Nuclear Membrane Fusion in Xenopus Egg Extracts

非洲爪蟾卵提取物中的核膜融合

• 批准号: 7413638
• 负责人: Martin W Hetzer
• 金额: $ 35.33万
• 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/7413638
• 关键词: Affinity; Affinity Chromatography; Binding; Binding Proteins; Biochemical; Biogenesis; Biological Assay; Caenorhabditis elegans; Cell division; Cellular Membrane; Chromatin; Chromosomes; Color; Complex; Condition; Cytosol; Data; Defect; Endoplasmic Reticulum; Eukaryotic Cell; Event; Exclusion; Fingers; Fractionation; Gene Expression Alteration; Genomic Instability; Glass; Golgi Apparatus; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Image; In Vitro; Intracellular Membranes; Kinetics; Laboratories; Lead; Learning; Life; Mediating; Membrane; Membrane Fusion; Methods; Microscopy; Mitosis; Mitotic spindle; Molecular; Molecular Target; Monomeric GTP-Binding Proteins; Nature; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Pattern; Personal Satisfaction; Play; Process; Proteins; Proteomics; RNA Interference; Recombinants; Role; Running; Slide; Surface; Techniques; Testing; Time; Transmission Electron Microscopy; Tubular formation; Ubiquitin; Vesicle; Xenopus; analog; egg; inorganic phosphate; insight; mutant; novel; nucleocytoplasmic transport; p97 ATPase; rab GTP-Binding Proteins; research study; seal; tool; xanthosine

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会导致基因表达模式的改变和基因组的不稳定性。我们已经生成了关于核组装的两个主要调节因子 GTPase Ran 和 AAA-ATPase p97 的新信息。我们的具体目标是： 1. 了解 Importin b（一种主要的 RanGTP 结合蛋白）如何调节 NE 融合并识别与其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 如何抑制染色质结合囊泡的融合。 NE 小管形成和重组的动态将通过染色质包被的载玻片上的实时成像来可视化。我们将使用生化分级分离方法和亲和层析来鉴定 Importin b 的结合伴侣。 2. 表征 NE 膜密封并识别 Ufd1/Np14 调节的 NE 融合机制。为了了解 p97/Ufd1/Np14 依赖性闭合 NE 的形成，我们将使用 TEM（一种新型核排除测定）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 Ufd1/Np14 复合物的重组形式作为亲和层析的基质。 3. 表征 NE 形成中第二个 GTPgS 敏感步骤。我们将使用 2 色融合测定和 TEM 来表征该膜融合事件。我们将分析染色质结合囊泡上的膜相关 GTP 酶活性。为了确定额外 GTP 酶的性质，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内膜融合的 GTP 酶（例如 Rab GTP 酶）是否参与 NE 形成。作为替代方法，我们将使用光亲和方法、叠加分析和蛋白质组学方法来鉴定第二个 GTP 酶。