InteractiveFly: GeneBrief

transforming acidic coiled-coil protein: Biological Overview | References

BIOLOGICAL OVERVIEW

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) is 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 (Giet, 2002; Pascreau, 2005). TACC proteins stabilize spindle MTs in flies (Gergely, 2000; Lee, 2001), humans (Gergely, 2003), worms (Bellanger, 2003; Le Bot, 2003; Srayko, 2003), and frogs (O'Brien, 2005) 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 (Giet, 2002). 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).

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

The Aurora A-dependent localization of D-TACC to centrosomes prompted an examination of whether the two proteins show any physical interactions. To this end anti-Aurora A antibodies were used to immunoprecipitate Aurora A protein from Drosophila embryo extracts and the precipitate was analyzed for the presence of both Aurora A and D-TACC proteins by Western blotting. The presence of D-TACC in the Aurora A immunoprecipitate indeed suggests that the proteins interact either directly or via intermediate proteins. The reciprocal experiment was performed and D-TACC was immunoprecipitated from the extract, no Aurora A was detected in the precipitate. One possible interpretation of this result is that the anti-D-TACC antibody might interfere with the D-TACC-Aurora A interaction. Therefore, the experiment was repeated using a Drosophila line expressing a GFP-D-TACC fusion protein under the control of the polyubiquitin promoter and to perform immunoprecipitation using anti-GFP antibodies in an attempt to avoid possible antibody interference. GFP-tagged D-TACC protein can be immunoprecipitated from the extracts of such embryos, and Aurora A kinase is present in this immunoprecipitate. However, Aurora A was not found in the immunoprecipitate obtained using the same anti-GFP antibody on the extract from embryos expressing a Tau-GFP fusion protein. Together, these experiments demonstrate an interaction between the Aurora A and D-TACC proteins in extracts of wild-type embryos (Giet, 2002).

If this interaction has functional significance, it might be expected to have been conserved through evolution. Therefore, whether the human counterpart of Aurora A shows association with HsTACC3, the closest human counterpart to D-TACC, was examined. Just as in Drosophila cells, an immunoprecipitate of Hs Aurora A contained HsTACC3. Moreover, immunostaining has revealed both proteins to colocalize around centrosomes in cultured human cells (Giet, 2002).

The syncytial Drosophila embryo is provided with a rich dowry of maternal proteins that enable it to undertake the 13 rapid cycles of nuclear division before cellularization. Therefore, any collection of embryos will have not only representation of different cell cycle stages, but also of differing extents of development to which the maternally provided mitotic proteins make an increasing contribution. Thus, it was necessary to determine whether the interaction between Aurora A and D-TACC was mirrored by a major multisubunit complex that could represent either a maternal store or some other functional unit. Therefore, an extract was fractionated from a 2-h collection of predominantly syncytial embryos by sedimentation on a 5%-40% sucrose gradient. Western blotting of the fractions revealed that D-TACC sediments at 6-8 s, suggesting it is present in a complex with MSPS as shown by Lee (2001). The small, previously described complex of gamma-tubulin [thought to be a heterotetramer of two molecules of gamma-tubulin, and one molecule each of Dgrip84 and Dgrip91], by comparison sediments slightly ahead at 7 s. Aurora A, in contrast, sediments predominantly at 3-4 s, showing only a small proportion cosedimenting with the D-TACC complex. Thus, it seems that there are substantial maternal pools of both proteins, but they are not stockpiled in the same multiprotein complex, suggesting that only a small fraction of Aurora A interacts with the D-TACC complex (Giet, 2002).

Nevertheless, since immunoprecipitation experiments have suggested that the two proteins can associate, it was of interest to see whether Aurora A is also able to phosphorylate D-TACC. This was tested in two ways. In the first, GFP-tagged D-TACC protein was immunoprecipitated from Drosophila embryos and then the fusion protein was subjected to a heat treatment, to inactivate any endogenous protein kinases before incubating it with a preparation of active recombinant Aurora A-(His)6 protein kinase and radiolabeled ATP. The products of the kinase reaction were analyzed by SDS-PAGE on either a 10% or a 6% gel, followed by autoradiography. Western blotting was performed on the same membrane where the kinase reaction products were analyzed by autoradiography. When Aurora A kinase is added, the appearance of several phosphorylated bands was detected. These bands comigrate exactly with the GFP-D-TACC protein and its degradation products when observed by autoradiography. In a second test of whether D-TACC is an Aurora A substrate, bacterially expressed and purified fusion proteins between Maltose Binding protein and different domains of the D-TACC protein were used. It was found that Aurora A kinase phosphorylates the NH2- or the COOH-terminal domains of D-TACC only very poorly. In contrast, the middle fragment of the protein is a highly favored substrate. Thus, it appears that not only does D-TACC require active Aurora A kinase for its localization to the centrosome, but it is also a good in vitro substrate of the enzyme (Giet, 2002).

TACC is required for normal spindle function in the early Drosophila embryo

Drosophila TACC (D-TACC) is a novel protein that is concentrated at centrosomes and interacts with microtubules. D-TACC is essential for normal spindle function in the early embryo; if D-TACC function is perturbed by mutation or antibody injection, the microtubules emanating from centrosomes in embryos are short and chromosomes often fail to segregate properly. The C-terminal region of D-TACC interacts, possibly indirectly, with microtubules, and can target a heterologous fusion protein to centrosomes and microtubules in embryos. This C-terminal region is related to the mammalian transforming, acidic, coiled-coil-containing (TACC) family of proteins (Gergely, 2000b). The function of the TACC proteins is unknown, but the genes encoding the known TACC proteins are all associated with genomic regions that are rearranged in certain cancers. At least one of the mammalian TACC proteins appears to be associated with centrosomes and microtubules in human cells. It is proposed that this conserved C-terminal 'TACC domain' defines a new family of microtubule-interacting proteins (Gergely, 2000a).

D-TACC is a novel centrosomal protein that can associate with microtubules. Although it binds strongly to microtubules in embryo extracts, full-length D-TACC, translated in vitro, binds only weakly, if at all, to purified microtubules. Purified fusion proteins containing the N-terminal, middle or C-terminal regions of D-TACC also do not bind strongly to purified microtubules, although a small fraction of the C-terminal fusion proteins reproducibly co-pellets with the microtubules. If, however, the C-terminal fusion proteins are mixed with embryo extract, then they strongly interact with microtubules, suggesting that D-TACC interacts with microtubules via its C-terminal region, and that this interaction requires other factors present in the embryo extracts. These factors in the extract may modify D-TACC to enable it to bind to microtubules, or they may bind to microtubules in a complex with D-TACC. The latter possibility is favored, since gel filtration and velocity sedimentation analysis of the endogenous D-TACC in embryo extracts suggest that D-TACC may be part of a larger complex (our unpublished observations) (Gergely, 2000a).

Although D-TACC can interact with microtubules, its concentration at centrosomes may be independent of this interaction; it remains concentrated at centrosomes in embryos where the microtubules have been depolymerized with colchicine. It cannot, however, be excluded that some short microtubules remain associated with centrosomes under these conditions. Moreover, the same C-terminal region of D-TACC that interacts with microtubules in embryo extracts can also target a heterologous fusion protein to centrosomes. Thus, whether D-TACC associates with centrosomes independently of microtubules is still uncertain (Gergely, 2000a).

To investigate the function of D-TACC, a mutation was isolated in the D-TACC gene (D-TACC¹). Flies homozygous for D-TACC¹, or transheterozygous for D-TACC¹ and a deficiency that uncovers d-tacc, are viable but female sterile. Centrosomal microtubules at all stages of the cell cycle are often shorter than normal in D-TACC1 embryos. Pronuclear fusion, nuclear migration and chromosome segregation are also often defective in these embryos; since all of these processes are thought to require centrosomal microtubules, they are thought to be defective as a secondary consequence of the abnormally short centrosomal microtubules (Gergely, 2000a).

Since D-TACC1 embryos almost always develop severe mitotic defects prior to the migration of the nuclei to the cortex, it is not possible to observe microtubules directly at the cortex of living D-TACC1 embryos. Therefore labelled anti-D-TACC antibodies were injected into syncytial blastoderm embryos that expressed a tau-GFP fusion protein, allowing the behaviour of the antibodies and microtubules to be followed in real-time in living embryos. In these embryos, the injected antibodies bind to the centrosomes, and the spindles that form around the injection site are shorter than normal. Although chromosomes can move to the poles on these spindles, the chromosomes are often insufficiently separated on the short spindles and polyploid nuclei often form. These effects appear to be specific, since injecting labelled antibodies against CP60 (another centrosomal protein that binds to microtubules does not cause similar defects, even though the antibodies bind strongly to centrosomes (Gergely, 2000a).

Taken together, these results suggest that D-TACC is normally required to organize or stabilize centrosomal microtubules. Although it is not clear how D-TACC functions, the interaction of the protein with microtubules and its concentration at centrosomes suggest that this effect is likely to be direct. One possibility is that D-TACC stabilizes microtubules that are nucleated from the centrosome. It is currently thought that microtubules are nucleated from γ-tubulin ring complexes. There is increasing evidence, however, that at least some of these microtubules can be released from their centrosomal nucleation sites, and, in many cell types, the released microtubules can be held in the vicinity of the centrosome by the action of microtubule motor proteins. It is not clear how the minus ends of such free microtubules are stabilized in the cell. Perhaps D-TACC is involved in this stabilization. In support of this possibility, D-TACC-GFP becomes slightly concentrated in the area of the mitotic spindle where the minus ends of the microtubules are clustered near to, but slightly detached from, the centrosomes. γ-tubulin has a similar distribution in methanol-fixed embryos, raising the possibility that D-TACC might interact preferentially with the minus ends of microtubules, as is thought to be the case for γ-tubulin. No direct evidence, however, is availabel that D-TACC interacts preferentially with the minus ends of microtubules, and there are several other possible explanations for these results. D-TACC could, for example, be required to nucleate centrosomal microtubules, although D-TACC is unlikely to be a component of the γ-TuRC as there are apparently no proteins of the appropriate size in these complexes. Alternatively, D-TACC could somehow serve to prevent the premature release of microtubules from the centrosome, perhaps by regulating the activity of centrosomal microtubule-severing activities (Gergely, 2000a).

Is D-TACC only required in the early embryo? The d-tacc¹ mutation is viable but female sterile, and several other female sterile alleles of d-tacc have been isolated independently by other groups on the basis of their mitotic and pronuclear fusion defects. These findings suggest that D-TACC may only be essential in the early embryo, perhaps because the rapid mitoses and large spindles of the early embryo impose a more stringent requirement for long spindle microtubules. In support of this possibility, the abnormally short spindles in the antibody-injected embryos are always delayed in exiting mitosis, suggesting that they transiently activate the spindle assembly checkpoint. Eventually, however, these spindles complete mitosis, although chromosome segregation often fails on the short spindles. Perhaps, at other stages of development, when mitosis is slower and the spindles no longer occupy a common cytoplasm, the activation of the spindle assembly checkpoint may allow the short spindles time to compensate for the loss of D-TACC function and complete mitosis successfully. In contrast, d-tacc¹ mutation is unlikely to be a functional null since the mutants produce an apparently full-length protein but at only 10% of normal levels, and the protein is concentrated at centrosomes. The D-TACC protein is detectable at all stages of fly development, and it is possible that a null mutation would be lethal (Gergely, 2000a).

The C-terminal ~200 amino acids of D-TACC are predicted to form a coiled-coil structure, and this region is related to the mammalian transforming, acidic, coiled-coil-containing family of proteins. Although the sequence similarity between D-TACC and the three known mammalian TACC proteins is relatively weak (~25-30% identity in the ~200 amino acid C-terminal region), it is believed that it is functionally significant for three reasons.(1) D-TACC is significantly more homologous to the known TACC proteins than to any other coiled-coil protein in the databases. (2) Although the mammalian TACC genes appear to have been generated by gene duplication events, the sequences of the known TACC proteins outside of this putative coiled-coil region are poorly conserved between family members. Thus, it is perhaps not surprising that D-TACC is only related to the mammalian proteins in this region. (3) TACC2 appears to have a very similar distribution in human cells to that of D-TACC in Drosophila cells, and the conserved C-terminal region of D-TACC appears to be important in determining this distribution in Drosophila cells. The simplest interpretation of these results is that this conserved region targets D-TACC and TACC2 to centrosomes and microtubules, and it is speculated that the TACC proteins will all interact with microtubules via this conserved C-terminal domain. Currently investigations are underway to determine whether this is the case (Gergely, 2000a).

Although the functions of the mammalian TACC proteins are unknown, the known TACC genes all map to regions of chromosomes that are rearranged in certain cancers, and it has been proposed that alterations in TACC gene function may contribute to tumorigenesis. All of the known TACC genes, however, are closely linked to fibroblast growth factor receptor (FGFR) genes (presumably the consequence of linked gene duplication events), and these genes are also attractive candidates for any gene(s) in these regions whose disruption could contribute to tumorigenesis. Thus, a direct link between the TACC genes and tumorigenesis remains to be established (Gergely, 2000a and references therein).

Recently, much attention has focused on the role of genetic instability in cancer progression. Since centrosomes organize the mitotic spindle, it has been suggested that defects in centrosome or microtubule function could contribute to the large-scale genetic instability that is usually associated with aggressive cancers. Many cancer cells have extra centrosomes, and some overexpress specific centrosomal or microtubule-associated proteins. While it is unclear whether alterations in TACC gene expression can contribute to cancer, the data demonstrate that D-TACC is involved in regulating microtubule behaviour in Drosophila, and a mutation in the d-tacc gene causes severe chromosome segregation defects in the early embryo. If the mammalian TACC proteins play similar roles in human cells, TACC defects could contribute to increased levels of genetic instability and thus to the development and progression of cancer. Preliminary results suggest that the overexpression of TACC2 in human cells does perturb the microtubule cytoskeleton (Gergely, 2000a).

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. 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. 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. 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. Here it is shown 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, 2000a; 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 affect by 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 exclude (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).

Drosophila Ajuba is not an Aurora-A activator but is required to maintain Aurora-A at the centrosome

The LIM-domain protein Ajuba localizes at sites of epithelial cell-cell adhesion and has also been implicated in the activation of Aurora-A (Aur-A). Despite the expected importance of Ajuba, Ajuba-deficient mice are viable, which has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insights into the function of Ajuba, this study investigated its role in Drosophila, where a single gene (jub) encodes a protein closely related to Ajuba and LIMD1. A key function were identified in neural stem cells, where Jub localizes to the centrosome. In these cells, mutation in jub leads to centrosome separation defects and aberrant mitotic spindles, which is a phenotype similar to that of aur-A mutants. In jub mutants Aur-A activity is not perturbed, but that recruitment and maintenance at the centrosome is affected. As a consequence the active kinase is displaced from the centrosome. On the basis of studies in Drosophila neuroblasts, it is proposed that a key function of Ajuba, in these cells, is to maintain active Aur-A at the centrosome during mitosis (Sabino, 2010).

The regulation of kinase activity in time and space is crucial for the coordination of cellular events. Aurora-A (Aur-A), one of the three members of the Aurora family of kinases in mammals, is a serine/threonine kinase that functions as a key regulator of several events. The kinase Aur-A was first identified in Drosophila as a mitotic kinase. In flies, mutations in aur-a cause severe developmental defects and pleiotropic phenotypes, which include abnormal centrosome and spindle behavior, lack of astral microtubules (MTs), defects in chromosome segregation, spindle positioning, cortical targeting of cell fate determinants and neural stem-cell self-renewal (Sabino, 2010).

In vertebrate cells, Aur-A also plays a major role in mitosis, and recently an unexpected role for this kinase has been described in non-mitotic cells. Aur-A phosphorylates and activates the tubulin deacetylase HDAC-6 to promote disassembly of cilia and cell cycle re-entry (Pugacheva, 2007). The large spectrum of functions attributed to the kinase Aur-A is thought to be, at least in part, regulated by different cofactors or activators (Carmena, 2009). TPX2, a MT-associated protein (MAP), binds Aur-A, thereby promoting Aur-A autophosphorylation and targeting it to the mitotic spindle (Wittmann, 2000). Hef-1 (also known as Nedd9) binding and activation of Aur-A is required for HDAC-6 phosphorylation (Pugacheva et al., 2007). In Drosophila, a single Aur-A activator, Bora, has been described so far. In bora mutants, defects in centrosome behavior and spindle assembly, together with defects in the asymmetric cell division of sensory organ precursors (SOPs), have been identified (Sabino, 2010).

Ajuba (Jub) is a LIM-domain protein that localizes at the sites of cell-cell adhesion in epithelial cells and has also been implicated in the activation of Aur-A (Hirota, 2003). Surprisingly, however, Jub-deficient mice are viable (Pratt, 2005); this has been attributed to functional redundancy with the related LIM-domain protein LIMD1. To gain insight into the function of Jub, its role was investigated in Drosophila, where a single gene encodes a protein closely related to mouse Jub and LIMD1. A mutation was generated in ajuba (jub) and jub mutants were found to die at the larval-pupal transition. No defects were detected in cell adhesion or epithelial polarity. However, a key function was detected in neural stem cells, where Jub localized to the centrosome. In these cells, mutation of jub led to centrosome separation defects and abnormal mitotic spindles. Surprisingly, It was found that in jub mutants Aur-A activity was not perturbed, but that Aur-A recruitment and maintenance at the centrosome was affected. As a consequence the active kinase was ectopically displaced into the cytoplasm, which resulted in abnormalities of the mitotic spindle. On the basis of these studies, it is proposed that a major function of Jub in Drosophila neuroblasts is to restrict active Aur-A to the centrosome during mitosis, but that Jub does not function as an Aur-A activator (Sabino, 2010).

This study generated mutations in the jub gene in Drosophila in order to examine its functions within an intact animal without the complications of potential redundancy with closely related genes. Unexpectedly, it was discovered that Jub had an essential role in just a subset of cells within the animal, namely the neural stem cells. Although not all cell types were exhaustively examined, cell cycles that are normally very sensitive to centrosome or MT perturbation, such as the nuclear divisions of the early embryo and the meiotic divisions of the male germline, occurred normally in the absence of Jub. Thus, Nbs are especially dependent on Jub to generate normal centrosomes and spindles, consistent with the clearly detectable levels of Jub-GFP on the centrosomes in these cells, but not in other cell types (Sabino, 2010).

Within the Nbs lacking Jub, three related, but distinct, phenotypes were detected: defects in the separation of centrosomes following mitosis, defects in spindle assembly, and defects in cortical targeting of determinants and orientation of the mitotic spindle. These phenotypes are shared by Nbs lacking Aur-A, consistent with previous work demonstrating that Jub and Aur-A proteins bind to each other and function together (Hirota, 2003). However, it was not possible to detect a biochemical interaction between these two proteins in Drosophila brains. In addition, the loss of Aur-A causes a number of additional defects that were not observed in jub mutant Nbs, such as defects in centrosome maturation and increased levels of genomic instability, demonstrating that Jub is not required for the majority of Aur-A functions (Sabino, 2010).

It is worth mentioning that no Nbs were obserged with supernumerary centrosomes in jub mutants. Defects in centrosome separation should result in the generation of daughter cells without centrosomes (which was see in 10% of the cells in jub mutant brain cells) and in those with two centrosomes, which should undergo duplication during the following cell cycle to produce extra centrosomes. Future work will be required to explain the absence of Nbs with supernumerary centrosomes (Sabino, 2010).

The results show that, in the absence of Jub, Aur-A is not as concentrated at the centrosome, and hence Tacc recruitment is affected. However, even in the absence of Jub, Tacc (transforming acidic coiled-coil protein), a MT-associated protein, can be phosphorylated by Aur-A, which further supports the idea that Jub is not an Aur-A activator, at least in Drosophila. Furthermore, it appears that loss of Jub results in a displacement of Aur-A from the centrosome. Thus, the key question is whether the defects caused by loss of Jub are due to diminished Aur-A activity on the centrosome, elevation of activity in the cytoplasm or both. The defects in centrosome separation, just after cell division, might be due to diminished levels of Aur-A on the centrosome, whereas the loss of astral MTs might be explained by diminished levels of P-Tacc or other MAPs. The manipulation of Aur-A levels in the presence or absence of Jub suggests that it is the elevated cytoplasmic Aur-A activity that is causing the defects in spindle assembly. Elevated cytoplasmic Aur-A is also likely to account for the defects in spindle positioning during asymmetric cell division (Sabino, 2010).

One possible explanation for the Nb-specific requirement for Jub is that the centrosome cycle is significantly different in these cells. In Nbs, just after centrosome duplication and migration to the apical cortex, one of the centrosomes moves away from the other. This dynamic centrosome continues to move throughout the S and G2 phases, which means that centrosome separation in Nbs takes place substantially before mitosis, in contrast with the timing in other cell types. It is therefore possible that Jub is only required for centrosome separation in cells where centrosomes separate earlier in the cell cycle, substantially before the following mitosis, when Aur-A activity is still present (Sabino, 2010).

Finally, no evidence has bee obtained to support a role for Jub as an Aur-A activator, since no reduction was seen in the phosphorylation of the Aur-A substrate Tacc. Many cell types require Aur-A function, including embryos and male spermatocytes. No Jub-GFP was observed at the centrosome in these cells and no jub mutant phenotypes were seen in the early embryo or male germline. In addition, no co-immunoprecipitate was seem of Jub and Aur-A in brain extracts. It is therefore proposed that the main function of Jub is to bind Aur-A at the centrosome, not to activate the kinase, but rather to restrict its activity in time and space. Too much active Aur-A in the cytoplasm during mitosis seems to perturb astral MT nucleation and centrosomal spindle assembly. Alternatively, Jub might also help to recruit and/or maintain Aur-A at the centrosome, so that it can be activated by another protein concentrated there, and, most crucially in Nbs, ‘hold’ the active Aur-A away from the cytoplasm. Unfortunately, such a candidate protein has not yet been identified in flies. Flies do not have an obvious TPX2 orthologue and the only Aur-A activator identified so far lacks a function in the fly brain. The failure of Jub to regulate Aur-A in Drosophila could also just reflect differences in the way Aur-A is regulated between vertebrates and invertebrates. In human cells, Jub is also associated with kinetochores and spindle MTs, and it has been shown that Jub, together with BubR1 and Aurora B, plays a role in the regulation of the metaphase-to-anaphase transition. However, the lack of a mitotic phenotype in Jub-knockout mice also strongly suggests that it might not play an essential role in Aur-A activation (Sabino, 2010).

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal-D

Bicaudal-D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. As BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize them to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles, and the tandem spindles during female meiosis II. Furthermore, BicD is required to correctly localize clathrin and the microtubule-stabilizing factors, D-TACC and Msps, to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, right after the establishment of the female pronucleus, D-TACC and C. elegans BicD, tacc, and Chc are also needed for pronuclear fusion, pointing to the possibility that the underlying mechanism might be more widely used (Vazquez-Pianzola, 2022).

Encoded by a single gene, the Drosophila Bicaudal D (BicD) protein is part of a family of evolutionarily conserved dynein adaptors responsible for the transport of different cargoes along microtubules (MTs). The founding member of this protein family, Drosophila BicD, was identified because of its essential role during oogenesis and embryo development, in which it transports mRNAs that control polarity and cell fate. This process is mediated by its binding to the RNA-binding protein Egalitarian (Egl). Since its initial discovery, BicD and its orthologs have been shown to control a diverse group of MT transport processes through binding to different cargoes or adaptor proteins (Vazquez-Pianzola, 2022).

BicD can alternatively bind to Clathrin heavy chain (Chc) and this interaction facilitates Chc transport of recycling vesicles at the neuromuscular junctions and regulates endocytosis and the assembly of the pole plasm during oogenesis. The best-known function of Chc is in receptor-mediated endocytosis, in which it forms part of clathrin, a trimeric scaffold protein (called a triskelion), composed of three Chc and three Clathrin light chains (Clc). Aside from this, clathrin was shown to localize to mitotic spindles in mammalian and Xenopus cells and to have non-canonical activity by stabilizing the spindle MTs during mitosis. This function depends on clathrin trimerization and its interaction with Aurora A-phosphorylated Transforming Acidic Coiled-Coil protein 3 (TACC3) and the protein product of the colonic hepatic Tumor Overexpressed Gene (ch-TOG). This heterotrimer forms intermicrotubule bridges between kinetochore fibers (K-fibers), stabilizing these fibers and promoting chromosome congression. Recently, TACC3 and a mammalian homolog of Chc (CHC17) were shown to control the formation of a new liquid-like spindle domain (LISD) that promotes the assembly of acentrosomal mammalian oocyte spindles (Vazquez-Pianzola, 2022).

In order to transport its cargos along MTs, BicD interacts with the dynein/dynactin motor complex, a minus-end-directed MT motor. This complex is involved in different cellular processes, including intracellular trafficking of proteins and RNAs, organelle positioning and microtubule organization, some of which also require BicD. The dynein/dynactin complex also plays essential roles during cell division, in which it is required for centrosome separation, chromosome movements, spindle organization and positioning and mitotic checkpoint silencing (Vazquez-Pianzola, 2022).

Given that Drosophila BicD forms complexes with Chc and Dynein, both of which, as described above, perform essential activities during mitosis, this study set out to investigate possible BicD functions during cell division. Reducing BicD levels by specific protein-targeted degradation in freshly laid eggs revealed that BicD is essential for pronuclear fusion. In addition, it is required for metaphase arrest of female meiotic products after meiosis II completion. This activity appears to be mediated by the role of BicD in localizing the spindle assembly checkpoint (SAC) components. Furthermore, BicD interacts with its cargo protein, Chc, and they both localize to the mitotic spindles and centrosomes and the female tandem meiotic II spindles. In addition, BicD localizes D-TACC, clathrin, and Mini spindles (Msps; ch-TOG homolog) to the meiosis II spindles. The failure to localize these proteins accurately might also contribute to the SAC function defects observed in embryos with reduced BicD levels. D-TACC and Caenorhabditis elegans bicd-1, tac-1 and chc-1 are also needed after fertilization for pronuclear fusion, revealing an evolutionary conserved and essential role of these proteins in early zygote formation and suggesting that their mechanism of action on MTs might be widely used across species (Vazquez-Pianzola, 2022).

This study found that BicD localizes to the female tandem spindles and the central aster during MII. After fertilization, BicD also localizes to the mitotic spindles and the centrosomes. BicD^null mutants rarely survive and are sterile, but this study generated embryos with reduced levels of BicD at the beginning of embryogenesis (BicDhb-deGradFP embryos) by setting up a strategy based on the deGradFP technique. Consistent with BicD localization at the female MII spindles, it was discovered that BicDhb-deGradFP embryos arrest development, displaying aberrant meiotic products and no pronuclear fusion. Especially if combined with the CRIPSP-Cas9 strategy first to produce functional GFP-tagged proteins of interest, the construct designed in this study could be helpful for studying the role of female-sterile and lethal mutations during very early embryonic development (Vazquez-Pianzola, 2022).

In unfertilized BicDhb-deGradFP eggs, the female meiotic products were not arrested in metaphase as normally happens. Instead, they underwent additional rounds of replication. They failed to recruit or maintain the recruitment of the SAC pathway components BubR1 and Mad2, which are normally present at the kinetochores in the wild-type female meiotic polar bodies. Interestingly, in Drosophila eggs mutated for Rod, mps1 and BubR1, well-conserved orthologs of the SAC pathway, the polar bodies also cannot remain in a SAC-dependent metaphase-like state and decondense their chromatin. Furthermore, in these mutants, the polar bodies cycle in and out of M-phase, replicating their chromosomes similarly to those in BicDhb-deGradFP eggs. Thus, it appears that BicD functions to localize the SAC components to induce and/or maintain metaphase arrest of the polar bodies. Several mechanisms could explain the failure to maintain SAC activation observed in BicDhb-deGradFP embryos. BicD might be needed to recruit the SAC components to kinetochores directly. By contrast, during mitosis, the Rod-Zw10-Zwilch (RZZ) complex binds to the outer kinetochore region and recruits Mad2, Spindly and the dynactin complex. Spindly and dynactin act cooperatively to recruit dynein, which then transports the SAC components along the MTs away from kinetochores as a mechanism to trigger checkpoint silencing and anaphase onset. Given that the BicD N-terminal domain binds dynein and dynactin and promotes their interaction, it is also possible that BicD helps to move the SAC components away from the kinetochores. If this does not happen, the SAC remains persistently activated. It was also found that BicD activity in BicDhb-deGradFP embryos is insufficient to localize clathrin, TACC and Msps efficiently along the MTs of the spindle. During mitosis, impairment of MT motors, such as dynein, and treatments that prevent the TACC/clathrin complex from binding to the mitotic spindles and affecting K-fiber stability, also persistently activate the SAC. Thus, reduced levels of BicD in BicDhb-deGradFP embryos could additionally trigger SAC hyperactivation through its role in stabilizing the K-fibers. Although these data strongly suggest that the lack of BicD contributes to SAC defects through its role in localizing clathrin, D-TACC and Msps, further work is needed to elucidate whether BicD also acts more directly by binding to, and localizing, the SAC components, or indirectly by affecting the function of dynein (Vazquez-Pianzola, 2022).

Whereas persistent SAC activation leads to metaphase arrest and delayed meiosis (D-meiosis), this delay is known to be rarely permanent, at least during mitosis. Most cells that cannot satisfy the SAC ultimately escape delayed mitosis (D-mitosis) and enter G1 as tetraploid cells by a currently poorly understood mechanism. It is possible that, in BicDhb-deGradFP, the SAC pathway is constantly activated, delaying meiosis. However, at one point, the nuclei might escape metaphase II arrest, cycling in and out of M-phase, thereby replicating their chromosomes and decondensing their chromatin. The fact that female meiotic products over-replicate in BicDhb-deGradFP eggs and show no or only pericentromeric PH3 staining supports the notion that these nuclei are on an in-out metaphase arrest phase. That meiotic products in about half of the BicDhb-deGradFP embryos failed to stain for the SAC components BubR1 and Mad2 supports this hypothesis (Vazquez-Pianzola, 2022).

Chc, its partner Clc and BicD are enriched at mitotic spindles and centrosomes. Furthermore, these proteins and the clathrin-interacting partners D-TACC and Msps localize to the tandem spindles and the central aster of the female MII apparatus. The interaction of Drosophila Chc with D-TACC is conserved, and Chc interacts through the same protein domain directly with D-TACC and BicD. Moreover, BicD is needed for localizing D-TACC, Msps and clathrin throughout the MII tandem spindles. The TACC3/Chc interaction was proposed to form a domain in tandem to bind spindle MTs. It is hypothesize that BicD could help recruit Chc to the MTs by association with dynein. Given that Chc usually acts as a trimer with Clc (triskelion), each Chc might interact with either BicD or D-TACC. Thus, a mixed complex could be formed, and BicD might help to move, recruit or stabilize Chc and D-TACC along the spindles via the interaction of BicD/Chc in the same trimer. The fact that expression of D-TACC enhances the Chc/BicD interaction and that overexpression of Chc and D-TACC (tacc) arrested early development in a background in which BicD is reduced to a level that does not produce visible phenotypes on its own, supports this model. These results suggest that, with respect to BicD, the levels of D-TACC and Chc should be tightly balanced for these proteins to perform their normal function during early development, as has been shown previously for other BicD transport processes (Vazquez-Pianzola, 2022).

BicD has a role in pronuclear fusion that is conserved during evolution given that C. elegans eggs depleted for bicd-1 also failed to undergo pronuclear fusion. Moreover, Drosophila D-TACC and C. elegans chc-1 and tac-1 are also needed for pronuclear fusion. These genes might be required indirectly through their role in meiosis because preliminary data suggest that MII is also compromised in bicd-1 and chc-1 dsRNA-fed worms. Alternatively, they might play a more direct role in pronuclear migration, which depends on dynein and MTs in bovine, primate and C. elegans embryos. This would then suggest that the underlying mechanism may be used to build correctly or stabilize different types of MT. Determining their precise mechanistic involvement in pronuclear fusion is an interesting question for further studies (Vazquez-Pianzola, 2022).

Search PubMed for articles about Drosophila TACC

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(7): 1039-46. PubMed ID: 16186253

Bellanger, J. M. and Gonczy, P. (2003). TAC-1 and ZYG-9 form a complex that promotes microtubule assembly in C. elegans embryos. Curr. Biol. 13: 1488-1498. PubMed ID: 12956950

Carmena, M., Ruchaud, S. and Earnshaw, W. C. (2009). Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins. Curr. Opin. Cell Biol. 21: 796-805. PubMed ID: 19836940

Cassimeris, L. (1999). Accessory protein regulation of microtubule dynamics throughout the cell cycle. Curr. Opin. Cell Biol. 11: 134-141. PubMed ID: 10047516

Gergely, F., Kidd, D., Jeffers, K., Wakefield, J. G. and Raff, J. W. (2000a). D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo. EMBO J. 19(2): 241-52. PubMed ID: 10637228

Gergely, F., Karlsson, C., Still, I., Cowell, J., Kilmartin, J. and Raff, J. W. (2000b). The TACC domain identifies a family of centrosomal proteins that can interact with microtubules. Proc. Natl. Acad. Sci. 97(26): 14352-7. PubMed ID: 11121038

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

Giet, R., et al. (2002). Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules. J. of Cell Biol. 156: 437-451. PubMed ID: 11827981

Hirota, T., Kunitoku, N., Sasayama, T., Marumoto, T., Zhang, D., Nitta, M., Hatakeyama, K. and Saya, H. (2003). Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell 114: 585-598. PubMed ID: 13678582

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

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

Megraw, T. L., Kilaru, S., Turner, F. R. and Kaufman, T. C. (2002). The centrosome is a dynamic structure that ejects PCM flares. J. Cell Sci 115: 4707-4718. PubMed ID: 12415014

Moore, A. and Wordeman, L. (2004). The mechanism, function and regulation of depolymerizing kinesins during mitosis. Trends Cell Biol. 14: 537-546. PubMed ID: 15450976

O'Brien, L., et al. (2005). The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly. Mol. Biol. Cell. 16: 2836-2847. PubMed ID: 15788567

Ohkura, H., Garcia, M. A. and Toda, T. (2001). Dis1/TOG universal microtubule adaptors - one MAP for all? J. Cell Sci. 114: 3805-3812. PubMed ID: 11719547

Pascreau, G., Delcros, J. G., Cremet, J. Y., Prigent, C. and Arlot-Bonnemains, Y. (2005). Phosphorylation of maskin by Aurora-A participates in the control of sequential protein synthesis during Xenopus laevis oocyte maturation. J. Biol. Chem. 280(14): 13415-23. PubMed ID: 15687499

Pearson, N. J., Cullen, C. F., Dzhindzhev, N. S. and Ohkura, H. (2005). A pre-anaphase role for a Cks/Suc1 in acentrosomal spindle formation of Drosophila female meiosis. EMBO Rep. 6(11): 1058-63. PubMed ID: 16170306

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

Pratt, S. J., Epple, H., Ward, M., Feng, Y., Braga, V. M. and Longmore, G. D. (2005). The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration. J. Cell Biol. 168, 813-824. PubMed ID: 15728191

Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P. and Golemis, E. A. (2007). HEF1-dependent Aurora A activation induces disassembly of the primary cilium. Cell 129: 1351-1363. PubMed ID: 17604723

Rogers, G. C., et al. (2004). Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature 427: 364-370. PubMed ID: 14681690

Sabino, D., Brown, N. H. and Basto, R. (2010). Drosophila Ajuba is not an Aurora-A activator but is required to maintain Aurora-A at the centrosome. J. Cell Science 124: 1156-1166. PubMed ID: 21402878

Srayko, M., Quintin, S., Schwager, A. and Hyman, A. A. (2003). Caenorhabditis elegans TAC-1 and ZYG-9 form a complex that is essential for long astral and spindle microtubules. Curr. Biol. 13: 1506-1511. PubMed ID: 12956952

Tournebize, R., et al. (2000). Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts. Nat. Cell Biol. 2: 13-19. PubMed ID: 10620801

Vazquez-Pianzola, P., Beuchle, D., Saro, G., Hernandez, G., Maldonado, G., Brunssen, D., Meister, P. and Suter, B. (2022). Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal-D. Development. PubMed ID: 35723263

Venkei, Z., Gaspar, I., Toth, G. and Szabad, J. (2006). alpha4-Tubulin is involved in rapid formation of long microtubules to push apart the daughter centrosomes during early Drosophila embryogenesis. J. Cell Sci 119: 3238-3248. PubMed ID: 16847053

Wittmann, T., Wilm, M., Karsenti, E. and Vernos, I. (2000). TPX2, A novel Xenopus MAP involved in spindle pole organization. J. Cell Biol. 149: 1405-1418. PubMed ID: 10871281

Zhang, J. and Megraw, T. L. (2007). Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires Centrosomin Motif 1. Mol. Biol. Cell 18(10): 4037-49. PubMed ID: 17671162

date revised: 5 August 2023

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