Determining the role of Cullin9 in neuronal differentiation of human pluripotent stem cells (hPSCs)
Compared to somatic cells, hPSCs have a rapid cell cycle with abbreviated gap phases. As hPSCs differentiate, the G1 phase is lengthened, resulting in increased cell cycle duration. Neural stem cells (NSCs) have a lengthened G1 phase compared to hPSCs. Growing evidence indicates cell cycle progression, self-renewal and differentiation are related. Data from our lab indicates an E3 ubiquitin ligase Cullin9 (CUL9) may regulate cell cycle progression based on its localization and protein interactions in hPSCs. CUL9 levels increase during neuronal differentiation. Preliminary data indicates that CUL9 depletion in NSCs affects differentiation potential. However, the role of CUL9 during neuronal induction remains uncharacterized. My project focuses on examining CUL9 in hPSC-derived NSCs. I will first differentiate human stem cells into neural progenitor cells, then determine the subcellular localization of CUL9 in NSCs. Our data shows CUL9 localizes to mitotic spindles in hPSCs. I will determine if this localization is maintained during differentiation by immunofluorescent staining to determine subcellular localization in NSCs, hPSCs and control cells (e.g. fibroblasts). Additionally, we identified a novel interaction between the cell cycle regulatory protein, Anaphase Promoting Complex/Cyclosome (APC/C) and CUL9 in hPSCs. I plan to examine if this interaction is maintained in NSCs by co-immunoprecipitation of APC/C and CUL9. I hypothesize CUL9 will maintain mitotic spindle localization and interaction with APC/C in NSCs as in hPSCs. Insight into cell cycle control of NSCs may provide greater understanding of mechanisms underlying brain size control and its related disorders such as macrocephaly.