Failure of cell division, resulting in the formation of genetically unstable tetraploid cells with multiple centrosomes, may be a mechanism for the initiation of tumorigenesis. Interestingly, a protein that localizes to the maternal centriole and membrane vesicles called centriolin contains domains homologous to the budding- and fission-yeast genes (Nud1/Cdc11) that can induce aneuploidy. The Nud1/Cdc11 homologous domain has been shown to interact with a membrane-vesicle-tethering and -fusion complex called the exocyst. During mitosis centriolin localizes to the regulatory-protein-rich midbody within the intercellular bridge that forms between the mother and daughter cell. Centriolin is required for the final stage of cell division- abscission. My overarching hypothesis is that centriolin's interaction with the exocyst at the midbody is necessary for the completion of abscission. More specifically, the following aims will be examined: 1) whether centriolin is required for temporal delivery of exocyst- containing endocytic and secretory membranes to the midbody 2) when and where centriolin interacts with the exocyst, and 3) by what means this interaction occurs. Both endocytic and secretory vesicles are required for cell cleavage at the midbody via fusion to the plasma membrane and possibly each other. Under control conditions, endocytic vesicles arrive at the midbody before secretory vesicles. However, there is an accumulation of secretory and an inhibition of endocytic vesicles at the midbody in mammalian cells without a functional exocyst. Based on these findings, Aim 1 will determine whether loss of the exocyst binding protein centriolin by siRNAs affects the temporal relationship between endocytic and secretory vesicle accumulation at the midbody.
In Aim 2, membrane fractionation techniques will be used to determine the type of membrane vesicle centriolin localizes to and whether this localization is required for abscission.
In Aim 3, the mechanism by which centriolin arrives at the midbody will be examined. One possibility is that the maternal-centriole moves to the intercellular bridge during telophase in order to shuttle regulatory components, e.g. centriolin, to the midbody. This will be tested by photoactivating the centrosomal pool of centriolin and examining whether it can arrive at the midbody using time-lapse video confocal microscopy.
Centriolin is a component of the vertebrate maternal centriole that is required for the final stages of cell division. It can influence entry into S phase, and may play a role in oncogenesis. Mammalian cells without centriolin generate multicellular syncytia and undergo continuous attempts at cell cleavage, producing binucleate cells as intermediates, a hallmark for tumorigenesis. Elucidation of the molecular mechanisms involving centriolin may provide a functional link between cytokinetic abnormalities and cancer predisposition.
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