PROJECT SUMMARY Ultrastructural molecular/sub-molecular level aging-induced degradation in cellular assemblies makes them dys- functional leading to cellular dysfunction that causes and exacerbates the aging-related problems. However, no investigation has been carried out to explore such degradations due to the lack of imaging modalities that are sen- sitive enough to capture the sub-molecular level features inside the cells in situ. One such assembly is the Nuclear Pore complex which creates a conduit in the nuclear membrane essential for cytoplasm-nucleoplasm trafficking, chromatin silencing, transcriptional control, and other vital cellular processes. NPC is a highly dynamic assembly formed of extremely long-lived aging-affected proteins, whose composition, structure, and function deteriorate with aging. This deterioration then negatively impacts their critical cellular functions, causing a decay in the health and function of the cells, which is especially relevant for non-dividing, non-rejuvenating, and aging-prone cells like neurons, thereby contributing to neurodegeneration. Cryo-focused ion beam milling and electron tomography (cryo-FIB-ET) imaging is a novel and emerging imaging modality that allows us to peek into the cells at the sub-molecular level, enabling in situ structural biology. Complementing cryo-FIB-ET imaging, single-molecule imaging can be used to explore the identification of the building blocks of massive cellular assemblies and also the molecular mechanism of its assembly. Using cryo-FIB-ET, Dr. Singh recently identified the in situ architecture of the NPC (Cell (2022)). He has also contributed to the development and application of cryo-FIB-ET imaging with contributions to 4 publications in his postdoc. During Ph.D., Dr. Singh trained in single-molecule imaging to study the molecular mechanism of CRISPR enzymes and T4-bacteriophage motor with 11 publications, including 6 as first/co-first. By leveraging his dual expertise in cryo-FIB-ET and single-molecule imaging and a broad collaboration effort within UCSD, around UCSD and with his collaborator and co-mentor at Rockfeller, Dr. Singh will determine: • The structures and isoforms of aged NPCs to map its aging-accumulated degradations (K99 phase). • Aging-induced changes in composition of NPC and its building blocks (K99 to R00 transition). • The assembly process of the NPC to understand how aging affects it (R00 phase). Dr. Singh will receive the needed training under the mentorship of Drs. Elizabeth Villa and Michael Rout. These mentors have demonstrated excellence in research leadership and have trained multiple trainees who have transitioned to independent fulfilling careers in sciences. In addition, an excellent team of collaborators and advisory committee (Drs. Andrej Sali, Nan Hao, Elizabeth Villa, Michael Rout) has been assembled to assist Dr. Singh’s research and provide additional training and career support through the K99-R00 phase. The R00 Award phase will set...