Molecular mechanism of exocyst-centriolin complexes in abscission

描述（由申请人提供）：细胞分裂失败，导致形成具有多个中心体的遗传不稳定的四倍体细胞，可能是肿瘤发生的一种机制。有趣的是，一种定位于母体中心粒和膜囊泡的称为中心蛋白的蛋白质含有与出芽和分裂酵母基因（Nud1/Cdc11）同源的结构域，可以诱导非整倍体。Nud1/Cdc11同源结构域已被证明与称为外囊的膜-囊泡-系留和融合复合物相互作用。在有丝分裂过程中，中心蛋白定位于在母细胞和子细胞之间形成的细胞间桥内富含调节蛋白的中间体。中心蛋白是细胞分裂的最后阶段——脱落所必需的。我的主要假设是，中心蛋白与中间囊的相互作用是完成脱落所必需的。更具体地说，将研究以下目标：1)将含囊内吞膜和分泌膜临时递送到中体是否需要中心蛋白；2)中心蛋白何时何地与囊外相互作用；3)这种相互作用是通过什么方式发生的。细胞分裂需要内吞囊泡和分泌囊泡，它们通过与质膜融合，也可能相互融合。在控制条件下，内吞囊泡先于分泌囊泡到达体中部。然而，在没有功能性外囊的哺乳动物细胞中，存在分泌积累和体内内吞囊泡抑制。基于这些发现，Aim 1将确定sirna丢失囊泡结合蛋白中心蛋白是否会影响体内内吞和分泌囊积聚之间的时间关系。在Aim 2中，将使用膜分离技术来确定膜泡中心蛋白定位的类型，以及分离是否需要这种定位。在第3章中，我们将探讨中心蛋白到达中体的机制。一种可能性是母体中心粒在末期移动到细胞间桥，以便将调节成分（如中心蛋白）运送到中间体。这将通过光激活中心体中心蛋白池并使用延时视频共聚焦显微镜检查它是否可以到达中体来测试。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.