Nuclear Membrane Fusion in Xenopus Egg Extracts

描述（由申请人提供）：分离染色体周围的核膜（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性。我们已经产生了关于核组装的两个主要调节器的新信息，即GTATRan和AAA-ATPase p97。我们的具体目标是：1.了解主要RanGTP结合蛋白Importin B如何调节NE融合，并确定与其相互作用的分子靶点。我们将使用双色融合试验和透射电子显微镜（TEM）来表征输入蛋白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力学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色谱法来鉴定Importin B的结合伴侣。2.为了表征NE膜密封并鉴定Ufd 1/Np 14调节的NE融合机制。为了了解p97/Ufd 1/Np 14依赖的封闭NE的形成，我们将使用TEM，一种新的核排斥试验，和真实的时间显微镜。为了鉴定与Ufd 1/Np 14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Ufd 1/Np 14复合物作为亲和色谱的基质。3.为了表征NE地层中的第二个GTPGS敏感步骤。我们将使用双色融合试验和TEM来表征这种膜融合事件。我们将分析染色质结合囊泡上的膜相关GTdR活性。为了鉴定额外的GT3的性质，我们将在C. elegans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。作为一种替代方法，我们将使用光亲和方法，覆盖测定和蛋白质组学方法来确定第二个GTdR。

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.