Molecular mechanism of exocyst-centriolin complexes in abscission

外囊-中心蛋白复合物脱落的分子机制

• 批准号: 8003744
• 负责人: Heidi Hehnly
• 金额: $ 4.76万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-16 至 2012-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003744
• 关键词: Affect; Aneuploid Cells; Aneuploidy; Binding; Binding Proteins; Cell division; Cell membrane; Cells; Centrioles; Centrosome; Complex; Confocal Microscopy; Cytokinesis; Cytosol; Endocytic Vesicle; Excision; Failure; Fission Yeast; Fractionation; Genes; Giant Cells; Link; Malignant Neoplasms; Mammalian Cell; Membrane; Microtubule Bundle; Mitosis; Molecular; Mothers; Phase; Play; Predisposition; Proteins; Role; Secretory Vesicles; Small Interfering RNA; Staging; Techniques; Testing; Vesicle; base; daughter cell; endosome membrane; genetic regulatory protein; public health relevance; telophase; time use; tumorigenesis

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.

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，可能是肿瘤发生的启动机制。有趣的是，一种定位于母体中心粒和膜囊泡的蛋白质（称为中心素）包含与芽殖和裂殖酵母基因（Nud1/Cdc11）同源的结构域，可诱导非整倍性。 Nud1/Cdc11 同源结构域已被证明与称为外囊的膜囊泡束缚和融合复合物相互作用。在有丝分裂期间，中心蛋白定位于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。 Centriolin 是细胞分裂的最后阶段（脱落）所必需的。我的总体假设是中心蛋白与中体外囊的相互作用对于完成脱落是必要的。更具体地说，将检查以下目标：1) 是否需要 Centriolin 将包含外囊的内吞膜和分泌膜暂时递送至中体；2) Centriolin 与外囊相互作用的时间和地点；以及 3) 这种相互作用通过什么方式发生。内吞囊泡和分泌囊泡都是通过与质膜融合以及可能彼此融合而在中体进行细胞分裂所必需的。在控制条件下，内吞囊泡在分泌囊泡之前到达中体。然而，在没有功能性外囊的哺乳动物细胞中，中体处存在分泌囊泡的积累和内吞囊泡的抑制。基于这些发现，目标 1 将确定 siRNA 导致的胞外囊结合蛋白 centriolin 的丢失是否会影响中体内吞和分泌囊泡积累之间的时间关系。在目标 2 中，将使用膜分离技术来确定膜囊泡中心蛋白定位的类型以及这种定位是否需要脱落。在目标 3 中，将检查 centriolin 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，以便穿梭调节成分，例如centriolin，到中体。这将通过光激活中心粒蛋白的中心体池并使用延时视频共聚焦显微镜检查它是否可以到达中间体来进行测试。 公共卫生相关性：Centriolin 是脊椎动物母体中心粒的组成部分，是细胞分裂最后阶段所必需的。它可以影响进入S期，并可能在肿瘤发生中发挥作用。没有中心蛋白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，产生作为中间体的双核细胞，这是肿瘤发生的标志。阐明涉及 Centriolin 的分子机制可能会提供细胞因子异常与癌症易感性之间的功能联系。