Mechanisms of Nuclear Migration and Anchorage

描述（由申请人提供）：在多种细胞类型中，细胞核的位置受到仔细控制。核迁移在正常细胞迁移事件和转移中起作用;核迁移缺陷导致神经系统疾病无脑畸形。核锚定在神经肌肉接头的发育中起作用，并可能导致肌营养不良。一组三个保守的核膜蛋白是正确的核定位在C。优雅我们的目标是表征这些蛋白质如何控制核定位，并确定其他蛋白质的功能与他们或平行。我们的中心假设是，α-84在内核膜上起作用，将α-83和ANC-1募集到外核膜上。它们一起桥接核被膜，将核基质连接到细胞骨架。我们的第一个目标将确定这三种蛋白质的拓扑结构，使用体内蛋白酶保护试验和免疫EM。目的2将利用分子遗传学技术来检测我们的模型的中心环节，在SUN结构域之间的相互作用，SUN-84和KASH结构域的<$-83和ANC-1。在目标3中，我们期望通过使用新的结构域的必需部分，通过生物化学和分子筛选来识别相互作用的伴侣，从而将α-83连接到细胞骨架。在目标4中，我们采用遗传学方法通过克隆unc-83等位基因的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个保守的核膜组分中鉴定核定位所需的蛋白质。在发育系统中联合收割机结合遗传和分子方法的能力使C. elegans为这些研究提供了强大的系统。总之，这些研究将为细胞核如何在细胞质中定位这一基本问题提供机理性的见解。

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.