InteractiveFly: GeneBrief

mars: Biological Overview | References

Microtubule-associated proteins (MAPs) ensure the fidelity of chromosome segregation by controlling microtubule (MT) dynamics and mitotic spindle stability. However, many aspects of MAP function and regulation are poorly understood in a developmental context. This study shows that mars, which encodes a Drosophila member of the hepatoma up-regulated protein family of MAPs, is essential for MT stabilization during early embryogenesis. As well as associating with spindle MTs in vivo, Mars binds directly to protein phosphatase 1 (PP1) and coimmunoprecipitates from embryo extracts with Minispindles and Drosophila Transforming acidic coiled-coil (dTACC), two MAPs that function as spindle assembly factors. Disruption of binding to PP1 or loss of mars function results in elevated levels of phosphorylated dTACC on spindles. A nonphosphorylatable form of dTACC is capable of rescuing the lethality of mars mutants. It is proposed that Mars mediates spatially controlled dephosphorylation of dTACC, which is critical for spindle stabilization (Tan, 2008).

Because disruption of mitotic spindle formation can promote genomic instability, an understanding of MAP function and regulation is central to dissecting basic mechanisms of tumorigenesis and would be invaluable in designing new therapies for the treatment of cancer. Although much progress has been made in understanding the functions of spindle-associated MAPs in the last few years, many aspects of their role or regulation remain to be fully elucidated. Human hepatoma up-regulated protein (HURP) has been described as a highly charged MAP that can bind directly to MTs in vitro and enhance their polymerization (Santarella, 2007). In vivo, HURP is part of a Ran-dependent complex that stabilizes mitotic MTs and is required for the formation and function of bipolar mitotic spindles (Koffa, 2006; Sillje, 2006; Wong, 2006). However, it is not known how HURP-associated proteins functionally interact with one another in a developmental context to support normal cellular function (Tan, 2008).

Mars, a Drosophila sequence homologue of HURP, is a protein phosphatase 1 (PP1) binding protein, implicating reversible phosphorylation in the control of Mars or Mars-associated proteins (Bennett, 2004; Yang, 2005). This paper reports the essential role of mars during early embryogenesis, its interactions with other MAPs, and its key role in promoting protein dephosphorylation on mitotic spindles to ensure spindle stability (Tan, 2008).

HURP is a component of the mitotic spindle apparatus. To determine the cell cycle distribution of Mars, a Mars-specific antibody was generated and used to stain syncytial embryos undergoing nuclear division. In prophase, Mars antibody staining is predominantly around the centrosome. In metaphase and anaphase, Mars is localized on spindle MTs in a gradient along the pole-to-pole axis with more intense staining at the centrosome-proximal regions. This is distinct from the distribution of HURP, which has been shown to be predominantly at chromatin-proximal regions until telophase, when levels sharply decline (Koffa, 2006; Sillje, 2006; Wong, 2006). In telophase, a low level of discrete Mars staining is observed at the midbody, but the majority of Mars protein appears to be spread over the nuclear envelope where it persists during interphase. Double staining for Mars and either Klp10A, which is primarily localized at focused minus ends where it promotes depolymerization and poleward flux, or γ-tubulin, which marks the face of the centrosome and nucleates MT polymerization, confirmed that Mars is localized at MT minus ends but not at the centrosome. Mars localization during mitosis is completely disrupted upon treatment with colchicine to depolymerize MTs, indicating that Mars associates with spindle MTs (Tan, 2008).

To determine the in vivo role of mars, a null allele of mars, mars¹, was generated by imprecise excision of a P element transposon (referred to as mars^P hereafter), which was found to be inserted in the mars 5' untranslated region. mars^P flies express full-length Mars protein at a much lower level than wild type. mars¹ flies fail to produce Mars protein, consistent with molecular analysis revealing that ~0.84 kb of the coding region, including the translation start site, is deleted in this mutant. mars mutant flies are viable but female sterile. Notably, eggs laid by mars¹, mars^P, and mars^1/P flies show a greatly reduced ability to hatch. Since no sperm tails were visible in early-arrested embryos laid by mars mutant females, it is concluded that the sterility observed in mars mutants is caused by arrest in embryogenesis after fertilization. The viability of embryos laid by mars mutant mothers was restored by moderate overexpression of mars^WT (wild-type mars) in the female germline, indicating that the failure of mars mutant embryos to develop is caused by disruption of the mars transcription unit (Tan, 2008).

To determine the cause of the lethality of mars mutant embryos, embryos from wild-type and mars mutant females were fixed, and the distribution of nuclei and MTs was examined. Embryos lacking maternal mars arrested during early embryogenesis after no more than five nuclear divisions. 82% of 15-45-min embryos laid by mars¹ mothers exhibited at least two discrete DNA-containing regions. The first of these was localized to the embryonic cortex and resembled a polar body, most likely containing the unused products of meiosis II. One or more additional DNA-containing regions, each surrounded by a bipolar spindle, were also observed more centrally, indicating that the vast majority of mutant embryos pass through meiosis to form one or more mitotic figure. Notably, the spindle structures in mars¹ mutant embryos were very small and weak, albeit still bipolar. Most spindles had at least one detached centrosome, possibly because of weakened spindle-centrosome interactions. Unaligned chromosomes were also observed in the mars¹ mutant, indicative of insufficient MT attachment or tension. Embryos laid by mars^1/P mothers resembled those from mars¹ mothers, except the phenotypes were somewhat less severe. Quantitation of α-tubulin staining revealed no significant difference between astral MTs in embryos laid by wild-type and mars^1/P mothers, whereas cold-resistant kinetochore MTs were destabilized in embryos laid by mars^1/P mothers. Approximately 51% of embryos laid by mars^P homozygous mothers failed to develop beyond embryogenesis. Many showed terminal phenotypes at or shortly after gastrulation, presumably as a consequence of primary defects during the early cleavage divisions. Collectively, analysis of loss-of-function mutants suggest that mars has a role in spindle MT stabilization. HURP has bundling activity in vitro and in vivo (Koffa, 2006; Sillje, 2006; Wong, 2006). Correspondingly, strong overexpression of mars^WT in syncytial embryos resulted in enlarged spindles with ectopic MT fibers, suggesting that mars is limiting for MT bundling (Tan, 2008).

mars was originally isolated from a two-hybrid screen for putative PP1 binding proteins and contains a canonical PP1 binding motif (K/R,x,V,x,F; Bennett, 2004; Bennett, 2006). To verify interactions with PP1, pulldown experiments were performed between Mars and PP1α87B, the major Drosophila PP1 isoform. GST-tagged full-length Mars^WT interacted efficiently with HA-tagged PP1α87B from crude Drosophila embryo extracts, whereas cells expressing GST alone did not bind (not depicted). Efficient binding was also observed between endogenous PP1 and FLAG-Myc (FM)-tagged Mars^WT by coimmunoprecipitation from Drosophila embryo extracts. To assess the importance of the putative PP1 binding motif for interaction with PP1, a mutant form of Mars was tested in which phenylalanine 839 was replaced with alanine (Mars^F839A) in the pulldown assay. The ability of Mars^F839A to bind PP1 was greatly reduced compared with wild-type Mars, indicating that Phe839 is crucial for interaction with PP1. Immunoprecipitation with an HURP antibody followed by immunoblotting with PP1 antibody showed that endogenous human PP1 and HURP also interacted efficiently with each other in HeLa cell extracts, suggesting that binding to PP1 is an evolutionarily conserved property of HURP proteins (Tan, 2008).

Binding to PP1 prompted a test of functional interactions between mars and PP1 in vivo. The ability of embryos laid by mars or PP1a87B heterozygotes to hatch resembled that of the wild type. Embryos laid by flies transheterozygous for mars and PP1a87B showed a significantly reduced hatch ratio. PP1 is a pleiotropic enzyme. To test the specific role of Mars-bound PP1, FM-tagged Mars^F839A was introduced into flies to create Mars complexes lacking PP1. Ectopic expression of mars^F839A at comparable levels to those of mars^WT failed to rescue the embryonic lethality of embryos laid by mars¹ or mars^1/P mothers. Collectively, these data suggest that binding to PP1 is critical for mars function. Identical distributions of Mars^F839A and Mars^WT, as determined by FLAG antibody staining, indicate that PP1 binding is not necessary for normal Mars localization on the mitotic spindle (Tan, 2008).

HURP has been shown to interact with other MAPs, including TPX2 and XMAP215 in Xenopus laevis (Koffa, 2006), suggesting that its role in the stabilization of spindle MTs may be partly mediated via interactions with other spindle assembly factors. In immunoprecipitation assays, it was found that Mars associates with two proteins that are known to cooperate with each other to stabilize MTs during cell division (Lappin, 2002): the Drosophila XMAP215 homologue encoded by minispindles (msps) and Drosophila transforming acidic coiled-coil (dTACC) protein. In syncytial embryos, dTACC is concentrated at centrosomes during mitosis but is also found on spindle MTs (Gergely, 2000) where it colocalizes with Mars. Mars staining was not affected in embryos that produce no detectable dTACC protein. Conversely, the ability of dTACC to associate with MTs was largely unaffected in mars^1/P mutant embryos. Therefore, although Mars and dTACC associate with one another, they do not appear to be dependent on each other for their localization (Tan, 2008).

Phosphorylation of dTACC on Ser863 is critical for stabilization of the minus ends of centrosome-associated MTs during mitosis (Barros, 2005). Although dTACC is found on both the centrosome and mitotic spindle, phosphorylated dTACC (p-dTACC) is tightly localized to the centrosomes (Barros, 2005), suggesting that once phosphorylated, p-dTACC is either unable to exchange with the soluble pool of dTACC or is rapidly dephosphorylated when it leaves the centrosome. The role of dephosphorylated TACC is not known, but it may function to stabilize MTs through lateral interactions with MTs or interactions with MT plus ends. The localization of Mars toward the minus ends of spindle MTs and association with both dTACC and PP1 prompted an examination of the involvement of Mars in maintaining low levels of p-dTACC on the spindle (Tan, 2008).

To examine the effect of Mars on dTACC phosphorylation, mars mutant embryos were stained with an antibody that specifically recognizes dTACC phosphorylated at Ser 863 (p-dTACC). mars mutant embryos showed increased levels of p-dTACC on the mitotic spindles compared with the wild type. On careful examination of these mutants, it was noticed some spindles that looked normal but possessed elevated levels of p-dTACC, indicating that increased p-dTACC was not simply a secondary consequence of aberrant spindle structure. Levels and distribution of total dTACC appeared normal in mars mutants, although it cannot be ruled out that global ratios of DTACC/p-DTACC are affected. mars^F839A was used to examine whether Mars promotes the dephosphorylation of dTACC by binding to PP1. Embryos with moderate levels of ectopic mars^WT in embryos laid by either mars¹ or mars^1/P mothers were essentially wild type in appearance and had little or no p-dTACC staining on mitotic spindles. In contrast, mars mutant embryos ectopically expressing mars^F839A at comparable levels to those of ectopic mars^WT retained elevated p-dTACC staining on spindles (Tan, 2008).

To test whether promoting the dephosphorylation of dTACC is a critical function of mars, whether the sterility of mars mutants could be rescued by a nonphosphorylatable form of dTACC (dTACC^S863L) expressed under control of the dTACC promoter (Barros, 2005) was tested. dTACC^S863L, but not dTACC^WT(wild-type dTACC), restored a normal distribution of p-dTACC staining on mitotic spindles. Quantification of p-dTACC staining confirmed that dTACC^S863L restored a normal ratio of spindle/centrosomal p-dTACC staining in mars mutant embryos. When hatching of these embryos was examined, it was found that the lethality of embryos laid by mars^1/P mothers was rescued by dTACC^S863L but not dTACC^WT. Collectively, these data indicate that dephosphorylation of dTACC on the spindle is an essential function of mars. Homozygous mars¹ mutants were not rescued by dTACC^S863L, suggesting that residual Mars protein in mars^1/P embryos may play a dTACC-independent role, such as MT bundling, or that the level of ectopic dTACC^S863L was insufficient to compensate for elevated p-dTACC in a mars¹ background (Tan, 2008).

In summary, this study has shown that mars, which encodes a Drosophila sequence homologue of HURP, is critical for mitotic spindle structure and chromosome segregation during early embryogenesis. The primary defect in mars mutants appears to be loss of spindle MT stability, whereas overexpression of mars leads to the production of enlarged spindles with ectopic MTs. These data are consistent with a role for mars in MT bundling/stabilization similar to that described for its human homologue HURP. However, identification of Mars as an interacting subunit of PP1 suggests a novel mechanism by which this family of proteins can maintain normal spindle structure in vivo. Binding of PP1 to Mars implicates PP1 in dephosphorylation of Mars or a Mars-associated protein (Tan, 2008).

Although dTACC may be a substrate of PP1, it is also possible that Mars-bound PP1 may indirectly stimulate dephosphorylation of dTACC on the spindle by activating another protein phosphatase or inactivating a dTACC kinase such as Aurora-A, a known target of PP1 during mitosis (Tan, 2008).

Genetic experiments indicate that promoting dephosphorylation of dTACC on mitotic spindles is an essential role of Mars. Why is it important to maintain dephosphorylated TACC on the spindle? One possibility is that mars functions to ensure that MT stabilization mediated by p-dTACC occurs only at the centrosome, allowing a more dynamic spindle. This is hard to test however, because the mechanism by which p-dTACC stabilizes MTs is unclear. The effect of dTACC on MT assembly appears to be mediated by effector proteins, such as Msps, since TACC has not been described to possess MT stabilizing activity on its own (Kinoshita, 2005). Phosphorylation of dTACC might help activate effectors because dTACC mutated at Ser863 is able to recruit Msps to the centrosome but not promote MT assembly (Barros, 2005). However, it is not known which aspect of Msps activity is affected by dTACC phosphorylation or to what extent the effect of dTACC phosphorylation is context dependent. It is conceivable that dTACC stabilizes spindle MTs by establishing lateral interactions with MTs or interactions with plus ends and that these functions of dTACC are impaired when phosphorylated at Ser863 (Tan, 2008).

Is mars a functional homologue of HURP? It was confirmed that various aspects of mars and HURP function are conserved, including spindle stabilization and binding of Mars to Msps and PP1. However, Mars and HURP display apparently distinct spindle localizations, suggesting that there may be differences in how these proteins are used during cell division. This may reflect a wider difference in the organization of MAPs that control MT stability and the formation of bipolar spindles in flies and humans (Tan, 2008).

Spindle defects caused by lack of TACC phosphorylation or by alterations in TACC or HURP protein levels may lead to genetic instability and are implicated in cancer progression. The data indicate that spatially controlled dephosphorylation also plays a positive role in TACC function, suggesting that deregulation of either phosphorylation or dephosphorylation of TACC may also be involved in the molecular pathology of cancer by compromising the fidelity of chromosome segregation (Tan, 2008).

Mars, a Drosophila protein related to vertebrate HURP, is required for the attachment of centrosomes to the mitotic spindle during syncytial nuclear divisions

The formation of the mitotic spindle is controlled by the microtubule organizing activity of the centrosomes and by the effects of chromatin-associated Ran-GTP on the activities of spindle assembly factors. This study shows that Mars, a Drosophila protein with sequence similarity to vertebrate hepatoma upregulated protein (HURP), is required for the attachment of the centrosome to the mitotic spindle. More than 80% of embryos derived from mars mutant females do not develop properly due to severe mitotic defects during the rapid nuclear divisions in early embryogenesis. Centrosomes frequently detach from spindles and from the nuclear envelope and nucleate astral microtubules in ectopic positions. Consistent with its function in spindle organization, Mars localizes to nuclei in interphase and associates with the mitotic spindle, in particular with the spindle poles, during mitosis. It is proposed that Mars is an important linker between the spindle and the centrosomes that is required for proper spindle organization during the rapid mitotic cycles in early embryogenesis (Zhang, 2009).

The proper establishment and maintenance of the bipolar mitotic spindle is essential for the equal segregation of genetic material into the daughter cells. Any defect in this process can result in aneuploidy, which is often associated with tumorigenesis. Currently, two mechanisms have been proposed for the formation of the bipolar mitotic spindle in eukaryotic cells. The stochastic 'search and capture' model proposes that the centrosomes nucleate microtubules, which capture the kinetochores of chromosomes from both ends to establish the bipolar spindle. The second mechanism is microtubule nucleation and growth in the vicinity of condensed chromatin, in which Ran-GTP is required as a crucial regulator. These two mechanisms may operate in parallel to different extents in different types of cells (Zhang, 2009 and references therein).

Several microtubule-associated proteins have been identified in vertebrate cells that are required for the efficient assembly of the spindle. The nuclear mitotic apparatus protein (NuMA), together with cytoplasmic dynein and dynactin, accumulates at spindle poles at mitosis, focuses microtubule minus ends and tethers centrosomes to the body of the spindle. The targeting protein for Xenopus kinesin-like protein 2 (TPX2), targets Xklp2 to microtubule minus ends during mitosis and the kinase Aurora A to the spindle. TPX2 is also involved in spindle pole organization and centrosome integrity. Hepatoma upregulated protein (HURP), localized to kinetochore microtubules in immediate proximity to the chromosomes, increases the efficiency of chromosome capture by microtubule stabilization during mitosis. The activities of NuMa, TPX2 and HURP are all regulated by high Ran-GTP concentration around chromosomes, which liberates these factors from inhibition by binding to members of the importin β superfamily (Zhang, 2009 and references therein).

In Drosophila, the minus end directed microtubule motor cytoplasmic dynein is required for spindle pole organization and centrosome attachment to the nuclear envelope and to the mitotic spindle, as in vertebrate cells. By contrast, neither NuMa nor TPX2 have obvious structural homologs in Drosophila. The Mushroom body defect (Mud) protein shows limited sequence similarity to NuMa and was shown to bind Pins, the fly homolog of the NuMa binding partner Lgn. Mud is required for correct spindle orientation in neuroblasts and for meiosis II in female flies, but a function in spindle pole organization has not been demonstrated so far. The protein encoded by abnormal spindle (asp) localizes to microtubule minus ends at metaphase spindle poles and is required for focussing of spindle poles. Due to these properties, Asp has been discussed as a functional Drosophila homolog of vertebrate NuMa and TPX2 (Zhang, 2009 and references therein).

In order to get a more comprehensive picture of the microtubule-associated factors that are required for the proper execution of mitosis in Drosophila, focus was placed on Mars, the closest relative of vertebrate HURP (Bennett, 2004). Previous studies showed that Mars is enriched in mitotic cells and that overexpression of Mars in the eye imaginal disc caused mitotic defects (Yang, 2005). However, the precise subcellular localization and function of Mars were unknown. This study shows that Mars is a microtubule-associated protein that translocates from the nucleus to the mitotic spindle in mitosis and is enriched at spindle poles. Loss-of-function mutants of mars are viable and fertile. However, more than 80% of embryos laid by mars homozygous mutant females show severe mitotic defects during the synchronous nuclear divisions at early blastoderm stages. Based on these results, it is proposed that Mars is required for centrosome attachment to the mitotic spindle and to the nuclear envelope (Zhang, 2009).

In most cell types, centrosomes are tightly linked to the nuclear envelope in interphase and localize to the spindle poles in mitosis. The attachment of the centrosome to the nuclear envelope and to the mitotic spindle is generally thought to result from the interaction of microtubules nucleated at the centrosome with microtubule-associated proteins located either at the nuclear envelope or at the minus ends of spindle microtubuli. In mars mutant embryos at the syncytial blastoderm stage, centrosomes frequently detached from nuclei and from mitotic spindles, pointing to a function of Mars in linking centrosomal microtubules to the nuclear envelope and to spindle microtubules. Like attached centrosomes in wild type, the free centrosomes in mars mutant embryos showed immunoreactivity for γ-tubulin, Cnn, Aurora A and DTACC. The free centrosomes retained their capacity to nucleate microtubules and continued to duplicate and separate, resulting in numerous microtubule asters detached from nuclei. Similar observations have been reported for other situations that result in the formation of free centrosomes. Most likely as a secondary consequence of the centrosome detachment, different types of mitotic defects accumulated in mars mutant embryos, including monopolar spindles with circular condensed chromosomes, multipolar spindles and short anastral spindles that were probably organized by the nucleation of microtubules around chromosomes. Thus, the function of Mars is apparently not strictly required for the normal assembly and microtubule-nucleating activity of centrosomes, but rather for the interaction of the centrosomal microtubules with the nuclear envelope and the spindle microtubules (Zhang, 2009).

A very similar phenotype has been described for Dhc64C mutant embryos. In these mutants, centrosomes also detached from the nuclear envelope and from mitotic spindles. It has been proposed that dynein associated with the nuclear envelope might be required for attachment of centrosomal microtubules. During mitosis, dynein at the centrosome could be necessary to link spindle microtubules to astral microtubules. This study has shown that the spindle pole localization of Mars was unaffected in a hypomorphic allelic combination of Dhc64C mutants. This could either mean that dynein is indeed not required for localization of Mars to the minus ends of microtubules or that the levels of dynein still produced from the hypomorphic Dhc64C alleles are sufficient for proper localization of Mars. Nonetheless, the intriguing similarity of the mars and Dhc64C mutant phenotypes suggests the existence of a functional link between these two proteins (Zhang, 2009).

One surprising finding of this work is the fact that homozygous mars⁹¹ mutant flies are viable and even fertile, in spite of the dramatic mitotic defects in more than 80% of mutant embryos. This could be most easily explained if mars⁹¹ was a hypomorphic and not an amorphic or null allele. For several reasons this is thought to be very unlikely: (1) the phenotype of heterozygous mars⁹¹/Df(2R)CX1 embryos is indistinguishable from the phenotype of mars⁹¹ homozygous mutant embryos, which is a classical genetic criterion for its classification as an amorphic mutation; (2) the mars⁹¹ deletion removes the ATG start codon of the gene. Although apparently an N-terminally truncated form of Mars can be translated in this allele starting from an ATG downstream of the 3' breakpoint of the deletion, this truncated form lacks the N-terminal region of Mars required for spindle localization and thus is presumably nonfunctional. Consistent with this, no localized staining for Mars was detected in the mars⁹¹ homozygous mutant embryos. A second recently published null allele of mars causes phenotypes essentially identical to the ones reported in this study, but these embryos never develop beyond the fifth nuclear division cycle (Tan, 2008). Whether this apparent discrepancy in the lethality of the two alleles is caused by some minor residual function still preserved in the mars⁹¹ allele or by some differences in the genetic background of both alleles remains to be shown (Zhang, 2009).

Based on these results it is thought that Mars is specifically required for spindle organization during the rapid cleavage divisions in the early Drosophila embryo but becomes dispensable later in embryonic, larval and adult development. The same finding was made for centrosomes, which, quite surprisingly, are not essential for mitosis at later developmental stages. Consistent with this interpretation, this study and work of others (Goshima, 2007) did not observe any dramatic increase of mitotic spindle defects after knockdown of Mars by RNAi in S2 cells compared to controls. However, a recent study quantified defects in mitotic spindle formation after RNAi-mediated knockdown of Mars in S2r cells and found a statistically significant increase in spindles with abnormal kinetochore microtubules (Yang, 2008). Thus, although Mars does not appear to be essential for proper spindle formation after the rapid cleavage divisions, it may contribute to the efficient formation of kinetochore microtubules at later developmental stages (Zhang, 2009).

Homology searches using the BLAST algorithm revealed that the closest vertebrate relative of Mars is the spindle-associated protein HURP (Yang, 2005). However, analysis of Mars localization and mutant phenotype reveals that those two proteins may have at least partially different functions in spindle organization. HURP was identified as a component of a Ran-dependent complex in Xenopus egg extract, which also contains Eg5, TPX2, XMAP215 and Aurora A (Koffa, 2006). Upon depletion of HURP, HeLa cells showed a delayed transition from prophase to anaphase with frequent misalignment of chromosomes at the metaphase plate (Koffa, 2006; Sillje, 2006; Wong, 2006). These data indicate that HURP stabilizes K-fibers and is required for the efficient capture of kinetochores by spindle microtubules. Whether Mars has a similar function in chromosome alignment at the metaphase plate is difficult to answer due to the severe mitotic defects resulting from centrosome detachment (Zhang, 2009).

The subcellular localization of HURP is under control of the Ran-GTP gradient originating from the chromosomes. Ran-GTP negatively regulates the binding of HURP to the nuclear import receptor importin β, which in turn prevents its interaction with microtubules (Sillje, 2006). In mitosis, HURP is associated with the spindle and is enriched in the part of the spindle that is close to the chromosomes (Koffa, 2006; Sillje, 2006; Wong, 2006). During interphase, HURP levels are strongly reduced and the protein is mainly found in the cytosol, with low amounts detectable in the nucleus (Sillje, 2006). By contrast, Mars associates with spindle poles, is not enriched in proximity to the chromosomes in mitosis and is localized in the nucleus in interphase. These results suggest that the subcellular localization of Mars to the spindle poles may be independent from Aurora A, in contrast to HURP, for which phosphorylation of its C-terminal region by Aurora A is required for the association with microtubules (Wong, 2008). Again, the possibility cannot be excluded that the low levels of Aurora A activity present in embryos homozygous for the hypomorphic aurA²⁸⁷ allele are sufficient for proper localization of Mars. In spite of these differences, the microtubule-binding activity of both HURP and Mars resides in the N-terminal region of both proteins (Wong, 2008; Zhang, 2009).

The subcellular localization and loss-of-function phenotype of Mars shows striking similarities to the vertebrate Ran-GTP-regulated proteins TPX2 and NuMA. Both proteins are required to ensure normal spindle morphology and spindle pole integrity. Upon knockdown of TPX2, mitotic cells form multipolar spindles in HeLa cells. In Xenopus egg extract, the depletion of TPX2 causes less compact spindles and a variety of spindle pole defects. The regulation of TPX2 activity occurs via its binding to importin α, which is mutually exclusive with the binding to microtubules and is regulated by Ran-GTP. Very interestingly, TPX2 was found in a complex together with Aurora A, Eg5, XMAP215 and HURP (Koffa, 2006). TPX2 is required for targeting Aurora A to the spindle and HURP is a phosphorylation target of Aurora A (Yu, 2005; Wong, 2008), revealing a functional interaction between TPX2 and HURP (Zhang, 2009).

The second vertebrate protein that resembles Mars with respect to its subcellular localization and loss-of-function phenotype is NuMa. This protein interacts with the dynein-dynactin complex and is required for the focussing of spindle poles and for the tight attachment of centrosomes to the spindle. Because the phenotype of mars mutants is very similar to the phenotype of cytoplasmic dynein heavy chain mutants and no function in spindle pole focussing and centrosome attachment has been described for Mud, a potential NuMa homolog in Drosophila, it is speculated that Mars may be a Drosophila counterpart to NuMa and TPX2 with respect to its function in spindle organization (Zhang, 2009).

Due to its mutant phenotype and its subcellular localization, the Asp protein of Drosophila has been discussed as a potential functional equivalent to NuMa and TPX2 (Manning, 2008). In asp mutants, spindle poles are disorganized and centrosomes frequently detach from the mitotic spindle, leading to the formation of free centrosomes. The subcellular localization of Asp overlaps with Mars at spindle poles, but in contrast to Mars, Asp is also localized to centrosomes in mitosis and is enriched at the side of the centrosome facing the spindle microtubules. Thus, Mars and Asp may have related and possibly redundant functions in spindle pole focussing and attachment of centrosomes to the spindle. The genetic interaction studies strongly support this interpretation. Flies doubly mutant for mars and asp were never obtained, but one intact copy of either mars or asp is sufficient for development to adulthood (Zhang, 2009).

This work has identified Mars as an important regulator of mitotic spindle organization in Drosophila. Although by sequence similarity Mars is the closest homolog to vertebrate HURP, functional and immunohistochemical analysis suggests that Mars may also be required for functions provided in vertebrate cells by NuMa and TPX2. Future work on the regulation of Mars by Ran, mitotic kinases and microtubule-dependent motor proteins will shed more light on its function in the assembly of the mitotic spindle (Zhang, 2009).

Search PubMed for articles about Drosophila Mars

Barros, T.P., Kinoshita, K., Hyman, A. A. and Raff, J. W. (2005). Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules. J. Cell Biol. 170: 1039-1046. PubMed ID: 16186253

Bennett, D., and Alphey, L. (2004). Cloning and expression of mars, a novel member of the guanylate kinase associated protein family in Drosophila. Gene Expr. Patterns 4: 529-535. PubMed ID: 15261830

Bennett, D., Lyulcheva, E. and Alphey, L. (2006). Towards a comprehensive analysis of the protein phosphatase 1 interactome in Drosophila. J. Mol. Biol. 364: 196-212. PubMed ID: 17007873

Gergely, F., Kidd, D., Jeffers, K., Wakefield, J. G. and Raff, J. W. (2000). D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo. EMBO J. 19: 241-252. PubMed ID: 10637228

Goshima, G., Wollman, R., Goodwin, S. S., Zhang, N., Scholey, J. M., Vale, R. D. and Stuurman, N. (2007). Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316: 417-421. PubMed ID: 17412918

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

Koffa, M. D., et al. (2006). HURP is part of a Ran-dependent complex involved in spindle formation. Curr. Biol. 16: 743-754. PubMed ID: 16631581

Lappin, T. R., et al. (2002). AINT/ERIC/TACC: an expanding family of proteins with C-terminal coiled coil domains. Leuk. Lymphoma. 43: 1455-1459. PubMed ID: 12389629

Manning, A. L. and Compton, D. A. (2008). Structural and regulatory roles of nonmotor spindle proteins. Curr. Opin. Cell Biol. 20: 101-106. PubMed ID: 18178073

Santarella, R.A., et al. (2007). HURP wraps microtubule ends with an additional tubulin sheet that has a novel conformation of tubulin. J. Mol. Biol. 365: 1587-1595. PubMed ID: 17118403

Sillje, H. H., Nagel, S., Korner, R. and Nigg, E. A. (2006). HURP is a Ran-importin beta-regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes. Curr. Biol. 16: 731-742. PubMed ID: 16631580

Tan, S., Lyulcheva, E., Dean, J. and Bennett, D. (2008). Mars promotes dTACC dephosphorylation on mitotic spindles to ensure spindle stability. J. Cell Biol. 182(1): 27-33. PubMed ID: 18625841

Wong, J. and Fang, G. (2006). HURP controls spindle dynamics to promote proper interkinetochore tension and efficient kinetochore capture. J. Cell Biol. 173: 879-891. PubMed ID: 16769820

Wong, J., Lerrigo, R., Jang, C. Y. and Fang, G. (2008). Aurora A regulates the activity of HURP by controlling the accessibility of its microtubule-binding domain. Mol. Biol. Cell 19: 2083-2091. PubMed ID: 18321990

Yang, C.P., et al. (2005). Using Drosophila eye as a model system to characterize the function of mars gene in cell-cycle regulation. Exp. Cell Res. 307: 183-193. PubMed ID: 15922738

Yu, C. T., Hsu, J. M., Lee, Y. C., Tsou, A. P., Chou, C. K. and Huang, C. Y. (2005). Phosphorylation and stabilization of HURP by Aurora-A: implication of HURP as a transforming target of Aurora-A. Mol. Cell. Biol. 25: 5789-5800. PubMed ID: 15987997

Zhang, G., Breuer, M., Foörster, A., Egger-Adam, D. and Wodarz, A. (2009). Mars, a Drosophila protein related to vertebrate HURP, is required for the attachment of centrosomes to the mitotic spindle during syncytial nuclear divisions. J. Cell Sci. 122(Pt 4): 535-45. PubMed ID: 19174464

date revised: 5 September 2009

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