Centrosome biogenesis and number: mechanisms of control: determination of SAK/PLK4 interactors

• Author(s): Freixo, Francisco Duque Projecto Avó
• Date: 2009
• Persistent ID: http://hdl.handle.net/10451/1504
• Origin: Repositório da Universidade de Lisboa
• Subject(s): Biologia celular; Drosophila; Ciclo celular; Teses de mestrado
• Document Type: Master thesis
• Language: English
• Advisor(s): Dias, Mónica Bettencourt,; Gomes, Rui Artur Paiva Loureiro
• Contributor(s): Repositório da Universidade de Lisboa

Description

Tese de mestrado, Biologia (Biologia Molecular Humana), 2009, Universidade de Lisboa, Faculdade de Ciências

The centrosome is the major microtubule organizing center in animal cells and regulates cell adhesion, migration and polarity in interphase and the formation of the mitotic spindle. It is formed by a dense protein lattice that surrounds two centrioles, which grant the centrosome its ability to duplicate. Centriole duplication occurs only once per cell cycle in order to avoid multipolar spindle formation which can lead to genomic instability or loss of asymmetric cell division. In fact, centrosome amplification is often associated with early stages of tumorigenesis. In Drosophila melanogaster, centrosome duplication is triggered by a kinase from the Polo family, SAK/Plk4, and regulated by other proteins such as SAS-6, SAS-4 and Bdl10, that also play a role in centriole assembly. It is known that SAK/Plk4 depletion leads to centrosome loss both in Drosophila and humans, and that overexpression causes centriole overduplication in a single cell cycle. In cells that don't have centrioles, SAK/Plk4 overexpression causes de novo formation of multiple centrosomes, by a process that is not yet fully understood. Other than its degradation by the SCF/Slimb complex, there are no other known SAK interactors or substrates. Here we show the result of an RNAi screen for novel SAK substrates and interactors whose depletion could affect centrosome duplication. The candidates were chosen coupling previous immunoprecipitation results with bioinformatic analysis. Minispindles (Msps) and Yps depletion lead to an increase in cells with 0 centrosomes when compared to the negative control. The depletion of these genes does not seem to affect SAK localization. Msps can act as microtubule (MT) polymerase. The centrosome number and microtubule defect phenotypes generated by its depletion are not rescued by co-depletion of MT depolymerases. This suggests that centrosome loss may be related to a MT network disruption in interphase, possibly due to impairment of centrosomal proteins transport. Yps localizes to the cytoplasm and nucleus, which is consistent with its transcription factor and RNA binding activities. It may be involved in regulation of gene expression, for genes whose product is required for centrosome duplication or stability. Its human homologue, YB-1, has tubulin binding activity, suggesting that it may also exist a similar function for Yps. Together, these results bring a new perspective on how centrosome duplication and stability can be regulated. This suggests the existence of one or several novel protein interactions and pathways through which SAK is playing its role.

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