Abstract

Cytokinesis is the physical separation of a cell into two daughter cells through the ingression of a contractile ring. Failure of cytokinesis can alter cell fate or ploidy, leading to developmental abnormalities and disease. To ensure fidelity, cytokinesis is tightly regulated by multiple spatiotemporal pathways associated with the mitotic spindle and chromatin. However, much of our current understanding of human cytokinesis derives from HeLa cells, and the requirement for these regulatory pathways likely varies across cell types. Our group recently demonstrated such diversity by comparing the localization of cytokinetic proteins in five distinct human cell lines, highlighting the need to study cytokinesis in both cancerous and healthy contexts. This work aims to investigate how chromatin-sensing regulates cytokinesis in cells with different ploidy and to uncover the mechanisms that control cytokinesis in normal human cells. In Chapter 2, we examined whether importins respond to the chromatin-associated Ran-GTP gradient to regulate contractile ring assembly. Using endogenous tagging, live-cell imaging, optogenetic perturbations, FRET–FLIM, and computational modeling, we found that importin-β1 becomes equatorially enriched and is required for cytokinesis in hypotriploid HeLa cells but not in euploid HCT 116 cells. A predictive model of Ran-GTP and Ran-free importin-β1, validated experimentally, identified factors such as the chromatin-to-cell size ratio that modulate this chromatin-sensing pathway. These findings suggest that highly aneuploid cancer cells depend on importin-mediated anillin recruitment, revealing a potential vulnerability that distinguishes them from diploid cells. In Chapter 3, we investigated cytokinesis in human induced pluripotent stem cells (iPSCs) with endogenously tagged cytokinetic proteins to elucidate mechanisms of ring assembly. We found that ring constriction is asymmetric in iPSCs, with anillin enriched at the leading edge of the furrow. Astral microtubules were shown to play dual roles: an outer pool bundled by central spindle proteins promotes ring assembly, while asters restrict the contractile ring to a defined cortical zone. Comparison with differentiated endoderm cells revealed broader contractile rings, suggesting that cytokinesis regulation changes during differentiation. RNA sequencing further showed that spindle-associated pathways are downregulated as iPSCs differentiate into hepatoblast progenitors and hepatocyte-like cells, potentially leading to defective ring assembly and cytokinesis failure. Overall, this work provides new insight into the diversity of cytokinetic regulation in human cells and represents a first step toward building a comprehensive atlas of cytokinesis mechanisms across healthy and cancerous cell types.