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Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule depolymerizing drugs suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. While not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. The localization of DdINCENP at the cleavage furrow is modulated by myosin II. Loss of myosin II restricted the localization of DdINCENP to a narrow zone at the cleavage furrow. Kif12, a homolog of mitotic kinesin like protein (MKLP), was essential for relocalization of DdINCENP from the central spindle to the cleavage furrow. Furthermore, Kif12 was also localized at the cortex of the cleavage furrow and its localization during cytokinesis closely resembled that of DdINCENP, suggesting a possible interaction between them. The correct localization of DdINCENP during cytokinesis also required its N-terminal sequence. DdINCENP1-500 was found at the cleavage furrow and interacted with the actin cytoskeleton. Domain analysis of DdINCENP also revealed that its DdINCENP1-500 was sufficient to rescue the weak spindle defect of DdINCENP null cells. / text

Les septines sont des protéines conservées de la levure à l’homme qui sont impliquées dans divers processus cellulaires tels que la cytokinèse, le transport vésiculaire et l’organisation du cortex cellulaire. Il existe 13 gènes de septines retrouvés en plusieurs isoformes chez l’humain, et seulement cinq chez Drosophila melanogaster, Sep1, Sep2, Pnut, Sep4 et Sep5, ce qui en fait un modèle idéal vu son génome simple. Les septines sont composées d’un domaine de liaison au GTP très conservé entre les espèces, dont le rôle reste à ce jour ambiguë, ainsi que de régions N et C-terminales variables. Les septines s’assemblent entre elles pour former un hexamère, composé de Sep1, Sep2 et Pnut chez Drosophila melanogaster, via l’interface N-C et G des septines. Ces hexamères s’assemblent bout à bout afin de former les filaments de septines. Ces filaments peuvent ensuite se regrouper et s’assembler en structures hautement ordonnées telles que des anneaux, des tubes, des faisceaux de filaments, des cages et elles sont retrouvées au sillon de clivage durant la cytokinèse. Le but était de déchiffrer les mécanismes d’assemblage des filaments de septines qui mènent à la formation des différentes structures, afin de mieux comprendre les mécanismes d’interaction entre les septines. Au sein des cellules S2 de Drosophila melanogaster, les septines sont retrouvées à trois structures hautement ordonnées et dépendantes de Pnut endogène : des tubes cytoplasmiques, des anneaux cytoplasmiques et le sillon de clivage durant la cytokinèse. Notre hypothèse est qu’il existe plusieurs mécanismes qui régissent la formation des structures hautement ordonnées et que ceux-ci sont dépendants des régions N et C terminales variables des septines qui sont impliquées dans plusieurs interactions. Divers mutants de Sep1, Sep2 et Pnut tronqués en N et en C-terminal ont été fusionnés à une protéine fluorescente et caractérisés par microscopie confocale. La localisation de ces mutants a été répertoriée et analysée en présence des septines endogènes ou lors de la déplétion de celles-ci. Nos résultats suggèrent que le domaine de liaison au GTP est suffisant pour le recrutement des septines au sillon de clivage durant la cytokinèse, mais que la région N-terminale est requise la formation des tubes et des anneaux cytoplasmiques dépendants de Pnut. / Septins are conserved from yeast to humans and are implicated in diverse cellular processes such as cytokinesis, vesicular transport and cellular cortical organization. There are 13 known genes that encode for human septins, which also have many isoforms, while there are only five septin genes in Drosophila melanogaster: Sep1, Sep2, Pnut, Sep4 and Sep5, which makes it an ideal model system. Septins have a conserved GTP binding domain, whose role is still not fully understood, and variable N-C-termini. Septins assemble together, via N-C and G interfaces, to form a hexamer, that is composed of Sep1, Sep2 and Pnut in Drosophila melanogaster, which assemble end-to-end to form non polar filaments. These filaments can subsequently assemble together to form higher-ordered structures, such as rings, tubes, bundles, and gauzes. Furthermore, septins are recruited to the cleavage furrow during cytokinesis although their organization there is unclear. The aim of this project is to define septin assembly mechanisms that can lead to the formation of different higher ordered structures. In Drosophila melanogaster S2 cells, septins are recruited to three, readily observable septin dependent structures: cytoplasmic rings, cytoplasmic tubes, and the cleavage furrow during cytokinesis. Our hypothesis is that multiple mechanisms govern septin incorporation into these structures and that these mechanisms differentially depend on septin N-C variable termini. A panel of mutants of Sep1, Sep2 and Pnut truncated in N-C-termini were fused to fluorescent proteins and their localization in S2 cells monitored by confocal microscopy, with or without depletion of endogenous septins. My results suggest that the GTP binding domain is sufficient for septin recruitment to the cleavage furrow during cytokinesis, but that the septin N-termini are required for recruitment to the cytoplasmic tubes and rings.