Mechanisms of Nuclear Migration and Anchorage

在各种各样的细胞类型中，细胞核的位置受到仔细的控制。 核迁移在正常细胞迁移事件和转移中发挥作用；缺陷 核迁移会导致神经性疾病--无脑畸形。核锚地 在神经-肌肉接头的发育中发挥作用，并可能有助于 肌肉营养不良症。一组三个保守的核膜蛋白是必需的 线虫体内适当的核定位。我们的目标是描述这些 蛋白质起着控制核定位和识别其他起作用的蛋白质的作用 与它们一起或与之平行的。我们的中心假设是UNC-84在内部起作用 核膜向外核膜募集UNC-83和ANC-1。一起， 它们桥接核膜，将核基质与细胞骨架连接起来。我们的第一次 AIM将使用体内蛋白酶保护来确定这三种蛋白质的拓扑结构 化验和免疫-EM法。目标2将使用分子遗传技术来测试中心链接 在我们的模型中，UNC-84的SUN结构域和Kash结构域之间的相互作用 UNC-83和ANC-1。在目标3中，我们希望通过识别将UNC-83与细胞骨架联系起来 通过生物化学和分子筛查，使用关键部分 UNC-83的新结构域。在目标4中，我们采用遗传方法来识别其他 克隆现有的UNC-83或UNC-84增强子参与核定位的蛋白质 等位基因。我们将使用全基因组RNAi来筛选更多的增强子。有能力 在一个发育系统中结合遗传、生化和分子方法 使C，elegans成为这些研究的强大系统。总而言之，这些研究将提供 对原子核如何在宇宙中定位这一根本问题的机械论见解 细胞质。

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 or unc-84 alleles. We will use genome-wide RNAi to screen for more enhancers. The ability to combine genetic, biochemical, 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.