TheXenopusTACC Homologue, Maskin, Functions in Mitotic Spindle Assembly

Abstract

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosomeinteracting proteins. Members of this family share a ϳ200 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. INTRODUCTIONThe mitotic spindle is essential for the process of partitioning a complete set of chromosomes to each daughter cell during cell division. It consists of microtubules, microtubule-based motor proteins, and hundreds of other proteins that function together to produce the configurations and movements required for chromosome segregation (reviewed by Gadde and Heald, 2004;Kline-Smith and Walczak, 2004). Not surprisingly, mitotic spindle assembly is highly regulated and requires the precise spatial and temporal coordination of many processes, including microtubule dynamics, attachment and alignment of chromosomes, and focusing of spindle poles (Compton, 2000). A detailed molecular understanding of the components of the mitotic spindle, their interactions, and their regulation is ess...

CPEB Interacts with an Ovary-specific eIF4E and 4E-T in Early Xenopus Oocytes

“…Moreover, eIF4A, a component of eIF4F, similarly failed to efficiently co-precipitate with CPEB, attesting to the specificity of the interactions described above. We also probed with antibodies raised against Xenopus maskin (TACC3), obtained from three laboratories (15,51,52), and the deadenylase PARN (53) but did not observe significant binding (Fig. 1G) (see "Discussion").…”

The Microtubule-Associated Protein Tumor Overexpressed Gene/Cytoskeleton-Associated Protein 5 Is Necessary for Myelin Basic Protein Expression in Oligodendrocytes

“…CPE binds CPEB. In Xenopus, CPEB binds maskin, which in turn binds XMAP215, the homolog of TOG; TACC, the maskin homolog, also binds TOG (Conte et al, 2003;O'Brien et al, 2005); it is not clear, however, that TACC is functionally similar to maskin with regard to translation. The CPEB-maskin complex is known to inhibit translation of CPE-containing mRNAs in neurons (Huang and Richter, 2004).…”

“…TACC1 has been identified as a TOG binding partner in a yeast two-hybrid screen (Lauffart et al, 2002). However, there is no reported interaction between TACC1 and CPEB, and TOG has not been implicated previously in translational regulation (Conte et al, 2003;O'Brien et al, 2005).…”

Corrections

“…O'Brien et al 5 found that Maskin interacts with XMAP215 in a manner that seems similar to how other TACC proteins act: although XMAP215 can bind to microtubules in Maskin-depleted egg extracts, it fails to accumulate at spindle poles. The Maskin-depleted extracts form undersized asters in response to Ran-GTP and assemble highly disorganized spindles around added sperm chromosomes.…”

“…One possible mechanism is suggested by another recent study by O'Brien et al 5 , who investigated the role of Maskin in spindle assembly. Maskin is a member of the 'transforming acidic coiled coil' (TACC) protein family, and is the first TACC protein to have been found in Xenopus 6 .…”