oogenesis (Sato, 2011).

Initial polarization of the oocyte during the oocyte specification phase in the germarium requires replacement of the fusome by a polarized MT network, which correlates with the formation of the MTOC. Mael is concentrated in the centrosomal region and is colocalized with αTub and γTub during cyst cell divisions. γTub does not migrate to a developing oocyte in mael germariums, suggesting that Mael is required for the migration of centrioles from the cytoplasm of cysts to pro-oocytes in the germarium. Currently, the detailed mechanism by which Mael functions in MT organization is not clear. The simplest hypothesis is that Mael might serve as a platform that nucleates other MTOC components to form a functional MTOC. A previous report has shown that weaker mutant alleles of γTub affect the number of nurse cells and oocytes within the egg chamber. These γTub mutant defects are very similar to those found in mael mutants in this study. γTub is involved in the nucleation of MTs and is present in the centrosomes and MTOCs in many different systems. It was hypothesized that reduced activity of γTub could activate the oocyte determination program in one of the nurse cells by ectopically presenting MTOC material. The findings that γTub does not accumulate at centrosomes in the mael germarium and is ectopically expressed in the mael egg chamber suggest that Mael regulates localization of γTub at centrosomes through its complex formation and is thereby involved in properly organizing or positioning the MTOC (Sato, 2011).

PIWI proteins function in transposon silencing via association with piRNAs and maintain genome integrity during germline development. Recent studies have suggested that PIWI proteins in sea urchin (Seawi) and Xenopus (Xiwi) can interact with the MTs of the meiotic spindle, while fly ovarioles with mutations in any of several piRNA pathway genes, including spn-E, aub, and armi, have disorganized MTs. This raises the possibility for either a functional role of PIWI proteins in the machinery that impacts on MT organization (in addition to transposon silencing), a role of the MT cytoskeleton in piRNA generation, or both. The current findings further corroborate a link between components of the piRNA pathway and proper MT organization. Although it was found that Mael forms a complex with MTOC components, components of the piRNA pathway in this complex could not be identified. This is in contrast to observations of the mouse Mael homolog, which functions in the piRNA pathway (similar to fly Mael) and interacts with mouse PIWI proteins in the testes (Costa, 2006). Mouse Mael in the testes is almost exclusively cytoplasmic with accumulation at nuage (Soper, 2008). In contrast, fly Mael is located in both the nucleus and the cytoplasm in the ovary and is known to shuttle between them. Thus, one possibility is that in fly ovaries, there may exist nuclear Mael complexes involved in both piRNA generation and transposon silencing, which are distinct from the cytoplasmic complex containing MTOC components that were identified in this study (Sato, 2011).

Female flies with mutations in several genes in the piRNA pathway often lay eggs with axis patterning defects because of MT cytoskeletal changes that result in the mislocalization of bic, grk, and osk mRNAs within the egg chamber. These defects have been linked to the Chk2 DNA damage checkpoint that may be activated by increased retrotransposon transcript levels in mutants defective in piRNA biogenesis. However, because a mutation in mnk does not suppress the mislocalization of Osk and Grk in the mael oocyte, the axis specification defect of mael oocytes does not appear to be triggered by the activation of germline-specific DNA breaks and damage signaling through Chk2. In addition, a mutation in the mei-W68 locus, which encodes the Drosophila Spo11 homolog and induces meiotic double-strand breaks in chromosomes, cannot suppress the axis specification defect of mael oocytes. Therefore, these results suggest that the axis specification defects of mael oocytes are not a secondary consequence of DNA damage signaling. However, it has been shown that in mael mutant ovaries, Vas is post-translationally modified. These results together imply that, acting not only through Chk2, the functions of Mael in MT organization are in parallel with its function in piRNA generation and transposon silencing. There are mutants (including zuc and spn-E) that are piRNA pathway genes; their axis defects cannot be rescued by mnk mutations. Vas also appears modified in these mutants, although the relationship with activated checkpoint-modified Vas is unclear (Sato, 2011).

Given that Mael is a new component of the MTOC in the Drosophila ovary, identification of a domain within Mael that is responsible for binding to other MTOC components could aid in understanding how Mael nucleates and regulates MTOC formation. Because Mael contains an evolutionarily highly conserved domain of unknown function, termed the Mael domain (Zhang, 2008), determination of its crystal structure should prove valuable in elucidating mechanisms of both MTOC formation and piRNA generation processes (Sato, 2011).

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. This paper shows in vitro that XMAP215^msps has a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerates growth and also increases catastrophe frequency. When all three factors are combined, the growth rate is synergistically enhanced, and rescue events are observed most frequently, but frequent catastrophes restrain the lengthening of the MTs. It is propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215^msps polymerase and the EB1-Sentin duo (Li, 2012).

This study reveals the in vitro growth-accelerating and catastrophe-promoting activities of Drosophila EB1 and Sentin toward growing MTs, in the presence or absence of XMAP215^msps, which has been shown to be a potent MT growth promoter. The combination of these two machineries, which likely interact with each other at the MT plus ends, produced MTs with the greatest dynamicity; this observation is consistent with the fact that either of the two machineries is critical for the formation of dynamic MTs in S2 cells. Furthermore, the localization data may reinforce the proposal that Sentin, for which no homologous genes have been identified thus far outside the insect species, might be the functional homologue of the mammalian Slain2 protein that was recently shown to be important for MT growth in cells and was observed to interact with XMAP215 as well as EB1 (van der Vaart, 2011). Thus, this study takes a step forward in understanding the molecular mechanism underlying the regulation of MT dynamics by multiple nontubulin proteins and, moreover, suggests that the mechanism may be widely conserved in animal cells (Li, 2012).

The growth-promoting activity of XMAP215^msps is generally consistent with that of the best-characterized Xenopus orthologue. The approximately twofold increase in MT growth rate detected in this study might be an underestimation, given that pig brain tubulins were used for Drosophila proteins. However, together with the appearance of severe RNAi phenotypes, it is highly probable that XMAP215^msps is the major MT polymerase of Drosophila (Li, 2012).

EB1 and Sentin had two activities, one that overlaps with the activity of kinesin-13 (catastrophe promotion) and the other with that of XMAP215 (growth acceleration). It is believed that these activities would be associated with their in vivo functions because the RNAi of EB1 or Sentin in the S2 cells decreased both growth speed and catastrophe frequency. However, the data do not go against the view that XMAP215 and kinesin-13 are the primary growth- and catastrophe-promoting factors, respectively. In fact, it is proposed that the EB1-Sentin machinery would act as a supporter of the core XMAP215-kinesin-13 machinery by accelerating MT growth as well as MT aging for catastrophe and that S2 cells do need both mechanisms to make MTs fully dynamic. This hierarchical relationship might be conserved in vertebrates because MT phenotypes observed after EB1 depletion are rescued by the overexpression of XMAP215 in Xenopus egg extracts (Li, 2012).

The growth acceleration on addition of both EB1 and Sentin was comparable with that observed after addition of XMAP215^msps. However, the EB1-Sentin complex is not considered to be a processive polymerase like XMAP215 for a few reasons. First, growth-promoting activity is not seen at a low tubulin concentration in the presence of EB1 and Sentin. Second, unlike the case of XMAP215, data could not be obtained to indicate that Sentin stably binds to soluble tubulins. Third, the known residence time of XMAP215 and EB1 on the MT ends is very different. The average end residence time of a single XMAP215 molecule is 3.8 s, which is long enough to processively add 25 tubulin dimers to the end of each MT protofilament. In contrast, the residence time of yeast and mammalian EB1 family proteins is <0.4 s, which is too short to processively add multiple tubulins. A recent structural study showed that EB1 recognizes a structural feature on the MT surface near the plus end and catalyses the transition of tubulin from the recognized state at growing ends to the state tubulin adopts in the lattice distant from the ends, which is assumed to be the basis of the EB1 activity. Sentin bound to EB1 might modulate the plus-end structure for favorable tubulin incorporation, rather than acting as a polymerase. An interesting future experiment would be to determine the structural basis of EB1-recruiting Sentin in this context (Li, 2012).

When XMAP215^msps, EB1, and Sentin were all combined, the most dynamic MTs thus far were observed; growth rate and catastrophe frequency were maximized, and rescue events were occasionally observed. Interestingly, the dramatic increase in the growth rate and rescue frequency might be because of the synergistic effects of EB1-Sentin and XMAP215^msps. On the basis of the experiments using the truncated Sentin fragments, it is tempting to speculate that the physical interaction of Sentin with XMAP215^msps facilitates localization of both proteins at the MT ends and modulates their activities, thereby contributing to these synergistic effects. However, other models, including a model in which the XMAP215^msps and EB1-Sentin machineries work independently, cannot yet be excluded at this stage (Li, 2012).

The key features of MT dynamic instability (growth, shrinkage, catastrophe, and rescue) were obtained by in vitro combination of three plus-end factors. The reconstitution, however, still failed to precisely reproduce what happens in vivo. For example, pausing MTs are frequently observed in cells, particularly in interphase (-40%), but this behavior was rarely seen in any conditions in the in vitro assay. In S2 cells, at least two other factors, kinesin-13 depolymerase and the CLASP rescue factor, are known to significantly affect interphase MT dynamics probably in an EB1-independent manner. Although CLASP is mostly concentrated at the kinetochore during mitosis and perhaps does not significantly affect the astral MT dynamics, kinesin-13 acts on every MT in every stage of the cell cycle. A next step toward understanding the mechanism controlling MT plus-end dynamics would be to include these two factors in the assay and determine whether they can better reproduce the cellular MT behavior that has, for example, frequent pause events (Li, 2012).

TOG proteins are spatially regulated by Rac-GSK3β to control interphase microtubule dynamics

Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and their basic contributions to microtubule dynamics are understood, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. This study have identified a novel protein-protein interaction between the XMAP215 homologue in Drosophila, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. Microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on these findings, it is proposed that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics (Trogden, 2015).

Microtubules interact with the small GTPase Rac in a complex pattern of cross-talk at the leading edge of motile cells. Growing microtubules induce cortical Rac activation by locally activating a guanine exchange factor (GEF) to induce protrusion and directional migration. In what is thought to be a positive feedback loop, active Rac promotes persistent microtubule growth in the lamellae and lamellipodia by locally regulating the activity of microtubule-associated proteins (MAPs). At least three MAPs have been implicated in regulation of microtubule dynamics downstream of Rac. The first is CLIP-170, a microtubule plus end interacting protein (+TIP) that interacts with the Rac effector IQGAP1 to capture microtubule plus ends at the plasma membrane. The second is Stathmin/OP18, a microtubule destabilizing factor that is locally inhibited at the leading edge due to phosphorylation by the Rac effector kinase Pak. The third is CLASP, another +TIP that suppresses dynamics, leading to increased stabilization of microtubules at the leading edge of polarized fibroblasts. CLASP binds directly to microtubules through a central lattice-binding domain and localizes to growing plus ends through an interaction with EB1. In the cell cortex, CLASP is phosphorylated by GSK3β, which blocks its ability to bind to the microtubule lattice, thus targeting it to growing plus ends. At the leading edge, GSK3β is locally inhibited by Rac and dephosphorylated CLASP binds along the microtubule lattice. Although local inhibition of Stathmin and activation of CLASP seem to be necessary for persistent microtubule growth at the leading edge, neither factor is sufficient, suggesting that other regulatory mechanisms remain to be discovered (Trogden, 2015 and references therein).

The present study identified the XMAP215 homolog, Msps, as a downstream effector of the Rac pathway and describes a novel regulatory mechanism for Msps through a protein-protein interaction with the microtubule stabilizer Orbit and the scaffolding protein Sentin. In S2 cells, the Drosophila CLASP homolog Orbit localizes to microtubule plus ends, but binds to the microtubule lattice upon expression of active Rac1 or depletion of GSK3β. These observations are similar to the dynamics of CLASP in mammalian cells and suggest that this mode of regulation is conserved. Activation of Rac or depletion of GSK3β promotes Msps binding to the microtubule lattice and this localization requires Orbit. The data suggests that, like CLASP, Orbit is directly phosphorylated by GSK3β which prevents it from interacting with and recruiting Msps to the microtubule lattice. The Orbit-Msps interaction further requires another +TIP, Sentin. As the localization of Sentin at the microtubule plus ends is not regulated by Rac-GSK3β, it is likely serving a scaffolding function to promote interactions between Msps and Orbit. This study mapped the protein-protein interaction sites on Msps and Orbit to their C-termini and found that mutations that block their interaction severely perturb microtubule dynamics. Both a non-phosophorylatable Orbit mutant and a mutant that prevented the Msps-Orbit interaction lead to more persistent growth, with the non-phosphorylatable Orbit mutant also causing an increase in microtubule pause. This may indicate that this interaction is important for persistent microtubule growth downstream of Rac-GSK3β (Trogden, 2015).

A growing body of evidence indicates that +TIPs exhibit cross-talk with one another to regulate microtubule dynamics in response to upstream regulatory cues. The current results indicate that Msps and Orbit function together during interphase to regulate dynamic instability in response to Rac and GSK3β activity. It is well established that members of the XMAP215 family promote microtubule dynamics by catalyzing microtubule polymerization and depolymerization. These activities are conserved in Drosophila Msps; microtubules in S2 cells lacking Msps are less dynamic, spending most of their lifetime in a pause state. In contrast, Orbit acts to suppress microtubule dynamics and promotes their stability. Thus, Msps and Orbit would be seem to regulate microtubule dynamics antagonistically, a functional relationship supported by recent genetic studies. However, the current results indicate that the two proteins share a more complex interaction (Trogden, 2015).

Msps exhibits two distinct localization patterns on microtubules in S2 cells- at the plus ends of microtubules and along the distal microtubule lattice in the periphery. The data support the model that these different modes of microtubule association represent functionally distinct pools of Msps. First, the Msps-Orbit interaction sites were identified, they were mutated to ablate the interaction, and these mutants were used to rescue cells depleted of either endogenous Msps or Orbit. Expression of either Msps-GFP 3A or 3K3A was able to rescue microtubule dynamics as compared to Msps-depleted cells. However, microtubules in these cells exhibited abnormally high frequencies of rescue and low frequencies of catastrophe, spending more time in growth and less in shrinkage compared to control cells. Cells expressing GFP-Orbit with the GSK3β phospho-acceptor sites mutated to alanine (5S->A) suppressed Orbit RNAi-induced increases in dynamic instability, but microtubules in these cells also exhibited higher frequencies of rescue, lower frequencies of catastrophe, and more time in the pause state compared to control cells. These results indicate that Msps must interact with Orbit in order to properly regulate microtubule dynamics in the cell periphery. Second, when the growth rates of microtubules were examined by tracking EB1-GFP, it was noted that EB1 comets in the cell periphery exhibit slower velocities than those in the cell cortex. These differences likely reflect interactions between growing microtubules and lamellipodial actin undergoing retrograde centripetal flow in the cell periphery. Recent work has also shown that EB1 comets are structurally different in the cortex versus the periphery, so the differences may be explained by changes in the microtubule as well. However, when EB1 comets on microtubules were compared with Msps at the plus end to those that had Msps localized along the distal lattice, it was discovered that the latter exhibited a significantly slower rate of growth. Collectively, the results indicate that the Msps-Orbit interaction 'tunes' microtubule dynamics in response to Rac activation in the cell periphery. It is suggested that Msps could be shunted onto the lattice to act as a localized 'sink' that attenuates its activity as a microtubule polymerase. This inactive pool may serve as a mechanism to partially suppress Msps activity so that microtubules grow at specific rates upon reaching the edge of the cell. The mechanism of how Msps regionally governs microtubule dynamics presents an intriguing problem; future studies employing biochemical reconstitution of microtubule dynamics with recombinant +TIPs and their regulators will likely be required to address these models (Trogden, 2015).

One puzzling aspect of this study is that, despite the biochemical and functional evidence for the Msps-Orbit interaction, colocalization of Msps and Orbit on the microtubule lattice in the cell periphery was not detected under unperturbed conditions. It is speculated that this protein-protein interaction is transient, occurring at the plus end, but is required for some conformational change in Msps that unmasks its microtubule lattice-binding activity. Two lattice-binding sites have been detected in the inter-TOG linker regions that seem to be inactive while the protein is localized to plus ends. This results were also confirmed using in vitro reconstitution assays. It is possible, however, that Orbit does localize to the lattice in the cell periphery, but at levels so low it was not possible to detect in living cells using the available probes. A third possibility is that Orbit is able to alter the structure of the microtubule lattice proximal to the plus end in order to promote lattice binding of Msps. This alteration could represent a change in the local nucleotide state of the polymer as a recent study indicated that mammalian CLASPs are able to promote GTP hydrolysis by polymerized tubulin. Alternatively, it has been shown that EB1 family members promote structural transitions within the microtubule lattice that favor GTP hydrolysis and compaction of the lattice itself. Perhaps similar localized changes in microtubule structure signal to Msps to transition from tip-association to lattice binding. Further work will be required to understand how these proteins interact to regulate their respective functions and it is expected that in vitro reconstitution assays will prove valuable to advance understanding of this protein-protein interaction (Trogden, 2015).

It is interesting to note that the localization patterns for Msps and Orbit observed in Drosophila cells seem to exhibit the converse relationships to those described for XMAP215/CH-TOG and CLASP in mammalian cells. Msps also differs from XMAP215 and ch-TOG through its lack of ability to either bind directly to EB1 or independently recognize growing microtubule ends. It is possible that the interaction with Orbit developed to increase Msps' ability to target the microtubule plus end. This interaction may also be present in mammalian cells, where it may serve to modulate growth rates of microtubules. Although Msps and XMAP215/CH-TOG exhibit high degrees of identity overall, it will be interesting to compare their relative activities in living cells using Msps to replace CH-TOG, and vice-versa, using heterologous systems (Trogden, 2015).

The data point to an outstanding question about how this localized regulation of dynamic instability impact behavior at the level of the cell. Dynamic microtubules exhibit a complex, bidirectional cross-talk with the Rho family of small G proteins. It is suggested that Msps and other XMAP215 family members are critical components of these pathways. In migrating cells, for example, Rac activity promotes processive microtubule growth while microtubule dynamics also promote Rac activation. It is predicted that Msps/XMAP215 family members are likely to participate in this positive feedback look and are, therefore, likely to play crucial roles in cell motility. Microtubules are also essential for directed membrane traffic to the leading edge. Msps-induced microtubule growth may also contribute to this polarized delivery of cargo to the front of motile cells. In order to address these fascinating questions, the Msps-Orbit interaction will have to be addressed in the context of migratory cell lines or, better still, within the developing embryo (Trogden, 2015).

In the current model in the cortex of the cell, Rac activity is low and therefore GSK3β is active, leading to phosphorylation of Orbit on 5 serine residues. Both Orbit and Msps are at the plus end, but cannot interact with each other. Msps is localized to the plus end through its interaction with Sentin. Orbit can bind either Sentin or EB1 to target the plus end. In the periphery of an S2 cell (or the leading edge of a migrating cell), Rac is active, which leads to the local inactivation of GSK3β and dephosphorylation of Orbit. Orbit is still on the plus end, but is now able to interact with Msps, allowing Msps to bind to the lattice. How this interaction allows Msps to bind the lattice with Orbit remaining on the plus end remains to be determined. It is hypothesized that when Msps is at the plus end it is in a closed conformation where the C-terminus covers the Linker4-TOG5 region that can bind the microtubule lattice. When Msps and Orbit bind to one another, this causes Msps to adopt an open conformation, exposing the lattice binding region which allows Msps to diffuse along to lattice (Trogden, 2015).

Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins

Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. This study showed that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients (Lattao, 2021).

Autosomal recessive Chediak-Higashi syndrome (CHS) results from a mutation in the lysosomal trafficking regulator (LYST) or CHS1 gene and leads to partial albinism, neurological abnormalities, and recurrent bacterial infections. CHS cells have giant lysosome-related organelles (LROs), compartments that, in addition to lysosomal proteins, contain cell-type-specific proteins. LROs include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and pigment granules of Drosophila. Whether the giant LROs of CHS form through the excessive fusion of LROs or by inhibition of their fission is unclear (Lattao, 2021).

The compromised immune system in CHS is associated with enlarged LROs in natural-killer (NK) cells. NK cells normally become polarized with centrosomes close to their contact site with antigen-presenting cells, the immunological synapse (IS). Despite the formation of a mature IS in CHS NK cells, centrosomes do not correctly polarize and the enlarged LROs neither converge at the centrosome nor translocate to the synapse. Such findings could reflect defective microtubule (MT) organization by the centrosomes in CHS cells, and while some groups describe CHS centrosomes to nucleate fewer MTs, others report normal MT numbers, lengths, and distributions. Thus, the consequence of mutation in LYST for centrosome and MT function is unclear (Lattao, 2021).

Drosophila's LYST counterpart is encoded by mauve (mv) (CG42863). mv mutants show a characteristic eye color due to larger pigment granules, defective cellular immunity through large phagosomes, and enlarged starvation-induced autophagosomes, indicating several types of LRO are affected. The embryo's LROs are the yolk granules (YGs), which provide nutrition and energy during early development. YGs are produced and stored in the egg chamber when the yolk proteins (YPs) of follicle cells are internalized by clathrin-mediated endocytosis and trafficked through the endocytic pathway of the growing oocyte. YGs are present at the periphery of the egg until the early nuclear division cycles of the syncytial embryo, when they translocate to the interior as nuclei migrate to the embryo's cortex in nuclear division cycles 8 and 9. Nurse cells of the egg chamber also supply eggs with endoplasmic-reticulum-derived lipid droplets (LDs), which store maternally provided proteins and neutral lipids for energy and membrane biosynthesis (Lattao, 2021).

This study reveals Mauve's role in regulating LRO/YGs and MT nucleation from centrosomes through the maternal effect lethal (MEL) phenotypes of two new mutant alleles of mauve, mvrosario (mvros) and mv3. Embryos derived from mutant mv females have enlarged YGs that fuse with LDs, and this can be reverted by reducing Rab5 activity. mv-derived embryos also show compromised MT nucleation leading to defects in the embryo's mitotic cycles and cytoskeletal organization. Moreover, a requirement for Mauve in regulating MTs through the TACC/Msps pathway suggests a role for endosomal trafficking in the recruitment or maintenance of pericentriolar material (PCM) components at centrosomes (Lattao, 2021).

Previous studies of Drosophila mv mutants suggested a role for Mauve in suppressing the homotypic fusion of LROs (Rahman, 2012). This study has extended those observations by showing that Mauve also regulates heterotypic fusion between LROs and LDs and by showing that Mauve interacts with molecules that regulate the behavior of interphase and mitotic MTs. This study also shows that dominant-negative Rab5 not only rescues the LRO enlargement defect in mv-derived embryos but also ameliorates recruitment of Msps and PCM at centrosomes. The participation of LDs in LRO fusion that this study now describes could have been previously overlooked because of the lower numbers of LDs in other tissues compared with those in embryos or through specific differences in the mutant alleles under study (Lattao, 2021).

The finding that high levels of Mauve did not induce the formation of smaller sized vesicles together with live imaging of excessive fusion events of autofluorescent vesicles during oogenesis in mv mutant females are consistent with a role for Mauve as a negative regulator of vesicle fusion. The behavior of LDs and the incorporation of their content into the dramatically enlarged YGs of mv-derived embryos is also consistent with this model (Lattao, 2021).

Several lines of evidence support a role for Drosophila Mauve protein in regulating MT nucleation. First, this study found an enrichment of Mv-mCherry around the spindle and centrosomes during mitosis. Second, Mauve co-purifies with γ-tubulin and Msps. Third, the rosario phenotype of mauve-derived embryos is enhanced by mutations in d-tacc or msps, suggesting co-involvement of Mauve and the D-TACC:Msps complex in establishing and/or maintaining the MT-mediated organization of the syncytium that ensures dividing nuclei are at the cortex and endoreduplicating yolk nuclei in the interior. Fourth, embryos derived from mv mutant mothers have reduced amounts of both Msps and γ-tubulin at centrosomes, in accord with the diminished MT nucleating capacity of these centrosomes. Fifth, in line with the reduced amounts of MT nucleating molecules at centrosomes, the regrowth of de-polymerized MTs from centrosomes is compromised in mv-derived embryos (Lattao, 2021).

Mauve's co-purification with Msps, but not its D-TACC partner protein, is another indicator that Msps can exist independently of D-TACC. Indeed, Msps is present in several separate pools: independent of D-TACC at the centrosome; in complex with the D-TACC: Clathrin complex on the spindle; with the MT minus-end protein Patronin to assemble perinuclear non-centrosomal MTOCs (ncMTOCs); with the Augmin complex at kinetochores; and in complex with endosomal proteins such as Mauve. It is speculated that mutations affecting the constitution of Msps complexes at any one of these sites can affect another (Lattao, 2021).

The finding of defects in mitotic MT nucleation by centrosomes in mv-derived embryos suggests that there might be similar requirements at later developmental stages that may have been overlooked because flies can progress through most of the development without functional centrosomes (Lattao, 2021).

The increased NUF seen in mv-derived embryos is likely to be a secondary consequence of disruption to either or both membrane trafficking and mitosis. NUF was first described for the mutant of the nuf gene encoding an ADP ribosylation factor effector that associates with Rab11. Nuf protein is required to organize recycling endosomes in the coordinated processes of membrane trafficking and actin remodeling and embryos deficient for Rab11 also show a strong NUF phenotype. Together this suggests the possibility that NUF in mv mutants could result from the accumulation of endosomal components in the enlarged YGs, which would diminish numbers of recycling endosomes and their associated Rab11-Nuf complex. NUF can also occur as a Chk2 protein kinase-mediated response to DNA damage (DSBs), activated by DNA lesions at mitotic onset. However, this study found no evidence for DNA damage marked by the accumulation of phosphorylated γ-H2Av at DSBs. Finally, NUF also occurs in response to a wide range of primary or secondary mitotic defects. Indeed, failure of the sequestration of histone H2Av to LDs results in embryos that display mitotic defects, nuclear fallout, and reduced viability (Lattao, 2021).

Dominant-negative Rab5 suppresses enlarged YG formation and the mitotic defects of mv-derived embryos in accord with known roles of Rab5 at the early endosome and growing indications of a requirement for Rab5 in mitosis. Rab5 also mediates transient interactions between LDs and early endosomes that enable the transport of lipids between the two without resulting in their fusion. The possibility that Msps transiently localizes to LROs in wild-type embryos cannot be reuled out because LD-YG associations were observed in wild-type embryos and Msps is a component of LDs. The incorporation of Msps and LD markers into the enlarged YGs in mv-derived embryos is also rescued by a dominant-negative form of Rab5 and reciprocally, levels of Msps at centrosomes are restored. This suggests that mutation in mauve leads to mislocalization of Msps around YGs at the expense of its localization at the centrosome and so its availability for mitosis. Suppression of these mv phenotypes by dominant-negative Rab5 could therefore either reflect a passive restoration of the balance of Msps between YGs and spindle poles once YG fusion is prevented or a more active role of Rab5 in organizing the spindle poles (Lattao, 2021).

These findings add to a small but growing body of evidence for the roles of endocytic membrane trafficking in regulating centrosomal function. There are no reports of a membrane-independent role of Rab5, although other groups have reported examples of trafficking proteins involved in MT nucleation in a membrane-independent manner, such as ALIX, a PCM component in human and fly cells, whose recruitment depends on Cnn/Cep215 and D-Spd2/Cep192. The late endosome marker Rab11 also appears to be a part of a dynein-dependent retrograde transport pathway bringing MT nucleating factors and spindle pole proteins to mitotic spindle poles. It is not clear whether Rab5-associated structures mature to Rab11-associated structures in mitosis as they do in interphase but it seems that the two vesicle types might have overlapping functions at centrosomes in mitosis. It will be of future interest to put these current findings into context with these earlier demonstrations of roles of Rab5- and Rab11-containing endosomes in spindle function (Lattao, 2021).

The dynamic relationship between endosomal trafficking and recruitment of MT nucleating molecules onto centrosomes may all have relevance for the role of LYST at the IS and how this is affected in CHS. Thus, it is conceivable that there may be a convergence of the two functions of the LYST protein in lymphocytes, both in regulating the size of LROs and in facilitating the correct positing of centrosomes and membraneous structures. Further studies will be required to clarify the precise roles of LYST in regulating vesicle trafficking and MT nucleation in this particular cell type (Lattao, 2021).

Although the results strongly indicate Mauve to act as a negative regulator of vesicle fusion, this study did not directly assess the fusion ability of LROs. In part, this was limited by the autofluorescent nature of YGs and LDs that restricted the extent to which fluorescently tagged proteins could be used to visualize membrane components of these bodies in dynamic studies. Future work should aim to complement these findings in cell culture and in cell-free systems to determine whether the involvement of both LROs and LDs is widespread. In a similar vein, it will be important to assess whether the roles of LYST proteins in regulating MT dynamics are conserved as implied by these findings. This would require carrying out studies of MT dynamics in other cell types, particularly in mammalian cells (Lattao, 2021).

Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, this study showed that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. This demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies (Hahn, 2021).

Axons are the enormously long cable-like cellular processes of neurons that wire nervous systems. In humans, axons of ≤15μm diameter can be up to two meters long. They are constantly exposed to mechanical challenges, yet have to survive for up to a century; ~40% of axons are lost towards high age and far more in neurodegenerative diseases (Hahn, 2021).

Their growth and maintenance absolutely require parallel bundles of microtubules (MTs) that run all along axons, providing the highways for life-sustaining transport and driving morphogenetic processes. Consequently, bundle decay through MT loss or disorganisation is a common feature in axon pathologies. Key roles must be played by MT polymerisation, which is not only essential for the de novo formation of MT bundles occurring during axon growth in development, plasticity or regeneration, but also to repair damaged and replace senescent MTs during long-term maintenance. However, the molecular mechanisms regulating MT polymerisation in axons are surprisingly little understood (Hahn, 2021).

MT polymerisation is primarily understood in vitro, where MTs can undergo polymerisation in the presence of nucleation seeds and tubulin heterodimers; the addition of catalytic factors such as CLASPs, stathmins, tau, Eb proteins or XMAP215 can enhance and refine these events. However, it is not known whether mechanisms observed in reconstitution assays are biologically relevant in the context of axons, especially when considering that none of the above-mentioned factors has genetic links to human neurological disorders on OMIM (Online Mendelian Inheritance in Man), except Tau/MAPT which features primarily with dominant mutations relating to functions less likely to represent its intrinsic MT-regulatory roles (Hahn, 2021).

To identify relevant factors regulating axonal MT polymerisation, Drosophila primary neurons were used as one consistent model, which is amenable to combinatorial genetics as a powerful strategy to decipher complex regulatory networks. Previous loss-of-function studies of 9 MT plus-end-associating factors in these Drosophila neurons (CLASP, CLIP190, dynein heavy chain, APC, p150Glued, Eb1, Short stop/Shot, doublecortin, Lis1) have taken axon length as a crude proxy readout for net polymerisation, mostly revealing relatively mild axon length phenotypes, with the exception of Eb1 and Shot, which cause severe axon shortening (Hahn, 2021).

This study has incorporated more candidate factors and additional readouts to take these analyses to the next level. Three factors, Eb1, XMAP215/Msps and Tau, share a unique combination of mutant phenotypes in culture and in vivo, including reduced axonal MT polymerisation in frog and fly neurons. These data reveal that the three factors co-operate. Eb1 and XMAP215/Msps act interdependently at MT plus-ends, whereas Tau acts through a novel mechanism: it outcompetes Eb1-binding along MT lattices, thus preventing the depletion of Eb1 pools at polymerising MT plus-ends. By upholding these Eb1 pools, the functional trio also promotes the bundle conformation of axonal MTs through a guidance mechanism mediated by the spectraplakin Shot. This work uniquely integrates molecular mechanisms into understanding of MT regulation that is biologically relevant for axon growth, maintenance and disease (Hahn, 2021).

Understanding the machinery of MT polymerisation is of utmost importance in axons where MTs form loose bundles that run along the neurite throughout its entire length; these bundles are essential for axonal morphogenesis and life-sustaining cargo transport, and must be maintained in functional state for up to a century in humans. To achieve this, MT polymerisation is required to generate MTs de novo, repair or replace them. The underpinning machinery is expected to be complex, but deciphering the involved mechanisms will pay off by delivering new strategies for tackling developmental and degenerative axon pathologies (Hahn, 2021).

This study has made important advances to this end. Having screened through 13 candidates, it was found the three factors Eb1, Msps and Tau stand out by expressing the same combination of phenotypes, and by displaying functional interaction in both Drosophila and Xenopus neurons. It was found that their functions are not only important to maintain MT polymerisation, but also to align MTs into parallel arrangements, thus contributing in two ways to MT bundle formation and maintenance, both in culture and in vivo. The observed impact on MT organisation is also consistent with roles of XMAP215 during MT guidance in growth cones of frog neurons (Hahn, 2021).

The data reveal that various mechanisms observed in vitro or in non-neuronal cells, apply in the biological context of axons, which was unpredictable for two reasons: Firstly, of the three proteins only the human tau homologue has OMIM-listed links to human axonopathies, and these do not necessarily relate to MT polymerisation. Absence of such disease links might well be due to the fact that these proteins are functionally too important, causing embryonic lethality when dysfunctional. Secondly, axons and non-neuronal cells can display significant mechanistic deviations as shown for CLIP170/190 and for the MT localisation of Msps that is facilitated by Sentin or dTACC in non-neuronal cells, dendrites and in vitro but seemingly not in axons. However, other mechanisms observed in axons matched previous reports: (1) the complementary binding preferences of Eb1 and Tau for GTP-/GDP-tubulin; (2) the mutual enhancement of Eb1 and XMAP215/Msps; (3) the correlation of GTP cap size with comet velocity. Furthermore, it was observed that depletion of α1-tubulin in neurons mutant for αtub84B or stathmin (a promoter of tubulin availability) affects comet numbers but not Eb1 amounts: this is consistent with observations that MT nucleation in vitro is far more sensitive to tubulin levels than polymerisation (Hahn, 2021).

Apart from demonstrating the relevance of various molecular mechanisms in the context of axonal MT regulation, this work provides key insights as to how they integrate into one consistent mechanistic model of biological function (see A mechanistic model consistent with all reported data. The TOG-domain protein XMAP215/Msps is relevant for neuronal morphogenesis in fly and Xenopus, likely through its expected function as a MT polymerase. In contrast, Drosophila and vertebrate Eb proteins are only moderate promoters of MT polymerisation in vitro, but rather act as scaffolds. Conserved binding partners of Eb proteins are the spectraplakins, which can guide extending MT plus-ends along actin networks in axons and non-neuronal cells (Hahn, 2021).

It is proposed therefore that Eb1 is the key mediator of MT guidance into bundles (as supported by data throughout this work), and Msps is the key promoter of MT polymerisation. To execute these functions, both proteins depend on each other: MT plus-end localisation of Msps is reduced upon loss of Eb1 and vice versa. This mutual dependency is unlikely to involve their physical interaction, since MT plus-end localisation of Eb1 is known to occur tens of nanometres behind XMAP215, as seems to be the case also for axonal MTs. Furthermore, the data do not support an obvious role of the Sentin or dTACC adaptors in mediating Eb1-XMAP215 interactions (Hahn, 2021).

Potential indirect mechanisms explaining this co-dependency are provided by the promotion of MT polymerisation through XMAP215/Msps, which maintains a prominent GTP-cap which, in turn, mediates Eb1 binding. Restricted GTP-cap formation as a limiting factor for Eb1 binding would also explain why Eb1 over-expression fails to improve Msps-deficient phenotypes. Vice versa, Eb proteins promote lateral protofilament contacts which could assist in sheet formation at the very plus tip, thus facilitating the binding of XMAP215/Msps (Hahn, 2021).

Tau and Map1b/Futsch are known to bind along MT lattices, to promote MT polymerisation in vitro, and to enhance axon growth in mouse and fly neurons through mechanisms that remain unclear (Hahn, 2021).

In the cellular model used in this study, loss of the Map1b homologue Futsch has no obvious effects, whereas Tau shares all assessed loss-of-function mutant phenotypes with Msps and Eb1, although with weaker expression. Of these phenotypes, reduced MT plus-end localisation of Eb proteins upon loss of Tau function was likewise reported for frog neurons, N1E-115 mouse neuroblastoma cells and primary mouse cortical neurons (Hahn, 2021).

One proposed mechanism involves direct interaction where Tau recruits Eb proteins at MT plus ends, consistent with other reports that Tau can bind Eb1. However, further reports argue against overlap of Tau and Eb1 and rather show that Eb1 and Tau have complementary preferences for GTP- and GDP-tubulin, respectively; this is also consistent with the current data. Furthermore, a recruitment model is put in question by the finding that Eb1 lattice localisation increases rather than decreases upon loss of Tau, as similarly observed for mammalian tau and Eb1 in vitro (Hahn, 2021).

Therefore, a different mechanism based on competitive binding is proposed where Tau's preferred binding to GDP-tubulin along the lattice prevents Eb1 localisation, comparable to Tau's role in preventing other proteins including MAP6 and MAP7 from binding in certain regions of the MT lattice. Given the high density of MTs especially in small-diameter axons, lattice binding could generate a sink large enough to reduce Eb1 levels at MT plus-ends, and the experiments with Eb1::GFP overexpression strongly support this notion. In this way, loss of Tau generates a condition comparable to a mild Eb1 loss-of-function mutant phenotype, thus explaining why Tau shares its repertoire of loss-of-function phenotypes with Msps and Eb1, but with more moderate presentation. This competition mechanism might apply in axons with high MT density, for example explain the reduction in axonal MT numbers upon Tau deficiency in C. elegans. It might be less relevant in larger diameter axons of vertebrates where MT densities are low (Hahn, 2021).

This study has used a standardised Drosophila neuron system amenable to combinatorial genetics to gain understanding of MT regulation at the cellular level in axons. A consistent mechanistic model is proposed that can integrate all the data, mechanisms reported in the literature, and previous mechanistic model explaining Eb1/Shot-mediated MT guidance. This understanding offers new opportunities to investigate the mechanisms behind other important observations (Hahn, 2021).

For example, the presence of an axonal sleeve of cortical actin/spectrin networks was shown to be important to maintain MT polymerisation, likely relevant in certain axonopathies; the underlying mechanisms are now far easier to dissect. As another example, it was found that loss of either Eb1, XMAP215/Msps or Tau all caused a reduction in comet numbers, consistent with reports of nucleation-promoting roles of XMAP215 in non-neuronal contexts or reactivating neuronal stem cells. This might offer opportunities to investigate how axonal MT numbers can be determined through the regulation of local acentrosomal nucleation in reproducible, neuron/axon-specific ways, thus addressing a fundamental aspect of axon morphology (Hahn, 2021).

By gradually assembling molecular mechanisms into regulatory networks that can explain axonal MT regulation at the cellular level, i.e., the level at which diseases become manifest, these studies come closer to explaining axonal pathologies which can then form the basis for the development of remedial strategies (Hahn, 2021).

Golgi-dependent reactivation and regeneration of Drosophila quiescent neural stem cells

The ability of stem cells to switch between quiescent and proliferative states is crucial for maintaining tissue homeostasis and regeneration. In Drosophila, quiescent neural stem cells (qNSCs) extend a primary protrusion, a hallmark of qNSCs. This study found that qNSC protrusions can be regenerated upon injury. This regeneration process relies on the Golgi apparatus that acts as the major acentrosomal microtubule-organizing center in qNSCs. A Golgi-resident GTPase Arf1 and its guanine nucleotide exchange factor Sec71 promote NSC reactivation and regeneration via the regulation of microtubule growth. Arf1 physically associates with its new effector mini spindles (Msps)/XMAP215, a microtubule polymerase. Finally, Arf1 functions upstream of Msps to target the cell adhesion molecule E-cadherin to NSC-neuropil contact sites during NSC reactivation. These findings have established Drosophila qNSCs as a regeneration model and identified Arf1/Sec71-Msps pathway in the regulation of microtubule growth and NSC reactivation (Gujar, 2023).

This work has established Drosophila qNSC protrusion as a regeneration model. It shares two features with other regeneration models, including adult rat retinal ganglion cells (RGCs) and adult C. elegans motor neurons. First, Drosophila qNSCs exhibit age-dependent decline in regeneration capability. This is similar to age-dependent decline in axon regeneration capacity after axotomy in C. elegans mechanosensory neurons and in corticospinal and rubrospinal neurons in the adult mammalian CNS. Second, alterations in microtubule dynamics affect regeneration. The effects of microtubule dynamics on qNSC regeneration are in line with findings in the mammalian CNS neurons that microtubule manipulation promotes axon regeneration after injury (Gujar, 2023).

One interesting aspect of Drosophila qNSC regeneration is that the distal tip of the protrusion does not degenerate after severing but is capable of the regeneration similar to the proximal end. Likely, certain cues or signals from the neuropil to which the protrusion is attached play an important role in the regeneration. It would be worthwhile to understand how the damaged plasma membrane is repaired during qNSC regeneration. Interestingly, re-sealing of the plasma membrane during axon regeneration is dependent on actin and calcium (Gujar, 2023).

This study shows that Golgi functions as a MTOC in qNSCs. The centrosomes may take over Golgi as the MTOC when NSCs re-enter the cell cycle as the proximity between the immature centrosomes and the apical Golgi likely facilitates this transition. Golgi is required for the regeneration of qNSC protrusions upon injury, likely via microtubule growth. Similarly, moderately stable microtubules are required for efficient axon regeneration in central neurons following spinal cord injury. The anterograde transport of cargo, such as mitochondria and vesicles, is also important for their delivery to lesioned axonal tip for axon regeneration. Although it was previously thought that qNSCs retract their protrusions before cell-cycle re-entry, recent work has shown that qNSCs can retain their protrusions throughout the first post-reactivation division (Gujar, 2023).

We also observed that the primary protrusion of larval brain NSCs persists during reactivation and subsequently the first division, although it remains unknown if this applies to most of the reactivating NSCs. Observations also suggest that regeneration of the protrusion precedes NSC reactivation at the event of injury (Gujar, 2023).

This study has identified Arf1 and Sec71 as key regulators of acentrosomal microtubule nucleation, growth, and orientation in the primary protrusion of Drosophila qNSCs. The role of Arf1 in regulating microtubule growth is unexpected, as it is well established that Arf1-6 proteins are critical for membrane trafficking, while Arf-like (Arl) proteins such as Arl2 are important for microtubule functions. Interestingly, mammalian Arf1 recruits tubulin-binding cofactor E (TBCE), which is known to associate with the microtubule regulator Arl2 during microtubule polymerization, to the Golgi, and Arf1 overexpression increases tubulin abundance in motor neurons (Gujar, 2023).

It will be particularly interesting to investigate a similar microtubule regulatory role for Arf1 in other Drosophila cell types as well as in different organisms (Gujar, 2023).

This study reports that in Drosophila qNSC regeneration, the distal tip is capable of the regeneration similar to the proximal end, and focus was placed on the intrinsic role of Arf1 and Msps in regeneration. It remains to be tested whether they could also function in neuropil to facilitate the regeneration of the distal protrusion. EB1-GFP comets going into PIS Golgi and coming out from it in a few seconds were assigned as 'not from Golgi,' which may underestimate the number of Golgi-derived EB1-GFP comets. Furthermore, due to technical limitations, it was not possible to pinpoint the changes in microtubule dynamics or cargo transport in qNSCs after injury. Further studies are warranted to better understand how microtubule growth and dynamics enhance qNSC regeneration (Gujar, 2023).

A pre-anaphase role for a Cks/Suc1 in acentrosomal spindle formation of Drosophila female meiosis

Conventional centrosomes are absent from a female meiotic spindle in many animals. Instead, chromosomes drive spindle assembly, but the molecular mechanism of this acentrosomal spindle formation is not well understood. This study screened female sterile mutations for defects in acentrosomal spindle formation in Drosophila female meiosis. One of them, remnants (rem), disrupted bipolar spindle morphology and chromosome alignment in non-activated oocytes. It was found that rem encodes a conserved subunit of Cdc2 (Cks30A). Since Drosophila oocytes arrest in metaphase I, the defect represents a new Cks function before metaphase-anaphase transition. In addition, it was found that the essential pole components, Msps and D-TACC, are often mislocalized to the equator, which may explain part of the spindle defect. The second cks gene cks85A, in contrast, has an important role in mitosis. In conclusion, this study describes a new pre-anaphase role for a Cks in acentrosomal meiotic spindle formation (Pearson, 2005).

Spindle formation in female meiosis is unique in terms of the absence of conventional centrosomes. Instead, chromosomes have a central role in the assembly of spindle microtubules. This acentrosomal (also called acentriolar or anastral) spindle formation is common in female meiosis for many animals including mammals, insects and worms. Despite potential medical implications, this spindle formation is much less studied than centrosome-mediated spindle formation in mitosis (Pearson, 2005).

Drosophila provides a valuable tool to study the acentrosomal spindle formation in vivo. Unlike many other species, mature non-activated Drosophila oocytes arrest in metaphase of meiosis I until ovulation, which coincides with fertilization. This provides a unique opportunity to study spindle formation, without interference from chromosome segregation or meiotic exit (Pearson, 2005).

Two components of acentrosomal spindle poles, Msps and D-TACC, physically interact and are crucial for spindle bipolarity. Other studies have identified essential components for spindle formation, such as kinesin-like proteins (Ncd and Sub, γ-tubulin, and a membrane protein surrounding the spindle (Axs). Some of these spindle components are probably modulated by cell-cycle regulators, but knowledge of the regulation is limited. To identify essential components and regulators, a cytological screen was performed for mutants defective in acentrosomal spindle formation of non-activated oocytes (Pearson, 2005).

Through the screen, remnants was identified and identified as a mutant of a Drosophila Cks/Suc1 homologue, Cks30A. Cks is the third subunit of the Cdc2 (Cdk1)-cyclin B complex, but the role of Cks is less clearcut than that of other subunits of the complex. It is implicated in entry into mitosis/meiosis, metaphase-anaphase transition, exit from mitosis/meiosis and inactivation of Cdk inhibitors. This study shows that Cks30A is required for spindle morphogenesis and chromosome alignment in the metaphase I spindle in arrested mature oocytes. This requirement of a Cks before metaphase-anaphase transition represents a new function that has not previously been identified. Furthermore, it was found that essential spindle pole components Msps and D-TACC mislocalize in the mutant, which may be partly responsible for the spindle defects (Pearson, 2005).

For molecular analysis of the acentrosomal spindle in Drosophila female meiosis, female sterile mutants were screened for spindle defects in non-activated oocytes. Female sterile mutants on the second chromosome have previously been isolated. This study focused on classes of mutants that lay eggs that do not develop beyond the blastoderm stage. This category of mutants includes known meiotic mutants affecting spindle formation, such as fs(2)TW1 (γ-tubulin 37C) and subito (a kinesin-like protein (Pearson, 2005).

The identity of the remnants (rem) gene was identified by positional cloning. The rem gene was previously mapped to 30A-C using a deficiency [Df(2L)30AC]. One missense mutation was identified in the gene CG3738 (cks, hereafter called cks30A; Finley, 1994). There were no other mutations within coding sequences and splicing junctions in the region. In addition, the amount and size of the transcripts that are known to be expressed in adult females was tested, and no differences were found between rem and wild type (Pearson, 2005).

Cks30A is one of two Drosophila homologues of Saccharomyces cerevisiae Cks1/Schizosaccharomyces pombe Suc1, a conserved subunit of the Cdc2 (Cdk1)/cyclin B complex, and has been shown to interact with Cdc2 (Finley, 1994). The mutation in rem¹ results in a conversion of the 61st amino acid from proline to leucine. This proline is completely conserved among all Cks homologues, further confirming that the mutation is not a polymorphism. Crystal structure analysis has indicated that this residue forms part of the interaction surface with Cdc2 (Bourne, 1996). Immunoblots using an anti-human Cks1 antibody indicated that this mutation disrupts the stability of the Cks30A protein (Pearson, 2005).

To explain the role of Cks30A, focus was placed on the rem¹ mutant in non-activated oocytes, which arrest in metaphase I. Non-activated oocytes were dissected from wild type and the rem¹ mutant, and chromosomes and spindles were visualized by immunostaining (Pearson, 2005).

In wild type, non-activated mature oocytes contain a single bipolar spindle around chromosomes. Bivalent chromosomes align symmetrically with chiasmatic chromosomes at the equator and achiasmatic chromosomes that are located nearer the poles. The rem¹ mutant was able to enter meiosis, condense chromosomes and assemble microtubules around chromosomes. However, only a minority of spindles showed normal spindle morphology and chromosome alignment (Pearson, 2005).

The most prominent defect in the rem¹ mutant was chromosome misalignment. This defect was observed in about a half of the spindles. Even in the cases in which the spindle remained well organized, chiasmatic chromosomes often moved away from the equator and lost overall symmetrical distribution. The second class of defect in the rem¹ mutant was abnormal spindle morphology. Although the abnormality varied from spindle to spindle in the rem¹ mutant, the most typical defect was the formation of ectopic poles near the spindle equator. The focusing of spindle poles seemed to be unaffected (Pearson, 2005).

Further quantitative analysis showed no significant difference between the phenotypes of rem¹ homozygotes (rem¹/rem¹) and hemizygotes (rem¹/Df). This indicates that the rem¹ mutation is genetically amorphic. A recent independent study has indicated that another weaker allele rem^HG24 shows similar abnormalities at a lower frequency. These results indicate that Cks30A is required before the metaphase-anaphase transition for spindle morphology and chromosome alignment (Pearson, 2005).

To gain an insight into the spindle defects in female meiosis, the localization of Msps was examined. Msps protein belongs to a conserved family of microtubule regulators, including XMAP215, and is the first protein identified at the acentrosomal poles in Drosophila. An msps mutation often leads to the formation of a tripolar spindle in female meiosis I (Pearson, 2005).

In wild type, Msps protein is accumulated at the acentrosomal poles of the metaphase I spindle in female meiosis, although the localization sometimes spreads to the spindle microtubules. In the rem¹ mutant, although the Msps protein is still concentrated at the poles, it is often accumulated around the equator of the spindle. Mislocalization of this important pole protein to the equator in the rem¹ mutant may sometimes lead to the formation of ectopic spindle poles near the equator (Pearson, 2005).

Msps localization is dependent on another pole protein D-TACC, which binds to Msps. To test whether D-TACC also mislocalizes, the localization of D-TACC was examined in the rem¹ mutant. In wild type, D-TACC is highly concentrated at the acentrosomal pole. In the rem¹ mutant, D-TACC often accumulates at the spindle equator, although it is still concentrated around the poles to some degree. In summary, Cks30A is required for correct localization of the essential pole proteins, Msps and D-TACC (Pearson, 2005).

To gain an insight into how the defect in the Cdc2 complex leads to Msps or D-TACC mislocalization to the spindle equator, the localization of cyclin B was examined. Cyclin B is considered to be the main determinant of the activity and cellular localization of the Cdc2 complex. Immunostaining in non-activated oocytes showed that cyclin B is localized to the metaphase I spindle, with a concentration around the spindle equator. This cyclin B localization could suggest a possible regulatory role of the Cdc2 complex in the transport of Msps and D-TACC from the spindle equator to the poles. The cyclin B localization is not affected in the rem mutant, suggesting that Cks30A mainly affects the substrate specificity of the Cdc2 complex, as shown in other systems (Pearson, 2005).

The Drosophila genome contains one more predicted cks homologue (CG9790), which is called cks85A. Although mammalian genomes also have two Cks genes, they are more similar in sequence to each other than to either of the two cks genes in Drosophila (Pearson, 2005).

The gene expression pattern of the two cks genes was examined during Drosophila development. RNAs were isolated from various stages of development and analysed by reverse transcription-PCR (RT-PCR) using primers that correspond to each of the cks genes. cks30A gave strong signals in adult females and embryos, whereas it gave only weak signals in adult males, larvae and pupae. This maternal expression pattern is consistent with the observed female sterile phenotype of the cks30A (rem¹) mutant. In contrast, cks85A signals were obtained more uniformly throughout the development without sex specificity in adults. In S2 cultured cells, which originated from embryos, both genes were well expressed (Pearson, 2005).

To identify the Cks proteins, an anti-human Cks1 antibody was used for immunoblots of protein extracts from embryos and S2 cells. Although the antibody recognized many proteins, two bands were detected within a range of molecular weights consistent with the Cks proteins. In embryos laid by the rem¹ mutant, the amount of the smaller band was greatly reduced. To further confirm their identity, S2 cells were subjected to RNA interference (RNAi) using doublestranded RNAs (dsRNAs) corresponding to the cks genes. It was found that both of the bands disappeared when both genes were simultaneously knocked down by RNAi. It indicated that, consistent with RT-PCR results, S2 cells produced both the Cks proteins and that RNAi effectively depletes them (Pearson, 2005).

Cytological analysis showed that cks85A RNAi results in a significant increase in chromosome misalignment/missegregation and spindle abnormality in mitosis after an extended time, whereas cks30A RNAi has a lesser impact on mitotic progression. About a half of anaphase or telophase cells had lagging chromosomes or chromosome bridges after cks85A RNAi. In some cases, spindles contained scattered chromosomes the sister chromatids of which were either attached or detached. The frequency of multipolar spindles was also increased. The genetic and RNAi results indicated that cks85A has an important function in mitotic progression, whereas cks30A mainly functions in female meiosis (Pearson, 2005).

This study has shown a new pre-anaphase function of a Cks protein in acentrosomal spindle formation during Drosophila female meiosis. Through a cytological screen, spindle defects in remnants among female sterile mutants. Cytological analysis showed that Cks30A is required for correct formation of the acentrosomal spindle and chromosome alignment in female meiosis I. The observation on mislocalization of the essential pole components, Msps and D-TACC, in the mutant provides a molecular insight into a role of Cks30A in spindle morphogenesis (Pearson, 2005).

Cks/Suc1 protein is the third subunit of the Cdc2-cyclin B complex, which is conserved across eukaryotes. Although it has been known to be essential for the cell cycle, the function seems to be less straightforward than that of the other subunits of the Cdc2 complex. One reason is that Cks also interacts with other Cdks and has Cdk-independent functions. Even if Cks is limited to roles in mitosis/meiosis, Cks proteins are implicated in entry into mitosis/meiosis, metaphase-anaphase transition and also exit from mitosis/meiosis. Furthermore, the roles of Cks were further complicated by the fact that animal genomes encode two Cks homologues (Pearson, 2005).

Studies in Caenorhabditis elegans and mice showed that one of two cks genes is required for female fertility (Polinko, 2000; Spruck, 2003). Similarly, the results indicated that one of two Drosophila cks homologues, cks30A, is expressed maternally and is required for female meiosis. Further analysis indicated that Cks30A is required for proper bipolar spindle formation and chromosome alignment in mature oocytes arrested in metaphase I. In C. elegans, depletion of one of the Cks proteins by RNAi results in a failure to complete meiosis I. Similarly, in mice, oocytes from a Cks2 knockout cannot progress past metaphase I and a small percentage of oocytes show chromosome congression failure. In both cases, the defects were interpreted mainly as post-metaphase defects. Since Drosophila non-activated oocytes are arrested in metaphase I until ovulation, pre-anaphase function of Cks30A can be distinguised from possible post-metaphase function. This study clearly showed that Drosophila Cks30A has a function in establishing metaphase I, in addition to later functions that have reported recently (Pearson, 2005).

At the moment, it is not known how the cks30A mutation disrupts spindle formation and chromosome alignment in female meiosis. It has been thought that a loss of Cks function affects the Cdc2 activity towards certain substrates. It was found that the essential pole components, Msps and D-TACC, mislocalize to the spindle equator in the mutant. Previously, it was hypothesized that Msps is transported by the Ncd motor and anchored to the poles by D-TACC. D-TACC localizes to the poles independently from Ncd, but may also be transported from the spindle equator along microtubules by other motors. Cks30A-dependent Cdc2 activity may be required for activating the transport system at the onset of spindle formation in female meiosis. Consistently, it was found that cyclin B is concentrated around the equator of the metaphase I spindle. Msps is the XMAP215 homologue and belongs to a family of conserved microtubule-associated proteins. It is a major microtubule regulator, both in mitosis/meiosis and interphase. The mislocalization of this microtubule-regulating activity could lead to the disruption of spindle organization in the mutant (Pearson, 2005).

Proper recruitment of γ-Tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires centrosomin motif 1

Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microtubule spindle apparatus at mitosis. Although the individual binding steps in centrosome maturation are largely unknown, Centrosomin (Cnn) is an essential mitotic centrosome component required for assembly of all other known pericentriolar matrix (PCM) proteins to achieve microtubule-organizing activity at mitosis in Drosophila. A conserved motif (Motif 1) has been identified near the amino terminus of Cnn that is essential for its function in vivo. Motif 1 has a higher degree of sequence conservation (40% identity/49% similarity) between Cnn and human CDK5RAP2 and is present in all homologues from S. pombe to human. Cnn Motif 1 is necessary for proper recruitment of γ-tubulin, D-TACC (the homolog of vertebrate transforming acidic coiled-coil proteins [TACC]), and Minispindles (Msps) to embryonic centrosomes but is not required for assembly of other centrosome components including Aurora A kinase and CP60. Centrosome separation and centrosomal satellite formation are severely disrupted in Cnn Motif 1 mutant embryos. However, actin organization into pseudocleavage furrows, though aberrant, remains partially intact. These data show that Motif 1 is necessary for some but not all of the activities conferred on centrosome function by intact Cnn (Zhang, 2007).

Previous studies showed that Cnn is required for centrosome assembly/maturation, for microtubule assembly from the centrosome at mitosis, and to organize actin into pseudocleavage furrows in the early embryo. It is shown in this study that Motif 1 of Cnn is required for specific and essential aspects of centrosome function. Centrosomes assembled in cnn^β1 embryos recruit some PCM components and are partially proficient to organize actin into pseudocleavage furrows, but do not properly recruit or maintain proteins with an established role in microtubule assembly: γ-tubulin, D-TACC, and Msps. Thus, although astral microtubules are produced at cnn^β1 mutant centrosomes, centrosome separation, a microtubule-dependent process, is severely affected. In addition, the less-understood process of satellite formation is inhibited at cnn^β1 centrosomes (Zhang, 2007).

Microtubule assembly at centrosomes is regulated by nucleation, where γ-Tub plays a key role, and by microtubule growth, which depends on a host of factors including Aurora A, D-TACC, and Msps, that promote stability. How these proteins are assembled and regulated is still largely unknown. This study shows that Cnn Motif 1 controls assembly of PCM proteins that are required for MTOC activity at centrosomes (Zhang, 2007).

γ-Tub is an essential component of MTOCs in eukaryotes for microtubule assembly. In cnn null mutant neuroblasts, imaginal disk cells, and cells depleted of Cnn by RNAi, neither γ-Tub nor astral microtubules are detected at centrosomes. However, in contrast to the above cell types, a Cnn-independent pool of γ-Tub is at the centrosome remnant in cnn null mutant early embryonic spindle poles. The small, sharp signal for γ-Tub at cnn null spindle poles implicates a centriolar pool of γ-Tub that is unique to the rapid divisions of early embryos. The level of γ-Tub at cnn^β1 mutant centrosomes is similar to the cnn null mutant, indicating that Motif 1 is required for recruitment of the Cnn-dependent pool of γ-Tub to the PCM in embryos. Drosophila Cnn and the S. pombe homolog Mto1p have been reported to coIP with γ-Tub, but a direct interaction with γ-Tub or any of the γ-TuRC proteins has not been demonstrated (Zhang, 2007).

D-TACC and Msps, and their counterparts in Xenopus (TACC3/maskin and XMAP215) and C. elegans (TAC-1 and ZYG-9) are direct binding partners required for centrosome-dependent growth of long microtubules (Gergely, 2000; Bellanger, 2003; Le Bot, 2003; Srayko, 2003; Kinoshita, 2005; Peset, 2005). Mutation or depletion of D-TACC or its homologues does not affect γ-Tub localization to centrosomes, but rather appears to function with Msps in the stability of microtubules that are nucleated by γ-Tub. D-TACC and Msps are partially recruited to centrosomes in cnn null and cnn^β1 mutants, accumulating at the centrosome periphery in cnn^β1 embryos. This incomplete assembly suggests that recruitment of D-TACC and Msps to centrosomes normally involves at least two steps and that Motif 1 of Cnn is required for a secondary step in the process subsequent to docking of D-TACC at the periphery of the centrosome. Thus, Cnn Motif 1 may be required for a later phase of recruitment to the centrosome or have a role in maintaining D-TACC and Msps once they are recruited (Zhang, 2007).

Aurora A kinase is required to localize D-TACC to centrosomes and directly phosphorylates D-TACC at Ser863 to activate its microtubule-stabilizing activity. The reduced recruitment of Aurora A to cnn null centrosomes further highlights the requirement for Cnn in PCM assembly. However, Aurora A localization did not appear affected in cnn^β1 embryos, indicating that, although Aurora A is necessary to recruit D-TACC/Msps, its localization at centrosomes is not sufficient to accomplish this. Aurora A binds directly to the C-terminal half of Cnn, which remains intact in the cnn^β1 mutant. Moreover, D-TACC is phosphorylated by Aurora A in cnn^β1 embryos; however, this activated pool of D-TACC is exiled to the centrosome periphery with the bulk pool of centrosomal D-TACC. This indicates that Motif 1 of Cnn is required for anchoring or maintaining D-TACC at centrosomes subsequent to its regulatory phosphorylation by Aurora A. Alternatively, because the immunofluorescence signal for P-D-TACC was weak and P-D-TACC levels were not quantified, an effect of cnn^β1 on Aurora A activity toward D-TACC cannot be excluded (Zhang, 2007).

In cnn^β1 and cnn null embryos microtubule asters are present, particularly at early cortical cycles (cycles 10 and 11). At later cycles asters are not detected at spindle poles in cnn null embryos, coinciding with centriole loss, which is evident from the absence of Nek2 kinase (a centriolar protein) signal. Centriole displacement from the spindle poles in cnn null embryos leads to centriole loss, resulting in anastral spindle poles (Lucas and Raff, personal communication to Zhang, 2007). By comparison to cnn null embryos, PCM integrity is restored to cnn^β1 mutant centrosomes, enough to retain centrosomes at spindle poles into later cleavage cycles and with retained ability to assemble astral microtubules. Nevertheless, centrosome separation failure indicates that microtubule-dependent processes are impaired at cnn^β1 centrosomes (Zhang, 2007).

Centrosome separation is a microtubule-dependent process that is coordinated by pushing forces from interpolar microtubules and forces supplied by molecular motors that include kinesin-5, kinesin-14 (Ncd), and dynein/Lis1/dynactin. The relative contributions of motor proteins and the pushing forces generated from the assembly of interpolar centrosomal microtubules have not been determined (Zhang, 2007).

A necessary role for microtubules in centrosome separation has been demonstrated using microtubule-depolymerizing drugs in cell culture and in early Drosophila embryos. Interpolar centrosomal microtubules may represent a specialized class of microtubules, an idea supported by the recent discovery of an α-tubulin variant, α4-tubulin, which is associated with faster-growing microtubules and is enriched in interpolar microtubules. α4-tubulin is required for centrosome separation in early embryos (Venkei, 2006). Cnn localized more strongly to interpolar fibers compared with spindle microtubules, suggesting that Cnn Motif 1 may regulate the organization of interpolar centrosomal microtubules to promote centrosome separation. In instances when cnn^β1 centrosomes separated, interpolar fibers formed, suggesting that interpolar fibers are obligatory to centrosome separation. Although the proposal that Motif 1 regulates microtubule assembly to achieve centrosome separation is favored, a role for Motif 1 in regulating molecular motors that are involved in this process cannot be ruled out. However, localization of the kinesin-5/Eg5 family member Klp61F to spindle poles and spindle microtubules was no different in cnn^WT and cnn^β1 embryos (Zhang, 2007).

Consistent with a role for γ-Tub and D-TACC recruitment to centrosomes by Cnn Motif 1 in centrosome separation, depletion or mutation of γ-Tub, γ-TuSC proteins, and D-TACC also perturbed centrosome separation. Thus, γ-Tub at reduced levels and also astral microtubules cannot be detected, embryonic cnn^β1 centrosomes have insufficient or inappropriate microtubule assembly activity to achieve centrosome separation (Zhang, 2007).

It has been shown by live imaging of GFP-Cnn embryos that centrosomal satellites are highly dynamic structures that traffic in a microtubule-dependent and an actin-independent manner (Megraw, 2002). Satellites, or 'flares,' emerge from the PCM and move bidirectionally at speeds of 4-20 µm min^-1 and are produced at highest numbers at telophase/interphase, coincident with the relative intensity of astral microtubules during the cleavage cycle. cnn^β1 mutant embryos produce significantly fewer satellites. Even incipient satellites, which are apparent on cnn^WT centrosomes and are present at colchicine-treated centrosomes, were nearly absent at cnn^β1 centrosomes. Satellite assembly may be an intrinsic function for Motif 1. Alternatively, fewer satellites may arise as a secondary consequence of altered MTOC activity at cnn^β1 centrosomes. Currently, it is not possible to distinguish between these two possibilities (Zhang, 2007).

The organization of actin into pseudocleavage furrows, an activity conveyed by centrosomes, is highly aberrant yet partially restored in cnn^β1 mutant embryos. This is in sharp contrast to cnn null embryos, where no apparent organization of cortical actin occurs. Although some studies have indicated that microtubules are required for cortical actin organization in the early Drosophila embryo, other evidence suggests that centrosomes organize actin and cortical polarity independent of microtubules. Because microtubule-dependent processes are disrupted in cnn^β1 embryos, the data support the model that centrosomes can organize actin independent of microtubules, but the possibility that cnn^β1 centrosomes produce sufficient astral microtubules to coordinate with actin in the assembly of furrows cannot be excluded (Zhang, 2007).

In summary, Motif 1, conserved among Cnn family members, is required for centrosome function in early embryos through the recruitment and anchoring of γ-Tub, D-TACC, and Msps, key factors in MTOC function in all eukaryotes where they have been examined. PCM architecture is partially restored in the cnn^β1 mutant compared with the cnn null, as shown by the normal distribution of CP60 and Aurora A. In addition, conspicuous yet aberrant pseudocleavage furrows assemble in cnn^β1 embryos but not in the cnn null, evidence that organization of actin by centrosomes is partially restored to cnn^β1 mutant centrosomes. This suggests that the activity to direct actin organization into cleavage furrows resides in another domain of Cnn. Identification of the direct binding partner for Cnn Motif 1 will be an important step toward understanding the relationship between Motif 1 and the MTOC functions that it governs (Zhang, 2007).

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

Microtubule plus end binding proteins (+TIPS) localize to the dynamic plus ends of microtubules where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1 and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. This study reports crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically-induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. It is proposed that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end (Slep, 2007).

This work analyzes the biochemical mechanisms of three plus end tracking protein families (EB1, CLIP-170 and XMAP215) using x-ray crystallography, in vitro tubulin binding/assembly assays, and in vivo plus end tracking assays. Five crystal structures are presented of +TIP tubulin binding domains across multiple species and families illustrating structural conservation within families and functional convergence across families. In vitro and in vivo analysis reveal an underlying general theme to these unrelated domains. While tubulin binding can be conferred by a single +TIP domain, the promotion of microtubule nucleation and polymerization in vitro and plus end tracking in vivo require multiple domains acting in concert. In the case of EB1 and CLIP-170 members, two domains are required for robust plus end localization and these two structurally unrelated domains can even cooperate if artificially joined together to achieve this activity. XMAP215 members are more complicated, perhaps due to the relative weaker association between TOG domains and tubulin in higher eukaryotic members. However, multimerization of TOG domains clearly facilitates tubulin nucleation in vitro and overall microtubule association in living cells. From these data, a model is presented in which unrelated tubulin binding domains have convergently evolved a similar mechanism for enhancing tubulin assembly on growing microtubule ends by acting as multivalent tubulin polymerization chaperones (Slep, 2007).

The tubulin binding domains of these three +TIP proteins are quite diverse in their architecture and show no evidence of a common evolutionary origin. However, the structural conservation within a +TIP class is very high, as evidenced by the near superimposition of yeast Bim1p with human EB1 and yeast Stu2p with Drosophila Mini spindles. The different architectures also suggest varying interactions modes with tubulin and potential synergy between the +TIPs. XMAP215 has a very flat binding interface created by a series of rigid, highly conserved loops on one face of the TOG domain; the length of the TOG domain is fairly closely matched to a tubulin monomer (α or β) and likely makes extensive contacts through a combination of hydrophobic and electrostatic interactions. In contrast to the elongated TOG domain, the CH domain of EB1 is spherical and approximately half the size. Mutagenesis suggests that several residues around one hemisphere contribute to tubulin binding. This data is consistent with recent work suggesting that EB1 might nestle in the groove between tubulin protofilaments (Sandblad, 2006). CLIP-170 also has a globular structure and the data suggests that it might bind tubulin’s disordered C-terminal tail as part of its binding interface (Slep, 2007).

Comparison of the +TIPS and protein engineering studies suggest a considerable structural variation in how +TIP proteins can be interconnected to achieve their activities in plus end tracking and promoting tubulin assembly. In some cases, tubulin binding domains are arrayed in tandem along a polypeptide chain with presumably unstructured linkers in between (e.g., Mini spindles, Ch-TOG, CLIP-170). The cis linking domains within a polypeptide appears to be critical, since it is shown that TOG domains added in trans do not reconstitute function. In other cases, single α/β tubulin binding domains are found in a polypeptide and multivalent tubulin binding is achieved by polypeptide dimerization [e.g., EB1 family and Bik1 (the CLIP-170 homologue from yeast)]. Hybrid strategies are also employed (e.g. CLIP-170, which has tandem Cap-Gly domains and is dimerized via a coiled coil and Stu2p which has tandem TOG domains, a dimerization domain and an additional C-terminal microtubule binding domain). It was also found that native dimerization sequences are not essential, as a variety of artificial dimerization strategies (e.g. GCN4 leucine zippers, gluthione S-transferase, and FKBP-rapamycin-FRB) are capable of reconstituting +TIP protein function (Slep, 2007).

Collectively, these studies reveal structural variation in how multiple +TIP tubulin binding domains can be combined to promote tubulin oligomerization for microtubule assembly. However, cis versus trans arrangements of +TIP domains may produce certain unique outcomes. For example, the linear arrays of TOG domains in the extended XMAP215 structure (Cassimeris, 2001) may generate a pseudo protofilament-like arrangement that is particularly effective for microtubule nucleation. Indeed, XMAP215 has a potent microtubule nucleation ability compared with CLIP-170, potentially related to an in vivo nucleation activity given XMAP215's TACC-dependent localization to the centrosome (Lee, 2001). Analogous mechanisms exist for the actin cytoskeleton where the Spire protein utilizes several arrayed actin binding domains to template the nucleation of an actin filament (Slep, 2007).

The data show that single +TIP tubulin binding domains do not promote microtubule nucleation or growth in vitro (and in some cases are inhibitory), nor do they localize to microtubule plus ends in vivo. In contrast, multimerized +TIP tubulin binding domains are potent microtubule nucleator in vitro, promote microtubule growth in vitro and are requisite in vivo for microtubule plus end localization. For EB1 and CLIP-170, it was dynamically shown that plus end tracking requires an ability to bind more than one tubulin subunit. These results suggest a model in which +TIPs bind multiple tubulin dimers in solution and then deliver these larger tubulin oligomers to the ends of microtubules. This general idea was first introduced two decades ago by (Gard, 1987) to explain the high rates of tubulin assembly induced by XMAP215. Multimerization overcomes the inherent polymerization barrier tubulin heterodimers face due to single longitudinal and lateral tubulin:tubulin affinities estimated to be on the order of mM and M respectively. +TIP-induced multimerization of tubulin would increase the effective affinity for the microtubule lattice through cooperative binding, thereby decreasing the critical concentration for polymerization. By stabilizing tubulin-tubulin interactions prior to their full incorporation into a mature, cylindrical lattice, the +TIPs would act as polymerization chaperones. Physiologically, such chaperones would become particularly important for enhancing the growth of microtubules when free tubulin dimers are below the critical concentration of microtubule assembly or when microtubule destabilizing proteins are active (e.g in mitosis). This mechanism also enhances the spatial and temporal regulation of microtubule assembly in the cell, in part by regulating the localization and/or activity of +TIPs without modifying tubulin, the basic unit of polymerization (Slep, 2007).

Support for this general model comes from a number of other laboratories. Tandem Cap-Gly domains in CLIP-170 have been shown to plus end track whereas single Cap-Gly domains of the homolog CLIP-115 show greatly reduced microtubule association. Optical trapping studies analyzing microtubule growth (Kerssemakers, 2006) showed step-wise growth that increased in size in the presence of full length XMAP215, a result they interpreted as XMAP215-facilitated incorporation of tubulin oligomers onto the microtubule end (Slep, 2007).

An apparent exception to the multiple tubulin binding rule for +TIPs is the CLASP family, which contains only one TOG domain at its N-terminus. However, informed by TOG structures and secondary structure predictions several TOG-like (TOGL) domains can be identified in the CLASP family. Two additional dodeca-helical domains are predicted with alternating loops that exhibit high homology to TOG domain intra-HEAT loops. If folded into a HEAT-like structure, the conservation profile of alternating loops would be localized to one face of the domain, suggesting that TOGL domains may bind tubulin by a mechanism similar to what is described in this study for the TOG domain. Thus, although poorly conserved at the primary structure level, XMAP215 and CLASP appear to be ancient +TIP relatives that may employ a similar general strategy for plus end tracking. Support for CLASP's possible polymerization chaperone role comes from studies that observed CLASP-dependent microtubule subunit incorporation into fluxing kinetochore fibres (Slep, 2007).

The model for +TIPs as multimeric tubulin chaperones undoubtedly oversimplifies the complexity of interactions that are occurring at the microtubule plus end in vivo. +TIP families show divergent effects on microtubule dynamics, some promoting growth while others act as anti-pause, destabilization, rescue and perhaps even nucleation factors. Some of these differences in activity may reside in variations in the affinity constants of +TIP domains with tubulin monomers, oligomers and microtubules. As an example, non-EB1 family microtubule-binding CH domain proteins, including CLAMP and HEC1 appear to have higher affinity for the microtubule lattice than EB1 and localize along the entire length of the microtubule rather than just at plus ends. The spatial arrangement of +TIP domains, arrayed versus dimerized, may lead to distinct effects on microtubule dynamics by preferentially stabilizing lateral versus longitudinal tubulin-tubulin interactions. Finally, the intricate web of +TIP-+TIP interactions may generate unique outcomes on tubulin assembly. Multi-component in vitro assays, high resolution EM analysis of microtubules, and x-ray structures of +TIP-tubulin complexes will be required to further understand how +TIPs regulate the microtubule lattice (Slep, 2007).

Hsp90 is required to localise cyclin B and Msps/ch-TOG to the mitotic spindle in Drosophila and humans

During mitosis, cyclin B is extremely dynamic and although it is concentrated at the centrosomes and spindle microtubules (MTs) in organisms ranging from yeast to humans, the mechanisms that determine its localisation are poorly understood. To understand how cyclin B is targeted to different locations in the cell, proteins were isolated that interact with cyclin B in Drosophila embryo extracts. Cyclin B interacts with the molecular chaperone Hsp90 and with the MT-associated protein (MAP) Mini spindles. Both Hsp90 and Msps are concentrated at centrosomes and spindles, and Hsp90, but not Msps, is required for the efficient localisation of cyclin B to these structures. Unlike what happens with other cell cycle proteins, Hsp90 is not required to stabilise cyclin B or Msps during mitosis. Thus, it is proposed that Hsp90 plays a novel role in regulating the localisation of cyclin B and Msps during mitosis (Basto, 2007; full text of article).

How might Hsp90 function in recruiting cyclin B to centrosomes and spindles? As Hsp90 is itself located at centrosomes and can bind to tubulin, it is possible that Hsp90 binds cyclin B and directly targets it to these locations. It is suspected that this is not how Hsp90 targets cyclin B to MTs, since only a small fraction of the total cyclin B is bound to Hsp90 in embryo extracts. Virtually all of the cyclin B in an embryo extract is capable of binding to MTs in MT spin-down experiments. Thus, it seems unlikely that Hsp90 could act as an essential co-factor that directly mediates the interaction between cyclin B and MTs. Similarly, it is suspected that Hsp90 does not directly target cyclin B to centrosomes, since the initial recruitment of cyclin B to centrosomes during prophase is only mildly disrupted in Drosophila cells (in HeLa prophase recruitment is not disrupted) when Hsp90 has been perturbed. Rather, Hsp90 seems to be involved in maintaining the centrosomal localisation of cyclin B during prometaphase and metaphase. Perhaps Hsp90 is essential for the proper folding or function of a specific domain of cyclin B that is required for the localisation of cyclin B on centrosomes and spindles. In such a scenario Hsp90 could even act indirectly to allow cyclin B to associate with other proteins that target it to centrosomes and MTs (Basto, 2007).

If the assumption that Hsp90 does not directly target cyclin B to centrosomes or MTs is correct, it raises the intriguing question of what targets cyclin B to these locations? It has previously been shown that cyclin B can interact with XMAP215 and it has been proposed that this interaction could target cyclin B to centrosomes and MTs. It was also found that cyclin B can interact with Msps/XMAP215 in Drosophila embryo extracts, suggesting that this interaction is conserved between frogs and flies. In msps mutant cells, or in human cells partially depleted of ch-TOG, however, it was found that cyclin B was still localised to centrosomes and MTs. Thus, it is concluded that Msps is not directly responsible for targeting cyclin B to centrosomes or MTs. Msps family members play an important role in regulating MT dynamics during the cell cycle so the interaction between cyclin B and Msps may simply reflect the fact that cyclin B/Cdc2 regulates Msps activity during the cell cycle. Indeed, it has been shown that Msps/XMAP215 is phosphorylated by cyclin B/Cdc2 in vitro (Vasquez, 1999; Basto, 2007 and references therein).

If Msps and Hsp90 do not directly target cyclin B to centrosomes and MTs, it remains unclear what does. A priori, it is expected that cyclin B in Drosophila embryo extracts would exist in a tight complex with any factor that would target it to MTs, since the vast majority of cyclin B binds to MTs in embryo extracts. Since cyclin B appears to localise at centrosomes and MTs in virtually all systems, it remains possible that cyclin B can directly bind to centrosomes and MTs. Indeed, bacterially expressed MBP-CBFL interacts strongly with purified MTs in MT-pelleting assays. In light of these results it seems that cyclin B is capable of interacting directly with MTs, although caution should b taken in interpretation of this in vitro experiment since fusion proteins containing cyclin B could have a tendency to aggregate in solution (Basto, 2007).

Nevertheless, the fact that no one has identified a factor that directly mediates the interaction between cyclin B and MTs or centrosomes, despite many years of effort in identifying cyclin B interacting proteins, suggests that there may be no other protein directly required for these interactions. In the favoured hypothesis, Hsp90 would serve simply to ensure that cyclin B was correctly folded to allow it to directly interact with MTs and with centrosomes. Interestingly it has been proposed that Hsp90 also contributes to increasing the association efficiency of Tau with MTs. Tau is a MAP with an important role in Alzheimer's disease. In the absence of Hsp90, Tau tends to aggregate and therefore less soluble Tau is available to bind to MTs. In this study, although it was also found that cyclin B and Msps require Hsp90 for their efficient recruitment to the spindle it is not thought that their activity, outside the spindle, is compromised (Basto, 2007).

Finally, it was found that Hsp90 was not only required to allow cyclin B to localise efficiently to centrosomes and MTs, it was also required to allow Msps to localise properly, and it was shown that the endogenous Hsp90 can interact with the endogenous Msps. Importantly, Hsp90 is not required for the localisation of several other proteins to centrosomes or MTs, demonstrating that its function in localising cyclin B and Msps is specific. Like cyclin B, the levels of Msps protein were not decreased in cells where Hsp90 function had been perturbed, suggesting that Hsp90 is not simply required to stabilise Msps protein. Thus, it is proposed that Hsp90 may act on several MT-associated proteins to ensure that specific domains of these proteins are in the correct conformation to allow these proteins to be targeted to different locations within the cell (Basto, 2007).

Parallel genetic and proteomic screens identify Msps as a CLASP-Abl pathway interactor in Drosophila

Regulation of cytoskeletal structure and dynamics is essential for multiple aspects of cellular behavior, yet there is much to learn about the molecular machinery underlying the coordination between the cytoskeleton and its effector systems. One group of proteins that regulate microtubule behavior and its interaction with other cellular components, such as actin-regulatory proteins and transport machinery, is the plus-end tracking proteins (MT+TIPs). In particular, evidence suggests that the MT+TIP, CLASP (Chromosome bows), may play a pivotal role in the coordination of microtubules with other cellular structures in multiple contexts, although the molecular mechanism by which it functions is still largely unknown. To gain deeper insight into the functional partners of CLASP, parallel genetic and proteome-wide screens for CLASP interactors were performed in Drosophila. 36 genetic modifiers and 179 candidate physical interactors were identified, including 13 that were identified in both data sets. Grouping interactors according to functional classifications revealed several categories, including cytoskeletal components, signaling proteins, and translation/RNA regulators. The initial investigation focused on the MT+TIP Minispindles (Msps), identified among the cytoskeletal effectors in both genetic and proteomic screens. This study reports that Msps is a strong modifier of CLASP and Abl in the retina. Moreover, Msps functions during axon guidance and antagonizes both CLASP and Abl activity. The data suggest a model in which CLASP and Msps converge in an antagonistic balance in the Abl signaling pathway (Lowery, 2010).

The in vivo functions of cytoskeletal effector and regulatory proteins have been studied very effectively in Drosophila, with particular success at the earliest stages of embryonic development prior to zygotic gene expression, when depletion of maternal stores of such proteins often results in disruption of mitosis, cellularization, or other aspects of cell biology. However, the functions of cytoskeletal effectors at late stages of development are often obscured by early embryonic functions. In this regard, the existence of maternal stores of some key effectors has been helpful for analysis of late events in nervous system development, such as axonal and dendritic patterning, because such maternal supplies of protein are sometimes exhausted only at late stages when axons and dendrites emerge. However, zygotic mutations in many key cytoskeletal components disrupt early stages, making screens based on neuroanatomical phenotypes problematic. For this reason, genetic interaction screens were used to explore the network of cytoskeletal regulators linked to key guidance signaling molecules as a means of identifying candidates for deeper analysis during axonal development. These screens for modifiers of Abl kinase phenotypes led to the identification of CLASP as an effector essential for accurate growth cone navigation. By using CLASP as a starting point for a new generation of screens, new functional categories and individual players of the CLASP interactome were identified, including cytoskeletal components, signaling proteins, and translation/RNA regulators. In addition, a microtubule regulatory protein (the MT+TIP Msps) not previously associated with axonal pathfinding decisions was identified (Lowery, 2010).

To build functional neural networks, axonal growth cones must accurately interpret and translate multiple guidance cues into directional movement by coordinating both microtubule and F-actin networks. There appears to be significant interplay between the two cytoskeletal components, but a sophisticated understanding of the signaling and effector mechanisms by which both systems are coordinated in response to guidance cues has not yet been obtained. The Abl tyrosine kinase is one of the few known signaling molecules shown to transduce guidance cue signals to both actin and MT networks, although far less is known regarding how it regulates MTs. This work, and that of others, suggests that CLASP may be an important player in the MT-actin crosstalk machinery (Lowery, 2010).

The largest thematic group of CLASP-interacting genes identified is the actin-binding proteins, including Shot, Zip, Capu, Pnut, Jar, Bif, Kst, and the uncharacterized CG13366 (ortholog of calponin-homology domain containing CYTSA/B). These are all known actin-associated factors that are also predicted to bind to MTs, and their presence in the screen points to a role for CLASP in mediating actin dynamics in coordination with the MT network. This supports previous observations that vertebrate CLASPs may function as actin-MT crosslinkers. CLASPs possess actin-binding activity (Tsvetkov, 2007), and CLASP-decorated MT tips track along actin filament bundles in the growth cone peripheral domain. Moreover, CLASP was recently shown to bind to the actin-binding protein, IQGAP1, and phosphorylation of CLASP controls linkage of MTs to actin through IQGAP1 for cell migration (Watanabe, 2009). From the current study, it appears that CLASP may have numerous other effector proteins that can modulate its interaction with the actin network. IQGAPs have not been found in Drosophila, and so, it is speculated that the novel interactors identified, as well as others, may allow CLASP to link MTs and actin in different contexts (Lowery, 2010).

The novel CLASP MT+TIP interactor that were identified, Msps, emerged from the screens as a high priority for future analysis. Msps interacts with CLASP in both the genetic and proteomic screens, and it antagonizes CLASP and Abl signaling. The antagonism seen between Msps and CLASP in the Drosophila retina is consistent with cell culture studies, which have shown that CLASP regulates MT dynamics by specifically promoting the pause state (Sousa, 2007) whereas Msps-family proteins function as MT antipause factors (Brittle, 2005). More specifically, CLASPs have MT-stabilizing effects, and depleting cells of CLASP protein results in highly dynamic, constantly growing or shrinking MTs (Sousa, 2007). Alternatively, Msps family members can have the opposite effect (Popov, 2003), catalyzing the addition and removal of multiple tubulin dimers at MT plus-ends (Brouhard, 2008), and depletion of Msps results in a dramatic increase in MT pausing with little or no growth (Brittle, 2005). These opposite effects on MT behavior in cell culture studies suggest reciprocal functions in the regulation of MT dynamics in vivo, and Msps could thus be a component of the Abl signaling pathway that provides an antagonistic counterbalance to CLASP in regulating the growth cone cytoskeletal output downstream of guidance cues (Lowery, 2010).

In the context of CNS, it seems likely that CLASP and Msps drive axon guidance decisions through reciprocal regulation of growth cone turning toward or away from the source of axon guidance factors at the midline. Accurate navigation of both ipsilateral and contralateral axon pathways requires a combination of cues, to regulate both midline crossing behavior and also the stereotyped positions of longitudinal axon tracts. The lateral specification model proposes that for longitudinal axons to find and maintain a correct trajectory at a specific distance from the midline, they must reach a balance of turning responses to the attractive Netrins and repellent Slit secreted by midline glia. Perhaps this balance requires antagonism of Msps and CLASP downstream of Abl, such that reduction of either protein would bias the growth cone or reduce the fidelity of the overall navigation process. Abl has been shown to mediate both Slit and Netrin activity, thus providing a potential point of integration (Lowery, 2010).

While it is anticipated that CLASP and Msps will influence the directionality of growth cone advance in response to guidance cues, the cellular mechanism by which these two effectors guide axons is not yet known. Reciprocal control over MT advance toward the growth cone peripheral domain could account for the effects of CLASP and Msps. However, there are alternatives. For example, reciprocal modulation of growth cone cell adhesion by Msps and CLASP might underlie the two phenotypes observed in the different mutants. Reduction of adhesion has already been shown to be key in the midline repulsive response to Robo, whereas an increase in adhesion has long been known to be vital for fasciculation. Interestingly, the Abl kinase that interacts with both Msps and CLASP was also implicated in Robo-dependent modulation of cell adhesion. If CLASP were to play a role in Robo-mediated suppression of adhesion, then the ectopic midline crossing that occurs in CLASP LOF mutants could be explained by an increase in growth cone adhesion toward the midline, which is exacerbated when Msps is overexpressed. Consistently, the fasciculation morphology defects that occur in msps LOF (and are exacerbated by CLASP GOF) could be explained if the role of Msps is to promote adhesion. Although the effects of CLASP-family proteins on cell adhesion have not been directly measured, studies in nonneuronal contexts suggest that CLASP helps to drive MT-cortical interactions, which would presumably promote, not suppress, adhesion (Lowery, 2010).

In conclusion, this is the first study demonstrating that Msps functions during axon guidance. Numerous studies have analyzed its role in the regulation of MT stability in several systems including the mitotic spindle and in centrosomes, but its potential role(s) in the nervous system has never been previously addressed. In fact, the growth cone functions of most MT+TIPs are unknown; however, previous discoveries that MT+TIPs CLASP and APC, and now Msps, are important for axon guidance demonstrates that the MT+TIPs are an exciting class of guidance effectors worthy of further exploration and understanding (Lowery, 2010).

The microtubule lattice and plus-end association of Drosophila mini spindles is spatially regulated to fine-tune microtubule dynamics

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, an in vivo structure-function analysis was conducted of the Drosophila homologue, Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNAi rescue experiments revealed that Msps' NH(2)-terminal four TOG domains function as paired units and are sufficient to promote microtubule dynamics and EB1 comet formation. TOG5 and novel inter-TOG linker motifs were identifed that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT-binding that underlies the ability of Msps to promote MT dynamic instability (Currie, 2011).

Growing evidence from several model systems indicates that members of the XMAP215/Dis1 protein family enhance microtubule dynamic instability and mitotic spindle assembly. These multi-domain proteins are highly conserved across taxa and there is particular interest in the human homologue, ch-TOG, as its overexpression has been documented in several cancer cell types. Despite the importance of these proteins, relatively little is known about their dynamics in living cells or about how their domain structure relates to their function in vivo. In this study, the Drosophila XMAP215 homologue, Mini spindles (Msps), was used as a model to address these questions in living S2 cells. It was found that Msps exhibits a complex and dynamic behavior in vivo. It localizes both to growing and shrinking microtubule plus ends. In the actin-rich cell periphery, Msps-GFP punctae exhibit a discontinuous and dynamic association with the microtubule lattice. Consistent with roles for both Msps and EB1 as key regulators of dynamic instability, it was observed that depletion of either of these proteins lead to alteration of the other' dynamics at microtubule plus ends (Currie, 2011).

Remarkably, microtubule dynamics and EB1 velocity could be partially restored by replacing endogenous Msps with the NH2-terminal four TOG domains TOG1-4. Structure/function analysis also revealed the presence of novel microtubule lattice-binding domains, which include sequences in two of the inter-TOG linker domains. These linkers contain novel motifs responsible for this interaction and are important for the normal dynamics of Msps as mutation of these motifs causes a loss of Msps from the lattice of peripheral microtubules and an alteration to the morphology and dynamics of the microtubule cytoskeleton. Thus, the data demonstrate that Msps is able to interact with microtubules through at least two mechanisms and that this bimodal interaction is required for normal dynamic instability (Currie, 2011).

This study, the first detailed examination of a Msps/Dis1 family member in a living animal cell, revealed that Msps exhibits a complex pattern of dynamics. Throughout the cell, Msps localizes to the plus ends of growing microtubules, consistent with other published descriptions of Msps behavior in early Drosophila embryos) and in vivo dynamics of homologues Stu2. Observations in S2 cells are in line with family members acting as microtubule polymerases that promote the incorporation of free tubulin at the plus end. Msps also exhibits dynamics similar to the in vitro behavior of XMAP215 as both proteins remain associated with microtubules during phases of growth and shrinkage. At present, the biological significance of Msps association with shrinking microtubules is not understood, however, Stu2 and XMAP215 do destabilize microtubules under specific in vitro experimental conditions. Persistent interaction with shrinking microtubules may reflect a physiologically important role for Msps during depolymerization in vivo. A model based on this interaction with both growing and shrinking microtubules is elaborated, in which Msps acts, not only as a polymerase, but as an anti-pause factor that is capable of 'catalyzing' the transition to either polymerization or depolymerization. Although it has primarily been shown to enhance the growth of microtubules, Msps is also necessary for their transition to disassembly in vitro and in vivo. These two seemingly opposing roles are mediated by 1) the TOG domains and plus end localization which enhance microtubule growth; and 2) microtubule lattice binding sites within linker2-TOG3 and linker4- TOG5 which enhance the transition between dynamic and non-dynamic states and also influence microtubule disassembly rates. Regulation and balance of these two domains maintain normal microtubule dynamics. In the future, it will be interesting to learn if these in vivo dynamics also apply to XMAP215 or human chTOG, which remain largely uncharacterized in cells (Currie, 2011).

One of the central observations from this work was that the NH2-terminal TOG domains of Msps are the key determinants of the protein's activity in vivo. Several previous studies have indicated a role for TOG domains as primary binding sites for tubulin in XMAP215/Dis1 proteins. Biochemical analysis of the TOG domains from Msps revealed that TOG1-2 was the minimal construct that was able to bind tubulin. Moreover, a construct consisting of a tandem array of TOG1212 functioned as a potent microtubule nucleator in vitro, suggesting that multiple pairs of TOG domains act in concert to promote polymerization. A fragment of Msps containing TOG1-4 was sufficient to rescue many aspects of microtubule dynamic instability in living S2 cells depleted of endogenous Msps, both in terms of interior EB1-GFP growth velocities and persistence as well as the frequencies of catastrophe and rescue. Consistent with the in vitro data, constructs embodying TOG1-2 imparted only a slight rescue of microtubule growth in S2 cells, while TOG3-4 did not. These data imply that individual pairs of TOG domains may contribute unequally to the activity of the protein, but do function cooperatively to promote persistent microtubule growth. This is supported by recent work that found that TOG1 and 2, specifically, play a key role in XMAP215 polymerase activity in vitro (Widlund, 2011). The lack of rescue by TOG1212 also indicates that each pair of TOG domains is uniquely suited to fulfill the cooperative function of tubulin addition. Although TOG1-4 exerted a strong effect on tubulin incorporation and loss from the plus end as measured by GFP-Tubulin and EB1-GFP growth rates, the TOG1-4 protein exhibited a predominantly cytoplasmic localization and did not accumulate at microtubule tips. These data suggest a mechanism in which TOG1-4 is able to associate with one or more tubulin heterodimers to transiently promote their addition or loss from the plus end without interacting in a processive manner or associating with the microtubule lattice (Currie, 2011).

Another key observation is the identification of novel microtubule interaction sites spanning the TOG3 and TOG5 and their preceding inter-TOG linkers. These sites are necessary for association with the microtubule lattice and for the lattice-associated diffusive movements exhibited by full-length Msps. This analyses identified the presence of conserved motifs in the inter-TOG regions that were enriched in basic amino acid residues, which are predicted form an electrostatic interaction with the negative COOH-terminus of tubulin. The linkers between Msps' TOG domains are predicted to be disordered stretches without secondary structure that were found to cooperate with the highly structured TOG3 or TOG5 to mediate microtubule binding (Currie, 2011).

The cooperation between ordered and disordered domains to mediate microtubule binding has become a common trend as more of the structural determinants of MAP-microtubule association have recently been elucidated. These disordered regions seem to allow a diffusion-capable attachment to microtubules in addition to the ability to 'titer' the interaction strength by charged residue addition or modification (i.e., phosphorylation) (Currie, 2011).

Beyond the structural basis for microtubule lattice-association, it was observed that Msps employs this mechanism in a spatially restricted manner in the actin-rich lamella of S2 cells. In other cell types, microtubules that enter this subcellular compartment exhibit decreased catastrophe rates, due to the small GTPase Rac1 regulating MAPs that influence microtubule dynamics. In this regard, the spatial transition from plus end binding to MAP lattice association has also been observed in mammalian epithelial cells for the +TIP CLASP. The Drosophila CLASP homolog, Orbit/MAST, when tagged with GFP displayed plus end dynamics similar to that of EB1 and did not differentially localize in the periphery of the cell. One possibility is that Msps functions in an orthologous manner to mammalian CLASP, which could be due to their shared structure as TOG domain-containing proteins. In any case, it is speculated that this transition from tip-tracking to lattice-binding reflects a regulated change in the conformation of Msps. Given the observation that TOG1-5 decorates the microtubule lattice constitutively, it is also hypothesized that the COOH-terminus of the protein is involved in this regulation and acts to 'gate' the microtubule binding activity of linker4-TOG5 in the full length protein. Future studies will address the basis of this regulation (Currie, 2011).

Ablating these microtubule lattice association sites in the full-length molecule had a dramatic effect on the dynamics and morphology of the microtubule cytoskeleton. Although lattice association is commonly a stabilizing property of most MAPs, it was surprising to find that without its normal lattice association, Msps Double Mutant produced very stable microtubules that exhibited fewer transitions and continuous growth after microtubules encountered the cell cortex. In contrast, although Msps dsRNA also produces very stable microtubules, these microtubules are seldom able to persistently grow against the actin retrograde flow in the peripheral lamella. These data, in conjunction with the increased shrinkage rates observed when cells expressed TOGs1-4 or TOG1-5, lead to a proposal that these lattice association sites may influence the catalysis between dynamic states and the shrinkage rates of peripheral microtubules. Microtubules in peripheral regions of S2 cells exhibit increased dynamic instability as compared to the interior of the cell and the data suggest a mechanism through which Msps may regulate these peripheral behaviors through its lattice association (Currie, 2011).

It is hypothesized that this influence on dynamics could be caused by three non-mutually exclusive mechanisms. Firstly, Msps lattice association could act to 'strip' Msps from the plus end, potentially taking heterodimers with it and lowering the concentration of this polymerase from the microtubule tip. Secondly, Msps- lattice binding domains could act to slightly perturb lateral interactions between heterodimers along the decorated protofilament. This cascade of 22 small perturbations could act to prime the microtubule lattice for depolymerization several microns distal to the plus end. Finally, lattice-bound Msps could be acting in concert with other +TIPs, such as kinesin-13 depolymerases, to influence catastrophe and shrinkage (Currie, 2011).

Msps dependence on EB1 for plus end localization in interphase has recently become more clear based on two recent studies identifying novel EB1 adaptor proteins, SLAIN/Sentin. In mammalian cells, SLAIN binds both chTOG and EB1 and is required for ch-TOG plus end tracking (van der Vaart, 2011). In Drosophila, a similar protein Sentin has also been described (Currie, 2011).

Although it has not yet been shown to bind Msps, RNAi depletion of Sentin alters the plus end localization of Msps. Depletion of EB1 removed Msps from both the plus end and lattice of microtubules. Although the mechanism for how Msps is lost from both plus end and lattice is not known, it suggests that the ability of full-length Msps to recognize the microtubule lattice is in some way tied to its plus end association. One possibility could be a whole-molecule conformational change that occurs when Msps interacts with EB1 at the growing end, which might be required to license Msps for association along the microtubule lattice. This most likely is concurrent with some other spatially regulated control that gives Msps a bimodal function between the cell interior and the periphery. EB1's plus end localization does not require Msps, as a population of EB1 remains on microtubule tips following Msps depletion, however, its dynamics are drastically altered. Rescue of EB1 velocities using the TOG domain region of Msps suggests that EB1 relies on the polymerase activity of Msps for its normal dynamics. This also seems true since Msps fragments that rescue EB1-GFP comet formation do not seem to localize to the microtubule plus end, although the possibility that there may be transient pools of TOG domain-fragments at the plus end cannot be ruled out. Instead, a model is favored where these TOG domain fragments act en masse to chaperone tubulin onto the plus end, subtly influencing the on and off rates of heterodimer addition. This creates a plus end structure with sufficient binding sites to support more normal EB1 comets (Currie, 2011).

Based on the data, Msps exhibits at least two modes of interaction with microtubules, both of which are essential to promote normal parameters of dynamic instability. A cycle of interactions is envisioned that begins upon association between soluble Msps and one or more tubulin heterodimers. The Msps-tubulin complex then recognizes the microtubule plus end and associates transiently. The molecular basis of this recognition is unknown, but may reflect a conformation of tubulin at the plus end (e.g., a growing sheet), a chemical signature (e.g., a cap of GTP tubulin), the presence of another +TIP such as EB1, or some combination of these. Upon binding, Msps delivers its tubulin 'cargo' to promote polymerization. In the cell interior, Msps then dissociates, thus behaving as a typical +TIP. In the cell periphery, however, following tubulin delivery, Msps receives a signal that causes it to engage its lattice-binding activity and to diffuse along the microtubule surface. Msps also associates with microtubule plus ends upon transition to catastrophe, perhaps working cooperatively with destabilizing factors such as kinesin-13 proteins and stathmin/OP18 (Currie, 2011).

The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array

XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed TOG domains that bind tubulin. Whether these TOG domains are architecturally equivalent is unknown. This study presents crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with alpha-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT polymerization. Drosophila S2 cells depleted of endogenous Msps were used to assess the importance of individual TOG domains. While a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. This work highlights the structurally diverse, yet positionally conserved TOG array that drives MT polymerization (Fox, 2014).

Solution NMR assignment of the cryptic sixth TOG domain of mini spindles

TOG domains contribute to the organisation of microtubules through their ability to bind tubulin. They are found in members of the XMAP215 family of proteins, which act as microtubule polymerases and fulfill important roles in the formation of the mitotic spindle and in the assembly of kinetochore fibres. A cryptic TOG domain was identified in the XMAP215 family proteins, chTOG and its Drosophila homologue, Mini spindles. This domain is not part of the well-established array of TOG domains involved in tubulin polymerisation. Instead it forms part of a binding site for TACC3 family proteins. This interaction is required for the assembly of kinetochore bridges in a trimeric complex with clathrin. This study presents the first NMR assignment of a sixth TOG domain from Mini spindles as a first step to elucidate its structure and function (Burgess, 2015).

Search PubMed for articles about Drosophila Msps

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Burgess, S. G., Bayliss, R. and Pfuhl, M. (2015). Solution NMR assignment of the cryptic sixth TOG domain of mini spindles. Biomol NMR Assign [Epub ahead of print]. PubMed ID: 25971232

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Currie, J. D., et al. (2011). The microtubule lattice and plus-end association of Drosophila mini spindles is spatially regulated to fine-tune microtubule dynamics. Mol. Biol. Cell 22(22): 4343-61. PubMed ID: 21965297

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Fox, J. C., Howard, A. E., Currie, J. D., Rogers, S. L. and Slep, K. C. (2014). The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array. Mol Biol Cell [Epub ahead of print]. PubMed ID: 24966168

Garcia, M. A., Vardy, L., Koonrugsa, N. and Toda, T. (2001). Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint. EMBO J 20: 3389-3401. PubMed ID: 11432827

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Gergely, F., Draviam, V. M. and Raff, J. W. (2003). The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells. Genes Dev 17: 336-341. PubMed ID: 12569123

Goshima, G., Wollman, R., Stuurman, N., Scholey, J. M. and Vale, R. D. (2005). Length control of the metaphase spindle. Curr. Biol. 15(22): 1979-88. PubMed ID: 16303556

Graf, R., Euteneuer, U., Ho, T. H. and Rehberg, M. (2003). Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number. Mol. Biol. Cell 14: 4067-4074. PubMed ID: 14517319

Gujar, M. R., Gao, Y., Teng, X., Deng, Q., Lin, K. Y., Tan, Y. S., Toyama, Y. and Wang, H. (2023). Golgi-dependent reactivation and regeneration of Drosophila quiescent neural stem cells. Dev Cell. PubMed ID: 37567172

Hahn, I., Voelzmann, A., Parkin, J., Fulle, J. B., Slater, P. G., Lowery, L. A., Sanchez-Soriano, N. and Prokop, A. (2021). Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons. PLoS Genet 17(7): e1009647. PubMed ID: 34228717

Holmfeldt, P., Stenmark, S. and Gullberg, M. (2004). Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis. EMBO J 23: 627-637. PubMed ID: 14749730

Kerssemakers, J. W., et al. (2006). Assembly dynamics of microtubules at molecular resolution. Nature 442: 709-712. PubMed ID: 16799566

Kinoshita, K., et al. (2001). Reconstitution of physiological microtubule dynamics using purified components. Science 294: 1340-1343. PubMed ID: 11701928

Kinoshita, K., Habermann, B. and Hyman, A. A. (2002). XMAP215: a key component of the dynamic microtubule cytoskeleton. Trends Cell Biol. 12: 267-273. PubMed ID: 12074886

Kinoshita, K., et al. (2005). Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis. J. Cell Biol. 170: 1047-1055. PubMed ID: 16172205

Lattao, R., Rangone, H., Llamazares, S. and Glover, D. M. (2021). Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins. Dev Cell 56(7): 1000-1013. PubMed ID: 33725482

Le Bot, N., Tsai, M. C., Andrews, R. K. and Ahringer, J. (2003). TAC-1, a regulator of microtubule length in the C. elegans embryo. Curr. Biol. 13: 1499-1505. PubMed ID: 12956951

Lee, M. J., et al. (2001). Msps/XMAP215 interacts with the centrosomal protein D-TACC to regulate microtubule behaviour. Nat. Cell Biol. 3(7): 643-9. PubMed ID: 11433296

Li, W., Moriwaki, T., Tani, T., Watanabe, T., Kaibuchi, K. and Goshima, G. (2012). Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin. J Cell Biol 199: 849-862. PubMed ID: 23185033

Lowery, L. A., et al. (2010). Parallel genetic and proteomic screens identify Msps as a CLASP-Abl pathway interactor in Drosophila. Genetics 185(4): 1311-25. PubMed ID: 20498300

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Peset, I., Seiler, J., Sardon, T., Bejarano, L. A., Rybina, S. and Vernos, I. (2005). Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis. J. Cell Biol. 170: 1057-1066. PubMed ID: 16172207

Polinko, E. S. and Strome, S. (2000). Depletion of a Cks homolog in C. elegans embryos uncovers a post-metaphase role in both meiosis and mitosis. Curr. Biol. 10: 1471-1474. PubMed ID: 11102813

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date revised: 5 August 2023

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