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）：围绕隔离染色体的核包膜（NE）的改革是有丝分裂结束时的关键事件。在此关键过程中的缺陷可能会导致基因表达模式和基因组不稳定性的改变。我们已经生成了有关核装配的两个主要调节因子GTPase RAN和AAA-ATPase P97的新信息。我们的具体目的是：1。了解Importin B（一种主要的RANGTP结合蛋白）如何调节NE融合并确定与之相互作用的分子靶标。我们将使用2色融合测定和透射电子显微镜（TEM）来表征Importin B如何抑制染色质囊泡的融合。 NE小管形成和重组的动力学将通过在染色质涂层的载玻片上实时成像来可视化。我们将使用生化分级方法和亲和力色谱法来识别Importin b的结合伴侣。 2。表征NE膜密封并确定UFD1/NP14调节的NE融合机械。为了了解闭合NE的p97/ufd1/np14依赖性形成，我们将使用TEM，一种新型的核排除测定法和实时显微镜。为了鉴定与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复合物作为亲和色谱的基质。 3。表征第二个GTPGS敏感的步骤。我们将使用2色融合测定和TEM来表征此膜融合事件。我们将分析与染色质结合囊泡上与膜相关的GTPase活性。为了确定其他GTPase的性质，我们将在秀丽隐杆线虫中执行RNAi筛选，以测试已知可以介导细胞内膜融合（例如RAB GTPases）的GTPases参与NE形成。作为另一种方法，我们将使用照片亲和力方法，覆盖分析和蛋白质组学方法来识别第二个GTPase。