RESUMO
Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.
Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Blastômeros/metabolismo , Blastômeros/patologia , Centríolos/genética , Segregação de Cromossomos , Cromossomos/genética , Cromossomos/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Técnicas de Silenciamento de Genes , Mitose , Proteínas de Neoplasias/genética , Oócitos/metabolismo , Oryzias/embriologia , Oryzias/genética , Oryzias/metabolismo , Proteômica , Proteínas Repressoras/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Xenopus laevis/genéticaRESUMO
Assembly of the mitotic spindle requires a global change in the activity and constitution of the microtubule-binding-protein array at mitotic onset. An important subset of mitotic microtubule-binding proteins localises to the nucleus in interphase and essentially contributes to spindle formation and function after nuclear envelope breakdown. Here, we used a proteomic approach to selectively identify proteins of this category and revealed 50 poorly characterised human gene products, among them the echinoderm microtubule-associated-protein-like gene product, EML3. Indirect immunofluorescence showed that EML3 colocalises with spindle microtubules throughout all mitotic stages. In interphase, EML3 colocalised with cytoplasmic microtubules and accumulated in interphase nuclei. Using YFP-fusion constructs of EML3, we located a nuclear localisation signal and confirmed the microtubule-binding domain of EML3. Functional analysis of EML3 using time-lapse fluorescence microscopy and detailed end-point analysis of phenotypes after siRNA knockdown demonstrates an important role for EML3 in correct metaphase chromosome alignment. Our proteomic identification screen combined with sensitive phenotypic analysis therefore provides a reliable platform for the identification and characterisation of proteins important for correct cell division.
