Mechanisms of Nuclear Migration and Anchorage

描述(申请人提供)：在各种各样的细胞类型中，细胞核的位置被仔细地控制。核迁移在正常的细胞迁移事件和转移中起作用；核迁移缺陷导致神经性疾病Lissephy。核锚定在神经-肌肉连接的发育过程中起作用，并可能导致肌营养不良。线虫的核定位需要一组三个保守的核膜蛋白。我们的目标是确定这些蛋白质如何发挥控制核定位的功能，并识别与它们一起或平行发挥功能的其他蛋白质。我们的中心假设是UNC-84在内核膜发挥作用，将UNC-83和ANC-1募集到外核膜。它们一起连接核膜，将核基质与细胞骨架连接起来。我们的第一个目标是使用体内蛋白酶保护试验和免疫-EM来确定这三种蛋白质的拓扑结构。目的2将使用分子遗传学技术来测试我们模型的中心环节，即UNC-84的SUN结构域与UNC-83和ANC-1的Kash结构域之间的相互作用。在目标3中，我们希望通过使用UNC-83新结构域的基本部分，通过生化和分子筛选确定相互作用的伙伴，将UNC-83与细胞骨架联系起来。在目标4中，我们采用遗传方法通过克隆现有的UNC-83等位基因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RNAi来筛选更多的增强子，并从蛋白质组学纯化的51个保守的核膜成分中鉴定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线虫成为这些研究的强大系统。总之，这些研究将为细胞核如何在细胞质中定位这一根本问题提供机械性的见解。

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.