PROJECT SUMMARY Nuclear pore complexes (NPCs) span the inner and outer nuclear membranes and allow for the regulated transport of macromolecules across the nuclear envelope. In addition, NPCs have important transport independent functions, including influencing nuclear envelope dynamics and integrity, contributing to chromosomal organization and regulating gene expression. Many features of NPC structure and function are conserved throughout eukaryotes; however, NPC number, distribution, composition and function can change dramatically during development and in response to environmental signals. Additionally, increased NPC density has been observed in human diseases including cancer. Work in numerous organisms has identified the conserved transmembrane nucleoporin Ndc1 as a key factor required for NPC assembly and insertion into the nuclear envelope. However, the exact mechanism by which NPC insertion occurs remains unclear. Additionally, attempts to dissect the role of Ndc1 and other putative insertion factors at NPCs in yeast have been complicated by their dual functions in the insertion of the yeast spindle pole body. To overcome these issues, we have developed innovative genetic and imaging approaches to characterize proteins that interact with the fission yeast Ndc1 ortholog, Cut11. Using high-throughput membrane yeast-two hybrid screens, we identified novel Cut11 interacting proteins involved in lipid metabolism and membrane organization that are conserved in vertebrates and have determined how these interactions are modulated by Cut11 mutations. We have also developed 3D structured illumination microscopy and image analysis tools to allow us to visualize and quantify NPCs in vivo. Importantly, this approach allows for determining NPC composition while maintaining single NPC resolution, and has revealed a region with heterogenous NPC composition near the spindle pole body. We will utilize this powerful imaging platform to determine whether the newly identified Cut11 interacting proteins localize to NPCs and have a function in NPC assembly and insertion. We will also examine the molecular mechanisms that regulate the localization of the conserved nuclear envelope protein Tts1 to the NPCs and clarify its role in NPC assembly and distribution. Last, we will define the composition of NPCs in the region near the spindle pole body and determine how this specialized pool of NPCs is established and maintained. These studies will expand our understanding of how NPC assembly is regulated and will provide valuable insight into novel proteins with potentially conserved functions in NPC assembly from yeast to mammals.