Mechanisms of Nuclear Migration and Anchorage

核迁移和锚定机制

• 批准号: 7034247
• 负责人: DANIEL A STARR
• 金额: $ 25.84万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7034247
• 关键词: Caenorhabditis elegans; RNA interference; cell component structure /function; cell migration; cell nucleus; cytoskeleton; helminth genetics; immunoelectron microscopy; membrane proteins; molecular genetics; nuclear matrix; nuclear membrane; protein localization; protein protein interaction; protein structure; protein structure function; protein transport

DESCRIPTION (provided by applicant): The position of the nucleus is carefully controlled in a wide variety of cell types. Nuclear migration plays a role in normal cell migration events and metastasis; defects in nuclear migration lead to the neurological disease Lissencephaly. Nuclear anchorage functions in the development of the neuro-muscular junction and may contribute to muscular dystrophy. A group of three conserved nuclear envelope proteins are required for proper nuclear positioning in C. elegans. Our objective is to characterize how these proteins function to control nuclear positioning and to identify other proteins that function with or in parallel to them. Our central hypothesis is that UNC-84 functions at the inner nuclear membrane to recruit UNC-83 and ANC-1 to the outer nuclear membrane. Together, they bridge the nuclear envelope to connect the nuclear matrix to the cytoskeleton. Our first aim will determine the topology of these three proteins using an in vivo protease protection assay and immuno-EM. Aim 2 will use molecular genetic techniques to test the central link of our model, the interaction between the SUN domain of UNC-84 and the KASH domains of UNC-83 and ANC-1. In aim 3 we expect to link UNC-83 to the cytoskeleton by identifying interacting partners through biochemical and molecular screens using essential portions of the novel domain of UNC-83. In aim 4 we take a genetic approach to identify additional proteins involved in nuclear positioning by cloning existing enhancer of unc-83 alleles. We will use genome-wide RNAi to screen for more enhancers and to identify proteins required for nuclear positioning from 51 conserved nuclear envelope components purified by proteomics. The ability to combine genetic and molecular approaches in a developmental system makes C. elegans a powerful system for these studies. Together, these studies will provide mechanistic insight into the fundamental problem of how the nucleus positions itself in the cytoplasm.

描述（由申请人提供）：细胞核的位置在多种细胞类型中仔细控制。核迁移在正常细胞迁移事件和转移中起作用。核迁移的缺陷导致神经系统疾病的脑脑。核锚定功能在神经肌肉结的发展中，可能导致肌肉营养不良。在秀丽隐杆线虫中，需要一组三个保守的核包膜蛋白才能进行适当的核定位。我们的目标是表征这些蛋白质如何控制核定位并鉴定与与之合作或并行作用的其他蛋白质。我们的中心假设是，UNC-84在内部核膜上起作用，将UNC-83和ANC-1募集到外核膜。他们一起桥接核包膜，将核基质连接到细胞骨架。我们的第一个目标将使用体内蛋白酶保护测定法和免疫EM来确定这三种蛋白质的拓扑结构。 AIM 2将使用分子遗传技术来测试我们模型的中心连接，UNC-84的太阳域与UNC-83和ANC-1的KASH域之间的相互作用。在AIM 3中，我们希望通过使用UNC-83的新型领域的生化和分子筛选来通过生化和分子筛选来识别相互作用的伴侣，将UNC-83与细胞骨架联系起来。在AIM 4中，我们采用一种遗传方法来通过克隆UNC-83等位基因的现有增强子来鉴定参与核定位的其他蛋白质。我们将使用全基因组的RNAi筛选更多增强剂，并确定从51个保守的核包膜成分纯化蛋白质组学所需的核定位所需的蛋白质。在发育系统中结合遗传和分子方法的能力使秀丽隐杆线虫成为这些研究的强大系统。总之，这些研究将提供有关核如何在细胞质中定位的基本问题的机械洞察力。