Exchange of molecules between the cytoplasm and the nucleus occurs through conduits called nuclear pore complexes (NPCs). NPCs consist of roughly 30 distinct proteins (nucleoporins), forming a central channel with filaments extending into the nucleus and cytoplasm. Besides macromolecular trafficking, nucleoporins participate in the control of gene expression, chromatin maintenance and mitotic progression. Their action in these processes offers a rich variety of possible mechanisms for biological regulation via nucleoporins and for coordination amongst cellular functions. However, understanding the roles of individual nucleoporins in vertebrate cells is limited because their manipulation by standard methods (e.g., RNAi) has been problematic due to their abundance and their multiple essential roles for cell viability: vertebrate nucleoporin depletion can cause highly pleiotropic phenotypes, many of which may be secondary consequences of extended incubations with sub-physiological nucleoporin levels. To circumvent this problem, we are systematically targeting nucleoporin genes using CRISPR/Cas9 gene editing to create cell lines wherein endogenous nucleoporins have Auxin Inducible Degron (AID) tags, allowing their degradation in a rapid and regulated manner. We are using this approach to analyze the function of individual nucleoporins in a variety of contexts. We are currently focused on three regions of the NPC. First, Nup153, Tpr, and Nup50 localize to nucleoplasmic filaments, and they are collectively called the basket nucleoporins. The nucleoplasmic filaments have been proposed to serve as a platform for RNA modification and export, as well as for chromatin remodeling. AID-tagged basket nucleoporins localize correctly, are functional within NPCs and are rapidly degraded upon Auxin addition (<2 hours). To assess the role of each nucleoporin, we followed cell growth in the absence and presence of Auxin, as well as nuclear trafficking and the immediate response in gene expression profile (RNA-sequencing). Moreover, we assessed the interdependence of the basket components, and associated with the basket proteins (SENP1, SENP2, MAD1) on each other, on the stability of the assembled nuclear pore, and ability to reform the nuclear pore post mitosis. Our data show that individual basket nucleoporins play distinct roles in nuclear function and gene expression, and that this system provides us the capacity to dissect these roles at a molecular level. Second, the central domain of NPCs consists of three co-axial rings that each display a lattice-like arrangement, and that are called the cytoplasmic ring, inner ring, and nucleoplasmic ring, respectively.The Nup107-160 complex contains nine core nucleoporins (Nup37, Nup85, Seh1, Sec13, Nup96, Nup107, Nup133 and Nup160), with a tenth subunit called ELYS required for chromatin recruitment. The Nup107-160 complex forms the scaffold underlying the cytoplasmic and nuclear rings. The Nup107-160 complex also associates with kinetochores in metazoan mitosis, where it plays a transport-independent role in spindle assembly and chromosome segregation. Earlier efforts at in vivo analysis of individual vertebrate Nup107-160 complex members during interphase and mitosis have been problematic because their abundance and stability makes them difficult to deplete by RNAi: The extended time required for depletion causes progressive defects in both interphase and mitotic functions that can produce adverse secondary consequences. Moreover, the levels of non-targeted subunits decrease during extended RNAi depletion, possibly suggesting that they become unstable when the larger complex is absent. AID-tagged Nup107-160 complex nucleoporins assemble into functional NPCs, and they are degraded rapidly (<4 hours) after auxin addition, with minimal impact on the stability of other Nup107-160 complex members. We have assessed the roles of Nup107-160 complex subunits in nuclear trafficking through comparison of nuclear import and export in the absence and presence of auxin. We are now examining how individual complex members contribute to the structural stability of NPCs, and the inter-dependence between subunits for Nup107-160 complex persistence at existing NPCs, as well as for spindle function and post-mitotic NPC assembly. Third, nucleoporins associated with the cytoplasmic filaments (CFs) include RanBP2 (also known as Nup358), Nup214, Nup88 and Aladin. RanBP2 binds the SUMO1-modified form of the Ran GTPase activating protein (RanGAP1-SUMO1), and the SUMO conjugating enzyme Ubc9 in a stable complex (RRSU complex). CFs interact with transport complexes as they enter and exit the nucleus, and CFs interact with components of the microtubule cytoskeleton. During mitosis, some CF components localize on mitotic spindles and play important roles in spindle assembly or function. In particular, the RRSU complex associates to mitotic kinetochores in a Crm1- and Ran-dependent manner, and this recruitment is important for the formation of spindle-kinetochore attachments. Nup214 and Nup88 have likewise been reported to have important mitotic roles. AID-tagged CF nucleoporins correctly local in functional NPCs. Upon auxin treatment, they are rapidly and specifically degraded, allowing us to assay changes in cellular functions. We are particularly investigating nuclear transport, gene regulation, mitotic progression, and post-mitotic nuclear envelope and NPC re-assembly. These experiments collectively indicate that we are now able to assess the function of individual nucleoporins in vital cellular processes during both interphase and mitosis, and to dissect these processes at a molecular level. This offers an excellent opportunity to assess novel mechanisms of cellular regulation, as well as to assess how the disruption of nucleoporins contributes toward developmental defects and human disease.
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