The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a approximately 200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to "rescue" experiments. Defects in the "early" step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the "late" step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.     

Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3-XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A-dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.     

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

The XMAP215/TOG family of proteins is a closely related set of MAPs (microtubule-associated proteins) found in animals, yeast, and plants . In yeast and animal cells, the XMAP215/TOG proteins are required for both mitosis and meiosis. Although effects of XMAP215/TOG proteins on cytoplasmic microtubules have not previously been shown in animal cells, in plants the Arabidopsis family member MOR1 is required for the organization of cortical microtubule arrays . The Drosophila family member, encoded by the mini spindles (msps) gene, is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles . Here we show that msps is also required during oogenesis for the structure and function of cytoplasmic microtubules. Localization of bicoid (bcd) mRNA in the oocyte is a microtubule-mediated event . We show that bcd RNA localization is defective in msps mutants. We also identify defects in cytoplasmic microtubules in both the germ and follicle cells of mutant ovaries and determine the expression pattern of msps mRNA and protein in developing egg chambers. Our findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions.     

Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis

The Xenopus protein Maskin has been previously identified and characterized in the context of its role in translational control during oocyte maturation. Maskin belongs to the TACC protein family. In other systems, members of this family have been shown to localize to centrosomes during mitosis and play a role in microtubule stabilization. Here we have examined the putative role of Maskin in spindle assembly and centrosome aster formation in the Xenopus egg extract system. Depletion and reconstitution experiments indicate that Maskin plays an essential role for microtubule assembly during M-phase. We show that Maskin interacts with XMAP215 and Eg2, the Xenopus Aurora A kinase in vitro and in the egg extract. We propose that Maskin and XMAP215 cooperate to oppose the destabilizing activity of XKCM1 therefore promoting microtubule growth from the centrosome and contributing to the determination of microtubule steady-state length. Further more, we show that Maskin localization and function is regulated by Eg2 phosphorylation.     

TACC3 Protein Regulates Microtubule Nucleation by Affecting γ-Tubulin Ring Complexes

Centrosome-mediated microtubule nucleation is essential for spindle assembly during mitosis. Although γ-tubulin complexes have primarily been implicated in the nucleation process, details of the underlying mechanisms remain poorly understood. Here, we demonstrated that a member of the human transforming acidic coiled-coil (TACC) protein family, TACC3, plays a critical role in microtubule nucleation at the centrosome. In mitotic cells, TACC3 knockdown substantially affected the assembly of microtubules in the astral region and impaired microtubule nucleation at the centrosomes. The TACC3 depletion-induced mitotic phenotype was rescued by expression of the TACC3 C terminus predominantly consisting of the TACC domain, suggesting that the TACC domain plays an important role in microtubule assembly. Consistently, experiments with the recombinant TACC domain of TACC3 demonstrated that this domain possesses intrinsic microtubule nucleating activity. Co-immunoprecipitation and sedimentation experiments revealed that TACC3 mediates interactions with proteins of both the γ-tubulin ring complex (γ-TuRC) and the γ-tubulin small complex (γ-TuSC). Interestingly, TACC3 depletion resulted in reduced levels of γ-TuRC and increased levels of γ-TuSC, indicating that the assembly of γ-TuRC from γ-TuSC requires TACC3. Detailed analyses suggested that TACC3 facilitates the association of γ-TuSC-specific proteins with the proteins known to be involved in the assembly of γ-TuRC. Consistent with such a role for TACC3, the suppression of TACC3 disrupted localization of γ-TuRC proteins to the centrosome. Our findings reveal that TACC3 is involved in the regulation of microtubule nucleation at the centrosome and functions in the stabilization of the γ-tubulin ring complex assembly.     

A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.     

XMAP215 is required for the microtubule-nucleating activity of centrosomes

Microtubules are essential structures that organize the cytoplasm and form the mitotic spindle. Their number and orientation depend on the rate of nucleation events and their dynamics. Microtubules are often, but not always, nucleated off a single cytoplasmic element, the centrosome. One microtubule-associated protein, XMAP215, is also a resident centrosomal protein. In this study, we have found that XMAP215 is a key component for the microtubule-nucleating activity of centrosomes. We show that depletion of XMAP215 from Xenopus egg extracts impairs their ability to reconstitute the microtubule nucleation potential of salt-stripped centrosomes. We also show that XMAP215 immobilized on polymer beads induces the formation of microtubule asters in egg extracts as well as in solutions of pure tubulin. Formation of asters by XMAP215 beads indicates that this protein is able to anchor nascent microtubules via their minus ends. The aster-forming activity of XMAP215 does not require gamma-tubulin in pure tubulin solutions, but it is gamma-tubulin-dependent in egg extracts. Our results indicate that XMAP215, a resident centrosomal protein, contributes to the microtubule-nucleating activity of centrosomes, suggesting that, in vivo, the formation of asters by centrosomes requires factors additional to gamma-tubulin.     

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform.     

The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2-cyclinB1 complex). It has previously been demonstrated that the p34cdc2-cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215-cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.     

RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability

The receptor for hyaluronan-mediated motility (RHAMM), an acidic coiled coil protein, has previously been characterized as a cell surface receptor for hyaluronan, and a microtubule-associated intracellular hyaluronan binding protein. In this study, we demonstrate that a subset of cellular RHAMM localizes to the centrosome and functions in the maintenance of spindle integrity. We confirm a previous study showing that the amino terminus of RHAMM interacts with microtubules and further demonstrate that a separate carboxy-terminal domain is required for centrosomal targeting. This motif overlaps the defined hyaluronan binding domain and bears 72% identity to the dynein interaction domain of Xklp2. RHAMM antibodies coimmunprecipitate dynein IC from Xenopus and HeLa extracts. Deregulation of RHAMM expression inhibits mitotic progression and affects spindle architecture. Structure, localization, and function, along with phylogenetic analysis, suggests that RHAMM may be a new member of the TACC family. Thus, we demonstrate a novel centrosomal localization and mitotic spindle-stabilizing function for RHAMM. Moreover, we provide a potential mechanism for this function in that RHAMM may cross-link centrosomal microtubules, through a direct interaction with microtubules and an association with dynein.     

The Centrosomal Adaptor TACC3 and the Microtubule Polymerase chTOG Interact via Defined C-terminal Subdomains in an Aurora-A Kinase-independent Manner

The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414–530) and CC2 (aa 530–630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.     

Epsilon-tubulin is required for centriole duplication and microtubule organization

Centrosomes nucleate microtubules and serve as poles of the mitotic spindle. Centrioles are a core component of centrosomes and duplicate once per cell cycle. We previously identified epsilon-tubulin as a new member of the tubulin superfamily that localizes asymmetrically to the two centrosomes after duplication. We show that recruitment of epsilon-tubulin to the new centrosome can only occur after exit from S phase and that epsilon-tubulin is associated with the sub-distal appendages of mature centrioles. Xenopus laevis epsilon-tubulin was cloned and shown to be similar to human epsilon-tubulin in both sequence and localization. Depletion of epsilon-tubulin from Xenopus egg extracts blocks centriole duplication in S phase and formation of organized centrosome-independent microtubule asters in M phase. We conclude that epsilon-tubulin is a component of the sub-distal appendages of the centriole, explaining its asymmetric localization to old and new centrosomes, and that epsilon-tubulin is required for centriole duplication and organization of the pericentriolar material.     

Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

Acomplex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3–clathrin binding. A dileucine motif and Aurora A–phosphorylated serine 558 on TACC3 bound to the “ankle” of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3–clathrin.     

XMAP215 regulates microtubule dynamics through two distinct domains

XMAP215 belongs to a family of proteins involved in the regulation of microtubule dynamics. In this study we analyze the function of different parts of XMAP215 in vivo and in Xenopus egg extracts. XMAP215 has been divided into three fragments, FrN, FrM and FrC (for N-terminal, middle and C-terminal, respectively). FrN co-localizes with microtubules in egg extracts but not in cells, FrC co- localizes with microtubules and centrosomes both in egg extracts and in cells, while FrM does not co- localize with either centrosomes or microtubules. In Xenopus egg extracts, FrN stimulates microtubule growth at plus-ends by inhibiting catastrophes, while FrM has no effect, and FrC suppresses microtubule growth by promoting catastrophes. Our results suggest that XMAP215 is targeted to centrosomes and microtubules mainly through its C-terminal domain, while the evolutionarily conserved N-terminal domain contains its microtubule-stabilizing activity.     

Phosphorylation of maskin by Aurora-A is regulated by RanGTP and importin beta

Mitotic spindle assembly in Xenopus egg extracts is regulated at least in part by importin beta and its regulator, the small GTPase, Ran. RanGTP stabilizes microtubules near the chromosomes during spindle assembly by selectively releasing spindle assembly factors from inhibition by importin alpha/beta in the vicinity of the chromosomes. Several spindle assembly factors are regulated in this manner. We identified maskin, the Xenopus member of the transforming acidic coiled coil family of proteins, as a potential candidate in a two-step affinity chromatography approach designed to uncover additional downstream targets of importin alpha/beta in mitosis. Here, we show that although maskin lacks a canonical nuclear localization sequence, it binds importin beta in a RanGTP-regulated manner. We further show that importin beta inhibits the regulatory phosphorylation of maskin by Aurora-A. This suggests a novel mechanism by which importin beta regulates the activity of a spindle assembly factor.     

TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.     

Hepatoma up-regulated protein is required for chromatin-induced microtubule assembly independently of TPX2

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.     

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.     

Caenorhabditis elegans TAC-1 and ZYG-9 form a complex that is essential for long astral and spindle microtubules

TACC (transforming acidic coiled-coil) proteins were first identified by their ability to transform cell lines [1], and links between human cancer and the overexpression of TACC proteins highlight the importance of understanding the biological function of this family of proteins. Herein, we describe the characterization of a new member of the TACC family of proteins in Caenorhabditis elegans, TAC-1. In other systems, TACC proteins associate with the XMAP215 family of microtubule-stabilizing proteins; however, it is unclear whether TACC proteins have microtubule-based functions distinct from XMAP215. We depleted both the XMAP215 ortholog ZYG-9 and TAC-1 via dsRNA-mediated interference (RNAi). We found that tac-1(RNAi) resulted in microtubule-based defects that were very similar to zyg-9(RNAi). Furthermore, TAC-1 and ZYG-9 are required for long astral microtubules in general and long spindle microtubules during spindle assembly. Loss of either protein did not affect the alpha-tubulin immunofluorescence intensity near centrosomes; this finding suggests that microtubule nucleation was not compromised. Both proteins localize to centrosomes and the kinetochore/microtubule region of chromosomes in metaphase and early anaphase. Furthermore, we found that ZYG-9 and TAC-1 physically interact in vivo, and this interaction is important for the efficient localization of the ZYG-9/TAC-1 complex to centrosomes.     

Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus

AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.