Nuclear Membrane Fusion in Xenopus Egg Extracts

非洲爪蟾卵提取物中的核膜融合

• 批准号: 7049668
• 负责人: Martin W Hetzer
• 金额: $ 36.39万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049668
• 关键词: Caenorhabditis elegans; RNA interference; Xenopus; Xenopus oocyte; adenosinetriphosphatase; affinity chromatography; animal extract; cell cycle; chromatin; enzyme activity; guanine nucleotide binding protein; guanosinetriphosphatases; intermolecular interaction; laboratory rabbit; membrane biogenesis; membrane fusion; molecular /cellular imaging; molecular assembly /self assembly; nuclear membrane; protein binding; protein structure function; proteomics; recombinant proteins; transmission electron microscopy; vesicle /vacuole

DESCRIPTION (provided by applicant): Reformation of the nuclear envelope (NE) around the segregated chromosomes is a key event at the end of mitosis. Defects in this key process may result in alteration of gene expression patterns and genomic instability. We have generated new information about the two major regulators of nuclear assembly, the GTPase Ran and the AAA-ATPase p97. Our specific aims are: 1. To understand how Importin b, a major RanGTP binding protein, regulates NE fusion and to identify the molecular targets with which it interacts. We will use a 2-color fusion assay and transmission electron microscopy (TEM) to characterize how Importin b inhibits the fusion of chromatin bound vesicles. The dynamics of NE tubule formation and reorganization will be visualized by live imaging on chromatin-coated glass slides. We will use biochemical fractionation methods and affinity chromatography to identify the binding partner(s) of Importin b. 2. To characterize NE membrane sealing and to identify the Ufd1/Np14 regulated NE fusion machinery. To understand p97/Ufd1/Np14-dependent formation of a closed NE, we will use TEM, a novel nuclear exclusion assay, and real time microscopy. To identify additional proteins that interact with Ufd1/Np14 and participate in NE sealing, we will use a recombinant form of Ufd1/Np14 complex as matrix for affinity chromatography. 3. To characterize a second GTPgS-sensitive step in NE formation. We will use the 2-color fusion assay and TEM to characterize this membrane fusion event. We will analyze a membrane-associated GTPase activity on chromatin-bound vesicles. To identify the nature of the additional GTPase(s) we will perform an RNAi screen in C. elegans to test if GTPases known to mediate intracellular membrane fusion (e.g. Rab GTPases) are involved in NE formation. As an alternative approach we will use photo-affinity methods, overlay assays and proteomic approaches to identify the second GTPase.

描述（由申请人提供）：分离染色体周围核膜（NE）的重组是有丝分裂结束时的关键事件。这一关键过程的缺陷可能导致基因表达模式的改变和基因组的不稳定。我们对核组装的两个主要调控因子GTPase Ran和aaa - atp酶p97有了新的认识。我们的具体目标是：1。了解主要的RanGTP结合蛋白导入蛋白b如何调节NE融合，并确定其相互作用的分子靶点。我们将使用双色融合实验和透射电子显微镜（TEM）来表征进口蛋白b如何抑制染色质结合囊泡的融合。NE小管形成和重组的动态将通过染色质涂层玻片的实时成像可视化。我们将使用生化分馏方法和亲和层析法来鉴定Importin b的结合伙伴。表征NE膜密封特性，鉴定Ufd1/Np14调控的NE融合机制。为了了解p97/Ufd1/ np14依赖性封闭NE的形成，我们将使用TEM，一种新的核排除试验和实时显微镜。为了确定与Ufd1/Np14相互作用并参与NE密封的其他蛋白质，我们将使用重组形式的Ufd1/Np14复合物作为亲和层析的基质。3. 表征NE地层中第二个gtpgs敏感步骤。我们将使用双色融合实验和透射电镜来表征这一膜融合事件。我们将分析染色质结合囊泡上的膜相关GTPase活性。为了确定额外的GTPase的性质，我们将在秀丽隐杆线虫中进行RNAi筛选，以测试已知介导细胞膜内融合的GTPase（例如Rab GTPase）是否参与NE的形成。作为一种替代方法，我们将使用光亲和方法，覆盖分析和蛋白质组学方法来鉴定第二个GTPase。