Grant: Discovery Grants Program - Individual

The goal of the proposed research is to reveal the mechanisms controlling cytokinetic diversity in human cells. Cytokinesis typically occurs at the end of mitosis to separate the daughter cells, which must occur properly to avoid pathologies. The core cytokinesis machinery is conserved, but the expression and regulation of this machinery differs with cell type. For example, we found that cells at the earliest state of fate acquisition in C. elegans embryos have distinct cytokinesis phenotypes. Our knowledge of human cell cytokinesis is derived from transformed cancerous cell lines and inferred from studies of model organisms. However, cells are extremely diverse in their morphology and function. While epithelial cells lining the intestine have apicobasal polarity for the uptake of nutrients from the lumen, liver hepatocytes gain ploidy for the secretion of enzymes required for metabolism. Thus, cytokinesis is expected to be regulated differently in epithelial cells where daughters need to retain apicobasal polarity, compared to hepatocytes which need to abort cytokinesis to gain ploidy. Given our lack of knowledge of how most human cells divide, the proposed research program will define a range of cytokinesis phenotypes in diverse human cell types, and the mechanisms controlling these phenotypes in three major aims. Cytokinesis occurs due to the ingression of a contractile ring that is controlled by multiple pathways. My group has made major contributions to our knowledge of how spindle-dependent and-independent pathways regulate the ring, and we speculate that the requirement for these pathways changes with cell fate, size and ploidy. Recently, we used CRISPR-Cas9 editing to endogenously tag cytokinesis genes with fluorescent proteins in different cultured human cells to reveal key differences in the ring, and built a human iPSC line amenable to tagging genes of interest with mNeonGreen. With these reagents, we will use microscopy and quantification methods with approaches to perturb pathways and cortical properties to: 1) define a range of ring phenotypes from different cultured cells and differentiated iPSCs, 2) determine the mechanisms controlling ring position by revealing how pathways synergize in response to cell fate, size and ploidy, and 3) determine the mechanisms controlling ingression by revealing how the shape of the ring influences the efficiency and symmetry of ring closure. These studies will reveal new fundamental knowledge of how cytokinesis occurs in diverse human cell types, including non-transformed, differentiated cells. By understanding how cytokinesis phenotypes are regulated in response to specific parameters, we can predict how cytokinesis is regulated in other cell types, and how changes in these parameters correlate with pathologies.