Molecular mechanism of exocyst-centriolin complexes in abscission

描述(申请人提供)：细胞分裂失败，导致遗传不稳定的具有多个中心体的四倍体细胞的形成，可能是启动肿瘤发生的一个机制。有趣的是，一种定位于母体中心粒和膜小泡的蛋白质称为中心素，它包含与发芽和分裂酵母基因(nud1/cdc11)同源的区域，可以诱导非整倍体。Nud1/CDc11同源结构域已被证明与称为胞外囊的膜-囊泡捆绑和融合复合体相互作用。在有丝分裂期间，中心素定位于母细胞和子细胞之间形成的细胞间桥梁内富含调节蛋白的中体。向心草素是细胞分裂-脱落的最后阶段所必需的。我的主要假设是，中心素与中体的外囊相互作用是完成脱落所必需的。更具体地说，将检查以下目标：1)中心素是否需要将含有外囊的内细胞膜和分泌膜暂时递送到中体；2)中心素与外囊相互作用的时间和地点；以及3)这种相互作用发生的方式。胞内囊泡和分泌囊泡都是细胞在中体通过融合到质膜并可能相互融合进行分裂所必需的。在控制条件下，内吞小泡在分泌小泡之前到达中体。然而，在没有功能性外囊的哺乳动物细胞中，中体有分泌的积累和内吞囊泡的抑制。基于这些发现，目标1将确定siRNAs丢失外囊结合蛋白中心素是否会影响内吞和分泌囊泡在中体聚集的时间关系。在目标2中，膜分离技术将被用来确定中心素定位于膜泡的类型，以及这种定位是否需要脱落。在目标3中，将研究中心素到达中体的机制。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，如中心素，运送到中体。这将通过光激活中心体池并使用延时视频共聚焦显微镜检查它是否能到达中体来进行测试。 公共卫生相关性：Centriolin是脊椎动物母体中心粒的一种成分，是细胞分裂的最后阶段所必需的。它可以影响进入S期，并可能在肿瘤发生中发挥作用。没有中心素的哺乳动物细胞会产生多细胞合胞体，并不断尝试细胞分裂，产生双核细胞作为中间体，这是肿瘤发生的标志。阐明涉及中心素的分子机制可能提供细胞动力学异常与癌症易感性之间的功能联系。

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.