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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Is TD-60 a chromosomal passenger protein, a Guanine exchange factor, or both?

Papini, Diana January 2014 (has links)
The Chromosomal Passenger Complex (CPC) is a major regulator of mitosis composed of the catalytic subunit Aurora B, the inner centromere protein INCENP, Survivin and Borealin/Dasra B. The CPC controls aspects of mitosis, ranging from chromosome and spindle structure to the correction of kinetochore-microtubule attachment errors, regulation of mitotic progression and completion of cytokinesis (Carmena et al., 2012). Knocking down any one CPC component induces delocalization of the others and disrupts mitotic progression (Adams et al., 2001 ; Carvalho et al., 2003; Lens et al., 2003 ; Gassmann et al., 2004; Vader et al., 2006). Telophase Disc (TD-60), also known as RCC2, is a putative Guanine Exchange Factor (GEF) that has been suggested to be involved in completion of cytokinesis through GTPase activation (Mollinari et al., 2003). However, its mechanism of action is still unclear. Interestingly, TD-60 has a typical Chromosomal Passenger Complex (CPC) localization (Andreassen et al., 1991) and its down-regulation alters CPC localisation during early mitosis. However, it is not a member of the CPC immunoprecipitated from mitotic cells (Gassmann et al., 2004). Here, I improved human TD-60 recombinant protein production by expressing a synthetic cDNA in the baculovirus expression system. This allowed me to characterize TD-60-associated GEF activity in vitro and study its possible influence on core CPC activity in vivo. I tested purified human TD-60 against a broad selection of GTPase targets, representing each GTPase family, in an established GEF assay. My data demonstrated that TD-60 has consistent high GEF activity in vitro towards the Ras-like protein A, RalA. To understand if TD-60 links RalA GTPase function to the CPC in vivo, I performed TD-60 and RalA RNAi experiments in HeLa cells. Interestingly, both TD-60 and RalA-depleted cells exhibit destabilized kinetochore fibers, a similar defective prometaphase-like bipolar spindle structure, and an abnormal centromeric accumulation of the CPC in early mitosis. In order to confirm that phenotypes seen after TD-60 depletion were due to lack of RalA activation in vivo, I generated a constitutively active RalA mutant that I transfected into TD-60- deficient cells. Strikingly, the RalA Q72L active mutant (mimicking the GTP-bound form) rescued the abnormal bipolar spindle structure, corrected the defective kinetochore-microtubules attachments, and rescued the atypical CPC distributions observed at centromeres after TD-60 depletion. These results suggest that TD-60-associated RalA GEF activity stabilizes kinetochore-microtubule attachments in early mitosis and that, TD-60 links RalA GTPase function to the CPC during mitosis.
2

Dynamique de localisation de la kinase mitotique Aurora-A et caractérisation de la protéine passagère TD-60 au cours de la mitose.

Sirot, Fabienne 12 December 2005 (has links) (PDF)
De nombreuses kinases participent au bon déroulement de chaque étape du cycle cellulaire. Chez les eucaryotes supérieurs, les kinases Aurora-A et Aurora-B, structuralement très proches, exercent des rôles fondamentaux durant la mitose. Aurora-A est une protéine localisée au niveau des centrosomes, impliquée dans le cycle de division du centrosome et la formation du fuseau mitotique. Aurora-B est une protéine passagère localisée sur les centromères et qui migre, en anaphase, sur le sillon de division et se concentre en cytocinèse sur le corps résiduel. Aurora-B est responsable de la phosphorylation massive, en mitose, du résidu Serine 10 de l'histone H3. Par un système de pseudo-génétique, j'ai ciblé, dans l'extrémité amino-terminale de Aurora-A, le domaine responsable de sa localisation centrosomique. Ces expériences ont montré que les domaines catalytiques de Aurora-A et Aurora-B possèdent tous deux un signal de localisation centromérique. Mais, à l'inverse de Aurora-B, le domaine catalytique de Aurora-A ne se transfert pas des centromères vers le sillon de division en anaphase. Ces travaux montrent également que Aurora-A est capable d'assurer une partie des fonctions mitotiques de Aurora-B. J'ai par ailleurs identifié et cloné la séquence de la protéine passagère TD-60 de Xenopus laevis. J'ai exprimé des domaines protéiques de la protéine xTD60, afin de générer un anticorps spécifique de TD-60 de xénope. Des expériences de co-sédimentation de complexes protéiques d'extraits mitotiques d'œufs de xénope et des expériences d'immunolocalisation cellulaire nous permettent d'envisager pour xTD-60 des fonctions plus larges que celles attribuées aux protéines passagères.

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