Elucidating the mechanism of nuclear pore complex assembly in intact nuclei of live cells

核孔复合物（NPCs）是所有真核生物穿透核膜（NE）以介导核胞质运输的基本结构。为了细胞的生长和分裂，新的npc必须被组装成完整的细胞核。间期NPC组装的机制尚不清楚，几乎没有研究过（Rabut et al., 2004b）。本项目的目的是通过先进的光学显微镜和RNAi在活的哺乳动物细胞中完整细胞核的背景下研究这种机制。我们将在表达荧光DNA复制标记和核孔蛋白的细胞系中建立NPC组装的定量分析（Easwaran等人，2004年；Rabut等人，2004a）。在这些细胞的整个间期以单孔分辨率成像NE，然后提供与s期进展相关的NPC数量和空间分布的测量。使用这个实验，我们将首先定义组装事件的序列。其次，我们将通过sirna耗尽一大批候选蛋白质来筛选NPC组装所需的蛋白质（Elbashir等人，2001年）。候选因子包括核孔蛋白、Ran系统组分、膜融合机制和膜涂层组分，所有这些都与NE重塑有关（Hetzeret al., 2001; Liu et al., 2003; Walther et al., 2003b）。一旦确定了离散的机械步骤，我们将从超微结构上分析它们，以定义组装中间体的拓扑结构。通过这种方式，间期NPC组装的机制和这一过程所需的分子机制将在完整的细胞中被定义。

Nuclear pore complexes (NPCs) are essential structures that perforate the nuclear envelope (NE) of all eukaryotes to mediate nucleocytoplasmic traffic. For cell growth and division new NPCs have to be assembled into the intact nucleus. The mechanism of interphase NPC assembly is unknown and almost not studied (Rabut et al., 2004b). The aim of this project is to investigate this mechanism in the context of the intact nucleus by advanced light microscopy and RNAi in live mammalian cells. We will set up quantitative assays for NPC assembly in cell lines expressing fluorescent DNA replication markers and nucleoporins (Easwaran et al., 2004; Rabut et al., 2004a). Imaging the NE at single pore resolution throughout interphase in these cells then provides a measure of NPC number and spatial distribution in relationship to S-phase progression. Using this assay we will first define the sequence of assembly events. Second, we will screen the requirements of a large group of candidate proteins for NPC assembly by depleting them with siRNAs (Elbashir et al., 2001). Candidates include nucleoporins, components of the Ran system, membrane fusion machinery, and components of membrane coats, all of which have been implicated in NE remodeling (Hetzeret al., 2001; Liu et al., 2003; Walther et al., 2003b). Once discrete mechanistic steps are identified, we will analyze them ultrastructurally to define the topology of assembly intermediates. In this manner the mechanism of interphase NPC assembly and the molecular machinery required for this process will be defined in intact cells.