1 Biology Department, Concordia University, Montréal, QC H4B 1R6, Canada.
2 Biology Department, Concordia University, Montréal, QC H4B 1R6, Canada. Electronic address: alisa.piekny@concordia.ca.

Cytokinesis, the final step of cell division, relies on ingression of a precisely positioned actomyosin ring. Chromatin-associated Ran-GTP fine-tunes ring position, although the mechanism remains unclear. We hypothesize that depletion of Ran-GTP between segregating chromosomes leads to equatorial enrichment of importins, promoting recruitment of the scaffold protein anillin. However, the role of importins during anaphase is not known. Here, we tested whether importins form a gradient in response to chromatin-associated Ran-GTP and regulate ring assembly in two cultured human cell lines. We endogenously tagged importin-ß1 with mNeonGreen in hypotriploid HeLa cells and euploid HCT 116 cells. Live-cell imaging revealed that importin-ß1 becomes transiently enriched between segregating chromosomes in anaphase HeLa cells, but not in HCT 116 cells. Using a newly developed optogenetic tool to rapidly disrupt importin-ß1 function, we found that importin-ß1 is required for ring ingression in HeLa cells. We speculated that the stronger requirement for importin-ß1 in HeLa cells reflects differences in chromatin-to-cytosol ratio compared with HCT 116 cells, which could determine whether the Ran-GTP gradient reaches the cortex. Consistently, FLIM-FRET imaging showed that equatorially enriched importin-ß1 is Ran-free in HeLa cells, but not in HCT 116 cells. A predictive model of the Ran-free importin-ß1 gradient identified factors that modulate gradient formation, including chromatin-to-cytosol ratio. Experimentally decreasing or increasing the chromatin-to-cytosol ratio in HeLa and HCT 116 cells, respectively, altered importin-ß1 and anillin localization to resemble the other cell type. Our findings suggest that highly aneuploid cancer cells may depend on importin-mediated anillin recruitment, representing a targetable weakness. VIDEO ABSTRACT.