Identification of a TPX2-Like Microtubule-Associated Protein in Drosophila

Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs). One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38) lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions

Xenopus Meiotic Microtubule-Associated Interactome

In metazoan oocytes the assembly of a microtubule-based spindle depends on the activity of a large number of accessory non-tubulin proteins, many of which remain unknown. In this work we isolated the microtubule-bound proteins from Xenopus eggs. Using mass spectrometry we identified 318 proteins, only 43 of which are known to bind microtubules. To integrate our results, we compiled for the first time a network of the meiotic microtubule-related interactome. The map reveals numerous interactions between spindle microtubules and the newly identified non-tubulin spindle components and highlights proteins absent from the mitotic spindle proteome. To validate newly identified spindle components, we expressed as GFP-fusions nine proteins identified by us and for first time demonstrated that Mgc68500, Loc398535, Nif3l1bp1/THOC7, LSM14A/RAP55A, TSGA14/CEP41, Mgc80361 and Mgc81475 are associated with spindles in egg extracts or in somatic cells. Furthermore, we showed that transfection of HeLa cells with siRNAs, corresponding to the human orthologue of Mgc81475 dramatically perturbs spindle formation in HeLa cells. These results show that our approach to the identification of the Xenopus microtubule-associated proteome yielded bona fide factors with a role in spindle assembly

Computer simulations reveal mechanisms that organize nuclear dynein forces to separate centrosomes

Centrosome separation along the surface of the nucleus at the onset of mitosis is critical for bipolar spindle assembly. Dynein anchored on the nuclear envelope is known to be important for centrosome separation, but it is unclear how nuclear dynein forces are organized in an anisotropic manner to promote the movement of centrosomes away from each other. Here we use computational simulations of Caenorhabditis elegans embryos to address this fundamental question, testing three potential mechanisms by which nuclear dynein may act. First, our analysis shows that expansion of the nuclear volume per se does not generate nuclear dynein-driven separation forces. Second, we find that steric interactions between microtubules and centrosomes contribute to robust onset of nuclear dynein-mediated centrosome separation. Third, we find that the initial position of centrosomes, between the male pronucleus and cell cortex at the embryo posterior, is a key determinant in organizing microtubule aster asymmetry to power nuclear dynein-dependent separation. Overall our work reveals that accurate initial centrosome position, together with steric interactions, ensures proper anisotropic organization of nuclear dynein forces to separate centrosomes, thus ensuring robust bipolar spindle assembly