BIOLOGICAL OVERVIEW

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to chromosomal kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Studies on the function of Orbit/MAST (FlyBase designation: Chromosome bows or Chb for short) in Drosophila and its human homolog CLASP1 (Maiato, 2003a: CLASP stand for CLIP-associated protein), have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizes to the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. CLASP1 is therefore required at kinetochores to regulate the dynamic behavior of attached microtubules (reviewed by Maiato, 2003b).

Axon guidance requires coordinated remodeling of actin and microtubule polymers. Using a genetic screen, the microtubule-associated protein Orbit/MAST has been identified as a partner of the Abelson (Abl) tyrosine kinase. Identical axon guidance phenotypes are found in orbit/MAST and Abl mutants at the midline, where the repellent Slit restricts axon crossing. Genetic interaction and epistasis assays indicate that Orbit/MAST mediates the action of Slit and its receptors, acting downstream of Abl. Orbit/MAST protein localizes to Drosophila growth cones. Higher-resolution imaging of the Orbit/MAST ortholog CLASP in Xenopus growth cones suggests that this family of microtubule plus end tracking proteins identifies a subset of microtubules that probe the actin-rich peripheral growth cone domain, where guidance signals exert their initial influence on cytoskeletal organization. These and other data suggest a model where Abl acts as a central signaling node to coordinate actin and microtubule dynamics downstream of guidance receptors (Lee, 2004).

In order to establish an intricate yet specific network of neuronal connections, axons are guided from intermediate to final targets by an assortment of attractive and repellent factors. The navigational response to such guidance cues depends on a complex and dynamic cytoskeletal machine within the growth cone that is linked via signaling pathways to receptors at the cell surface. In essence, the growth cone acts as an exquisitely sensitive molecular compass, translating the spatial asymmetry of extracellular cues into polarization of the cytoskeletal elements that determine the directional specificity of cell movement. Two major targets in this cell polarity machine are the microfilament networks that support the growth cone perimeter and the microtubule arrays that build the core of the nascent axon. While a comprehensive understanding of cytoskeletal signaling is still elusive, much progress has been made in identifying pathways and effector molecules that control the rapid and initial response of actin assembly to particular guidance factors. However, very little progress has been made in identifying microtubule-associated proteins (MAPs) that participate in specific pathways (Lee, 2004 and references therein).

The actin networks that propel membrane protrusion are thought to mount the initial response to guidance information. Actin remodeling in the growth cone peripheral (P) domain remains dynamic and unstable, allowing for rapid changes in direction or cell contact. Microtubules are also dynamic in the periphery, and their recruitment and subsequent bundling to establish the central (C) domain of the growth cone represent a hallmark of directional growth. Consequently, as growth cones make guidance decisions in situ and select particular filopodia to define the new direction of movement, it is the consolidation of microtubule structures and concomitant dilation of filopodia that are most predictive of a change in direction. Indeed, perturbation of microtubule dynamics has a dramatic effect on growth cone navigational behavior (Lee, 2004 and references therein).

In the transition zone between the P and C domains of the growth cone, a specialized class of microtubules extend and penetrate into the actin-rich perimeter. Recent studies in growth cones and nonneuronal cells show that such 'pioneer' microtubules enjoy an intimate relationship with specialized actin structures that support individual filopodia and define the region between P and C domains, supporting theoretical models that predict crosstalk between the two polymer networks. Consistent with this idea, pharmacological studies show that growth cone microtubule structures and guidance are dependent on actin dynamics. Interestingly, nonneuronal studies suggest that a reciprocal signaling relationship exists at this cytoskeletal interface and that molecules such as Rho family GTPases act to coordinate the dynamics of both actin and microtubules. Signaling molecules or 'nodes' that coordinate multiple downstream events are common in signal transduction pathways. While Rho family GTPases are well known for this capacity, additional classes of proteins are also likely to serve this function during axon guidance (Lee, 2004 and references therein).

One excellent candidate as an axon guidance signaling node in Drosophila is the Abelson (Abl) protein tyrosine kinase. Abl is required for the accurate guidance of both central and peripheral axon pathways and modulates the function of several axonal receptors. Study of these signaling pathways shows that Abl interacts genetically with a number of intracellular effector proteins, including Enabled, Profilin, Trio, and the cyclase-associated protein (Lee, 2004 and references therein).

So far, the majority of known Abl interactors control aspects of actin assembly. However, a new Abl interactor known to associate with microtubules has been discovered, suggesting a link that might coordinate actin and microtubule dynamics. Genetic analysis has identified a number of MAPs that are necessary for axonogenesis, including Futsch/MAP1b, Short stop, and Pod-1. Although some of these effector proteins are targets for intracellular kinases, none have been shown to act downstream of specific axon guidance factors. While the Semaphorin effector Collapsin response mediator protein (CRMP) has been shown to bind Tubulin heterodimers and influence polymer assembly, the primary role of the CRMP family is thought to involve membrane dynamics (Lee, 2004 and references therein).

A kinase-dependent gain-of-function (GOF) phenotype for Abl in the Drosophila retina has been described that displays sensitive genetic interactions with receptors in the Roundabout (Robo) family (Wills, 2002). Loss-of-function (LOF) studies confirm that Abl plays a complex role in axon guidance at the midline, where Robo receptors mediate the repellent action of Slit. Moreover, there is an endogenous role for Abl in the retina, suggesting that this neural tissue can be used as a tool to identify additional classes of effectors in the Abl pathway. One of the genes derived from an ongoing retinal screen for modifiers of AblGOF is described in this study, the MAP Orbit/MAST (also known as Chromosome Bows [Chb]; Fedorova, 1997; Inoue, 2000 and Lemos, 2000), ortholog of the vertebrate cytoplasmic linker protein (CLIP)-associated proteins (CLASPs; Akhmanova, 2001). Using zygotic null alleles to escape a requirement during oogenesis, Orbit/MAST was found to be necessary for accurate axon guidance at the midline choice point. Phenotypic characterization, genetic interactions, and genetic epistasis experiments suggest that this MAP acts downstream of Abl in the Slit repellent pathway, consistent with its localization to axons and growth cones. Parallel imaging studies in Xenopus growth cones show that vertebrate CLASP identifies a subset of axonal microtubules that extend into the peripheral domain, where actin dynamics are known to influence microtubule behavior. Elevation of CLASP activity in Xenopus neurons has been shown to reduce not only microtubule advance but also growth cone translocation (Lee, 2004).

Genetic analysis reveals a postmitotic requirement for CLASP during the guidance of axons in multiple contexts. At the midline, CLASP is found to be necessary for accurate growth cone orientation away from the source of Slit and for lateral positioning of longitudinal axon fascicles, suggesting a model in which CLASP acts positively downstream of Abl as part of the repellent response initiated by activation of Roundabout receptors. Genetic interaction and epistasis experiments support this model. Moreover, protein localization studies in Drosophila and Xenopus growth cones indicate that this role for CLASP is likely to occur near the leading edge, where guidance cues exert their initial influence on cytoskeletal remodeling. In contrast to CLIP-170, CLASP is enriched at microtubule plus ends within the growth cone itself, suggesting a specialized role in neuronal cell biology. Indeed, CLASP-positive plus ends penetrate the growth cone peripheral domain and track along microfilament bundles into individual filopodia proximal to the site of guidance receptor activation. Taken together, the genetics and cell biology suggest a model in which Slit activation of the Abl kinase leads to a CLASP-dependent inhibition of microtubule extension favoring growth cone advance toward regions of low signaling activity (Lee, 2004).

Growth cone repellents play a major role in patterning neuronal connectivity and restricting regenerative capacity within the CNS, making repellent signaling a high priority for functional dissection. One theme to emerge from many studies of growth cone guidance is that the cell biology of directional navigation requires a coordination of many different subcellular events. This predicts the existence of signaling proteins that can regulate the combined activities of multiple effectors. Receptor-proximal factors fitting this profile have been found in several repellent pathways. For example, the guanine-nucleotide exchange factor Ephexin mediates EphA4-dependent repulsion by activating RhoA and simultaneously inhibiting Rac and Cdc42. The adaptor protein Grb4 displays coordinate interactions with a different cast of players downstream of Ephrin-B, including the kinase Pak1, the Cbl-associated protein (CAP/Ponsin), and the Abl-associated protein-1 (Abi-1), highlighting the diversity of potential effectors (Lee, 2004).

Through genetic analysis in Drosophila, the Abl tyrosine kinase has emerged as another key signaling center that is capable of coordinating multiple outputs (reviewed by Moresco, 2003). Abl is both necessary and sufficient to define axon guidance behavior (e.g., Wills, 1999a; Wills, 1999b; Wills, 2002; Bashaw, 2000 and Hsouna, 2003), suggesting that it acts high in the signaling hierarchy. At the CNS midline, Abl interacts with Enabled and the cyclase-associated protein (Capulet) to control growth cone behavior (Bashaw, 2000 and Wills, 2002). These Abl effectors are actin binding proteins with different types of activity in cytoskeletal dynamics (reviewed by Lee, 2003). Abl is also likely to regulate ß-Catenin/Armadillo function, which may be important for the in vivo response to Slit. While many studies on the cell biology of Abl family kinases have focused on actin effectors and Abl's ability to bind directly to actin polymers, recent work reveals that the Abl-related gene (Arg) associates directly with microtubules as well, thus placing it at the interface between the two cytoskeletal arrays Miller, 2004). Genetic screen and analysis of CLASP function in Drosophila adds a new dimension to this picture, showing that Abl controls both actin and MAPs in parallel (Lee, 2004).

CLASP family proteins fall into an intriguing group of microtubule-associated plus end tracking proteins (+TIPs; reviewed by Carvalho, 2003). While little is known about their function in neurons, and their precise mechanism of action is still mysterious, accumulated evidence suggests that +TIPs act to regulate microtubule stability. For example, dominant-negative experiments with CLIP-170 in nonneuronal cells suggest that this +TIP acts to reduce the frequency of rapid microtubule depolymerization (or 'catastrophe'; Komarova, 2002). While the impact of CLASP family function has not been determined using dynamic assays, overexpression of CLASP in COS cells increased the number of stabilized microtubules (Akhmanova, 2001). Preliminary RNA interference to remove CLASP in Drosophila S2 cells indicates that this function has been conserved (A. Ghose, U.E, and D.V.V., unpublished data cited by Lee, 2004). However, if an increase in stable microtubules comes at the expense of dynamic microtubule segments, then a negative impact on the persistence of growth cone advance might be predicted, based on existing pharmacological data. This provides an attractive model to explain how CLASP can cooperate with Slit, Robo, Robo2, and Abl during midline repulsion (Lee, 2004).

A growing number of MAPs have been shown to localize to plus ends (reviewed by Carvalho, 2003), raising the possibility that +TIP protein complexes coordinate multiple activities. In addition to the CLASP localization in this study, the EB1 family member EB3 displays +TIP behavior within the growth cone (Stepanova, 2003). Interestingly, the +TIPs EB1 and APC associate with Short stop/Kakapo/MACF in Drosophila cells. Short stop is known to bind both actin and microtubule polymers, suggesting a role in mediating interactions between the two polymer networks. Moreover, short stop mutants display an ISNb motor axon phenotype that is nearly identical to orbit/MAST and Abl loss of function. Other MAPs have also been implicated in mediating interaction between microfilaments and microtubules in developing axons, suggesting that this interface will be complex and highly coordinated. Future experiments will address whether other +TIP-associated proteins also contribute to repellent signaling and whether all the +TIP proteins display similar activities in vivo (Lee, 2004).

With a combination of in vivo genetic analysis and dynamic imaging, a working model can be proposed for the role of CLASP in growth cone repellent signaling. Recent work indicates that Abl functions positively to support Slit/Robo-mediated axon repulsion at the midline (Wills, 2002 and Hsouna, 2003). This suggests a model in which the polarity of cytoskeletal advance within the growth cone reflects an asymmetry of Abl kinase activity in response to a graded distribution of Slit. Since multiple lines of genetic evidence show that CLASP cooperates with Abl and acts genetically downstream of the kinase, a model is favored where CLASP helps to induce cytoskeletal events that are needed to impede leading edge advance nearest the source of Slit, thus allowing relative advance at sites most distant from the source. This model predicts that elevation of CLASP activity will have a negative impact on growth cone extension and on microtubule advance. Xenopus overexpression experiments satisfy this prediction (Lee, 2004).

Since the entire growth cone perimeter must remain competent to respond to asymmetrically localized repellents in order to navigate through complex terrain, it is anticipated that CLASP activity rather than its localization will change in response to local kinase activation. Studies of the actin regulatory protein mammalian Enabled (Mena), which localizes to the tips of filopodia, suggest an analogous mechanism downstream of cyclic nucleotide-gated kinases to control filopodium formation. Abl regulates both CLASP and Enabled during axon guidance in Drosophila (Wills, 1999a and Bashaw, 2000), suggesting that independent cytoskeletal effectors must be coordinated to achieve accurate navigational choices. In this regard, it will be interesting to ask how CLASP interacts with key factors like the GTPase Rac1, which appears to be important in midline guidance and interfaces with CLIP-170 in nonneuronal cells. While many assume that the immediate effectors in axon guidance are actin regulators, data from nonneuronal systems suggest that microtubule dynamics can control actin assembly from the inside out (Lee, 2004 and references therein).

Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes

After centrosome duplication, centrioles elongate before the M phase. To identify genes required for this process and understand the regulatory mechanism, this study investigated the centrioles in Drosophila premeiotic spermatocytes, expressing fluorescently tagged centrioles. An essential microtubule polymerisation factor, Orbit/CLASP, was identified that accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically for determining centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. Loss of centriole integrity was observed, including fragmentation of centrioles and earlier separation of the centriole pairs in Cp110 null mutant cells either overexpressing Orbit or harbouring Klp10A depletion. Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they are unpaired. These findings contribute to understanding the mechanism of centriole integrity, leading to chromosome instability in cancer cells (Shoda, 2021).

The centrosome plays an indispensable role as the major microtubule-organising centre (MTOC) in a cell. During mitosis, centrosomes duplicated in the S phase move apart from each other and reach the opposite poles of the cell. Each centrosome is involved in the assembly of spindle poles, which enables construction of the bipolar spindle microtubule structure. A centrosome consists of two components: a pair of centrioles and the surrounding pericentriolar matrix (PCM). After replication of the centrioles, the longer centriole (mother centriole) engages with the shorter one (daughter procentriole) in a V-shape. When a cell enlarges in the G2 phase, the short daughter procentriole undergoes elongation to a certain length before the subsequent M phase. A single centriole consists of a microtubule doublet or triplet, which is equivalent to the cytoplasmic microtubule. Several factors localised on centrioles have been shown to be involved in the centriole elongation process. The most critical step in centrosome duplication is the duplication of centrioles, which requires stringent regulation. However, the entire mechanism underlying regulation of centriole elongation and the regulatory factors required for the process are not known. Among the centriole-associated proteins, those belonging to the kinesin-13 family are known to act as microtubule-severing kinesins. Klp10A, a Drosophila member of the family, has been shown to play an indispensable role in the regulation of centriole elongation. Based on these observations, it is speculated that some of the factors regulating microtubule length might overlap with those required for centriole elongation. It is possible that the production of centrioles of specific length requires a balance between polymerisation and depolymerisation of the triplet microtubules. However, the main factor(s) counteracting Klp10A and promoting centriole elongation remain to be identified (Shoda, 2021).

Another characteristic complex containing Cp110 is localised at the distal tip of the centriole, where it regulates the accessibility of the distal end to the shrinking and hypothetical lengthening factors, thereby regulating centriole elongation at this end. In the absence of Klp10A, the longer centrioles harbour incomplete ninefold symmetry at their ends in Drosophila cultured cells and tend to undergo fragmentation. Importantly, Cp110 depletion differentially affects centriole elongation in a species- and/or cell type-specific manner. In Drosophila S2 cultured cells, Cp110 depletion results in centriole length diminution. This effect is rescued by simultaneous depletion of Klp10A. In contrast, CP110 (also known as CCP110) depletion results in centriole elongation in mammalian cells. The centriolar microtubules were dramatically elongated in somatic cells, such as wing discs and larval brain cells, in the Drosophila Cp110-null mutant, whereas subtle elongation of the structure was observed in the premeiotic spermatocytes of the mutant (Shoda, 2021).

The premeiotic spermatocyte in Drosophila is a good model for investigating centrosomes and centrioles. Drosophila spermatogenesis involves four mitotic and two meiotic cycles for the formation of haploid spermatids. In the same spermatocyte cyst, each of the 16 cells undergoes synchronous cell growth, which can be divided into the S1 stage, corresponding to S phase, and five subsequent stages, S2 to S6, before initiation of meiosis I. The centrioles, in particular, can be studied more easily in this cell type, since these organelles dramatically elongate until the onset of meiosis and the centriole cylinder is composed of microtubule triplets. In early spermatocytes that possess a pair of centrioles initially, centrioles duplicate at S1 stage. As primary spermatocytes enter in the growth phase, centrioles migrate toward the surface where they assemble the primary cilium at the distal end of basal body. At the beginning of meiotic division I, centrioles move close to the nucleus with their associated 'membrane pocket' on the distal end of the cilium-like region (CLR). Between the CLR and the basal body there is the transition zone (TZ), which plays an important role in elongating the primary cilium of the spermatocyte. Centrioles are no longer duplicated between the two meiotic divisions. Primary spermatocytes hold two pairs of centrioles composed of nine triplet microtubules and engaged by a cartwheel structure at the proximal ends. The centriole pair is disengaged during prophase II, and, consequently, singlet centrioles organise the centrosomes of secondary spermatocytes (Shoda, 2021).

Previous studies have shown that Orbit (the Drosophila CLASP orthologue, encoded by chb) is essential for microtubule polymerisation, as it adds tubulin dimers to the plus end of the microtubules; however, its role in centriole elongation has not been examined. Hence, this study aimed to investigate whether Orbit was involved in centriole elongation in the mature premeiotic spermatocytes before male meiosis. As Orbit antagonises Klp10A, a severing factor determining the length of spindle microtubules in cultured Drosophila cells, whether Orbit was also involved in centriole length regulation was assessed (Shoda, 2021).

In addition, this study highlighted the importance of these regulators of centriole dynamics and the distal end capping proteins in the centriole elongation process using Drosophila spermatocytes. The importance of regulating the elongation of duplicated centrioles to a certain length for proper chromosome inheritance during male meiotic divisions is also discussed (Shoda, 2021).

Centrioles in Drosophila spermatocytes consist of ninefold triplets of microtubules. This study showed that the centrioles elongated to a certain length as the cells grew before male meiosis. Klp10A, which is a Drosophila kinesin-13 orthologue essential for shortening the microtubules, plays an indispensable role in regulation of centriole length. Microtubule length can be determined by the balance between polymerisation and depolymerisation of tubulin heterodimers and protofilaments. Thus, dynamic factors that promote microtubule elongation might also play critical roles in the determination of centriole length. This study has presented evidence suggesting that overexpression of Orbit results in excessively long centrioles in premeiotic spermatocytes. Conversely, shorter centrioles were observed in hypomorphic orbit mutants. These results are consistent with the idea that Orbit is essential for promotion of centriole elongation in spermatocytes. Orbit was initially identified as a microtubule-associated protein that adds tubulin heterodimers at the plus end of microtubules. Hence, the probability that Orbit might also elongate the triplet microtubules of centrioles by adding tubulin dimers, similar to its function in microtubule elongation, is high. In contrast, the kinesin-13 orthologue Klp10A acts as a microtubule depolymerising factor at the plus ends of microtubules in interphase and is an important regulator of centriole elongation. In Drosophila S2 cells, Klp10A antagonises Orbit in bipolar spindle formation and its maintenance. This study has shown that simultaneous overexpression of Orbit and depletion of Klp10A further enhanced centriole elongation. Based on these findings, it is proposed that these two factors act antagonistically to produce centrioles of specific length. Very long GFP-Orbit signals were observed that extended from basal bodies in spermatocytes overexpressing the protein. Accordingly, it is hypothesised that these are overly long axoneme microtubules produced as a consequence of excessively stimulated polymerisation of tubulins by the overexpression. Orbit, like other CLASP proteins, has a microtubule-binding activity to stimulate tubulin polymerisation at the plus end. Alternatively, a possibility cannot be excluded that Orbit polymerises itself to construct microtubule-like structures on the distal tip of the basal body. It would be interesting to investigate whether basal body and axonemal microtubules overly elongate in cells overexpressing Orbit without a fluorescent tag (Shoda, 2021).

In addition to these two factors regulating centriole microtubules, it is hypothesised that Cp110 plays a role as a cap to restrict these factors acting on the distal ends of the microtubules at an earlier stage. After Cp110 releases from the ends at mid-stage, Orbit can access the distal ends more easily and stimulate the centriole microtubules to extend to a certain length. Although the loss of the cap protein resulted in only a subtle extension of centrioles, Orbit overexpression in the absence of Cp110 can change the centriole microtubule dynamics significantly. Consequently, remarkably long centriole microtubules would be produced in the spermatocytes. Consistent with this hypothesis, Cp110Δ1 mutation also significantly enhanced the overly long centriole phenotype in Klp10A-depleted spermatocytes. By contrast, the Cp110-null mutation did not enhance the shorter centriole phenotype caused by Klp10A overexpression. Further experiments need to be performed to verify the model. The fact that Cp110 influences centriole length depending on the cell type and cellular context has been demonstrated in previous studies. It is likely that differential regulation of the conserved core components underlies ciliary basal diversity in different cell types. As argued previously, cellular-specific and tissue-specific regulation in centriole duplication may be indispensable to regenerate diverse centriole structures (Shoda, 2021).

Previous reports have not investigated whether Orbit and Klp10A are localised on centrioles or around the PCM in Drosophila mitotic cells; however, a considerable amount of Orbit accumulates in centrosomes in early embryos, Drosophila cultured cells and germline stem cells during Drosophila oogenesis and spermatogenesis. Whether the protein is localised in the PCM or in the centrioles is not clear. Similarly, studies on the cellular localisation of Orbit/CLASP orthologues in other species have also shown centrosome localisation of these proteins. Anti-human CLASP1 immunostaining in Hela cells has demonstrated that the protein is localised on centrosomes during M phase (Maiato et al., 2003). Furthermore, a CLASP orthologue in Caenorhabditis elegans has been observed in the centrosomes of its embryonic cells. These reports did not mention whether the orthologues were associated with the centrioles. Recently, it has been reported that Klp10A is dominantly localised in the TZ of the ciliary structures in spermatocytes, spermatids and sensory neurons (Persico, 2019). Centrosomal localisation of the protein has also been reported in mitotic cells and germline stem cells in both sexes. Whether Klp10A localises on the cylindrical microtubules of the centrioles or in the PCM of mitotic cells has not been demonstrated. Therefore, whether these antagonistic regulators are localised on centrioles in mitotic cells should be determined at a higher resolution. Furthermore, whether these two regulators are required for centriole length determination in somatic cells warrants further investigation (Shoda, 2021).

In addition to excessively elongated centrioles, this study observed several abnormalities in centriole organisation and structure in spermatocytes overexpressing Orbit and/or harbouring Klp10A depletion. In the absence of Cp110, the loss-of-centriole-integrity phenotypes were also enhanced. Small pieces of centrioles observed may be broken pieces of over-elongated centrioles, as observed in cancer cells. Alternatively, they may have been unpaired centrioles separated precociously from centriole pairs containing daughter procentrioles, which are smaller than normal centrioles. By contrast, the loss-of-centriole-integrity phenotypes were not observed in cells overexpressing a shortening factor, Klp10A. Hence, it is considered that improvised construction of the basal body microtubules may be associated with the loss-of-integrity phenotype; thereby, centriole engagement would be lost. Centriole fragments were found with reduced diameter in the centriole microtubules. The presence of disintegrated centrioles supports this idea, but further investigations are necessary to test the current hypothesis (Shoda, 2021).

Spermatocytes homozygous for loss-of-function mutation of Sas6 and of Ana2 commonly demonstrate premature centriole separation before meiosis. Hence, Sas6 and Ana2 are considered to be required for centriole engagement and/or maintenance of the pairs. Orbit overexpression and/or Klp10A depletion may affect centriole engagement through interfering with Sas6 (or Ana2) function. It is also possible that the premature disengagement can occur independently of Sas6 or Ana2. In addition, it has been reported that APC/C activation and activation of separate, thereby unexpected, cleavage of Scc1 (also known as RAD21) cohesin can take place in mammalian cultured cells. The possibility cannot be excluded that alteration of microtubule dynamics in centrioles by altered expression of Orbit and/or Klp10A led to unexpected APC/C activation. This hypothesis will be tested by several experiments in future work (Shoda, 2021).

Previous studies have also mentioned that Cp110-null mutant spermatocytes or syncytial-stage embryos do not show detectable defects in centrosome behaviour, spindle formation or chromosome segregation. By contrast, this study has show that disintegrated centrioles and multipolar spindle microtubules emanating from the centriolar fragments existed in Cp110-null mutant spermatocytes and cells overexpressing Orbit, as well as in Klp10A-depleted cells less frequently. Cells homozygous for the loss-of-function Klp10A mutation also display multipolar spindle structures. However, it cannot be excluded that the spindle phenotype would result from abnormal microtubule organisation caused by Klp10A mutation, rather than centriole disintegration. Surprisingly, in Cp110 mutants overexpressing Orbit and undergoing meiosis I, it was observed that unpaired single centrioles, even a part of them, could act as the MTOC. Centrosomes must be 'licensed' to function as an MTOC that nucleates microtubules, although the mechanism whereby the 'license' is granted remains unclear. Nevertheless, a recent study has reported that excessive elongation of centrioles in cancer cells is related to the generation of overduplicated, fragmented or hyperactive centrosomes that nucleate considerably more microtubules during cell division. Chromosome segregation is disturbed in cells harbouring these abnormal centrioles. Interestingly, generation of cells harbouring extra centrosomes has been suggested to be able to drive spontaneous tumorigenesis in mice. Additional studies have reported that excessively elongated centrioles in Drosophila spermatocytes affect spermatogenesis via the production of defective flagella. Consistently, immotile sperm production and significant decrease in male fertility were observed in the Cp110-mutant males with spermatocyte-specific Klp10A depletion. These observations suggested that production of excessively elongated centrioles can affect cell division and subsequent spermatogenesis. Once an abnormal spindle microtubule structure is constructed, the extra MTOC results in chromosome mis-segregation and eventually chromosome instability, as observed in cancer cells (Shoda, 2021).

Certain abnormalities such as mis-segregation of chromosomes and the resulting aneuploidy might appear in the subsequent division of cells containing abnormal centrioles. Conversely, excessively short centrioles may be inadequate as templates for the duplication of centrioles in S phase. Hence, regulation of centrosome length via the antagonistic activities of elongation and shrinking factors, such as Orbit and Klp10A, is important. However, the loss of centriole integrity and the resulting aneuploidy may occur in meiotic cells, but not in mitotic cells, which have stricter microtubule assembly checkpoints. Further investigations are required to understand how centrosome length is regulated in mitotic cells (Shoda, 2021).

Current findings suggest the presence of mutually antagonistic regulation to determine centriole length and the significance of the production of centrioles with a certain length for centriole integrity, and for assurance of proper chromosome segregation. It is believed that these findings may enable the identification of a mechanism whereby the loss of centriole integrity causes chromosome mis-segregation in cancer cells (Shoda, 2021).

GENE STRUCTURE

cDNA clone length - 5959 base pairs

Bases in 5' UTR - 769

Exons - 2

Bases in 3' UTR - 668

PROTEIN STRUCTURE

Amino Acids - 1402

Structural Domains

The sequence of orbit cDNAs has revealed that the gene encodes a protein of 1,492 amino-acids. The first ATG consistent with Drosophila translation initiation consensus is found at position 769 of the 5,959 nucleotide cDNA sequence. A poly(A) addition signal AATAAA lies at position 5,892. The novel protein contains a centrally located highly basic region (pI = 11.0) of 472 amino acids, flanked on both sides by short stretches of acidic residues. Within the basic domain are two consensus sites for phosphorylation by P34^cdc2, and two putative GTP-binding motifs. The motif GGGTGTG (residues 544-550) closely resembles the glycine-rich peptide that interacts with the guanine or phosphate groups of the bound GTP in ß-tubulin and in the Escherichia coli FtsZ protein. The sequence NKLD (residues 400-403) corresponds to the NKXD (X for any amino acid residues) consensus motif that can interact with the purine base of the bound nucleotide in the GTPase superfamily. A BLAST search with the Orbit protein sequence has revealed the presence of four closely related proteins from other organisms: two identified by the human putative open reading frame, KIAA0622 and KIAA0627, and two, R107.6 and ZC84.3, predicted from the C. elegans genome sequencing project. The homologies fall into two regions: HR1 lying between residues 290 and 1,068, and HR2 between zresidues 1,093 and 1,271. Since the regions of homology between the five proteins lie in register, it is likely that they are the signature of a family of related proteins. Moreover, the basic domain contained in the HR1 is a common feature of all five proteins, and the consensus sequences for cdc2 phosphorylation are found within or in the vicinity of this basic region in all except ZC84.3. The NKXD motifs are also conserved in the two human homologs. Basic domains are a characteristic of microtubule-binding proteins and in this context, it is of interest that one of the conserved motifs of HR1 (residues 326-350) shows considerable similarity to the sequence involved in the binding of human MAP4 to microtubules. Furthermore, another conserved sequence within the basic domain (residues 479-506) has similarity to a motif in Stu1, a MAP identified from budding yeast (Inoue, 2000).

Mast shares significant identity with proteins encoded by two human cDNAs (KIAA0622 and KIAA0627), three putative proteins in C. elegans (C07H6.3, R107.6 and ZC84.3) and also limited identity with Stu1p from S. cerevisiae (Pasqualone, 1994) and its putative homolog in S. pombe, termed SpStu1p. Multiple alignment of the Mast sequence with those most closely related from other species shows that all the proteins share identity throughout their sequence; however, three regions (CR-1, CR-2 and CR-3) are more highly conserved. These results suggest that Mast and its homologs define a new evolutionarily conserved protein family that has been named Stu1-Mast. Database searches also indicate that Mast shares identity with proteins from the dis1-TOG family, especially at the N-terminal half of the protein (amino acids 1-494), where they are 20%-25% identical and 40%-45% similar. Inside this region, there is a small domain of 18 amino acid residues that is highly conserved among these proteins and falls inside the first HEAT repeat of Mast. Phylogenetic analysis including all sequences from the two groups suggests that they are evolutionarily close, but distinct, since they are positioned in different branches of the dendrogram (Lemos, 2000).

date revised: 25 August 2021

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