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

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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.