Sunday, June 1, 2025
6/1/2025
Structure and Function of the Nuclear Pore Complex by Cryogenic-EM - Structure and Function of the Nuclear Pore Complex by Cryogenic-EM Abstract The nucleus is compartmentalized by double membranes of the Nuclear Envelope (NE) and macromolecules must cross this barrier during cell growth and differentiation. The Nuclear Pore Complex (NPC) forms a conduit for the bidirectional transport of proteins and RNA-protein complexes between the cytoplasm and nuclear interior. Regulated access to this gateway is used to control gene expression. Together, four co-axial rings form a cylindrical scaffold comprised of Nucleoporins (Nups). This scaffold anchors Nups with flexible FG-repeats (FXFG/GLFG) that project into the central channel to form a barrier in the constricted ground state, while FG-repeats in the dilated NPC provide binding sites for the transport of karyopherin/importin cargo complexes. A deeper understanding of this mechano-sensitive translocation channel requires a detailed knowledge of the core scaffold and the enigmatic central transporter. In a series of recent papers, we determined Nup domain models for isolated and in situ yeast NPCs with cryogenic electron tomography in their ground state and dilated conformations, respectively. We extended this work with cryogenic-EM and single particle methods to study the isolated NPC and provide a composite, multiscale structure of the 8-fold symmetric core scaffold. To date, we have analyzed NPCs from growing cells engaged in transport, whose plug-like feature in the central channel is comprised of cylindrically-arrayed FG repeats and cargo complexes. However, the dynamic core scaffold contracts to a ground state configuration during solubilization of the NE and purification, due to the loss of lateral membrane tension. To extend our previous studies, a one-step NPC purification has been refined to minimize aggregation and stabilize membrane protein anchors. We will use stationary cells to provide a more uniform NPC population for structure determination that will focus on the core scaffold, Nup connector networks and features of the central channel. In this analysis, we will leverage our experience and knowledge to visualize NPC components at higher resolution and determine structures with improved precision, due to the incorporation of refined Nup crystal structures and Alphafold2 computed structure models. We will also determine single particle structures of relevant Nup complexes that form the cytoplasmic RNA export platform and which stabilize the NPC within the pore membrane, including membrane anchors and Nup84 Y-complexes. This will provide additional data to create a more complete model of the ground state NPC. To probe dynamic features of the NPC, a biochemical treatment that enhances permeability will be used to alter the conformation of this mega-channel for structural studies. Collectively, our studies will provide further insights into the roles of Nups in core scaffold assembly, pore membrane anchoring and nucleocytoplasmic transport, while a comparison with NPC models from humans and frogs will clarify steps in the molecular evolution of this remarkable channel.
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