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Zusammenfassung:
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.
