Presenting Author University of Hawaii at Hilo, Hawaii
Cytokinesis, the physical division of one cell into two, requires accurate positioning of the cleavage furrow between segregating chromosomes. Failure to properly position the cleavage furrow can result in aneuploidy and is a hallmark of cancer cells. However, key aspects of the molecular mechanisms that govern where the cleavage furrow forms remain poorly understood. Furrow formation is specified by localization of the conserved Rho-activator RhoGEF, which leads to local membrane ingression. RhoGEF binds to the conserved motor protein complex centralspindlin, which performs numerous essential functions during cytokinesis. While in vivo experimentation has resulted in multiple proposed models for how the centralspindlin-RhoGEF interaction mediates furrow formation, detailed molecular analysis is limited by the predominance of different and/or redundant pathways in various cell types. Here, we directly address this issue by performing in vitro reconstitutions of RhoGEF positioning using full-length, recombinant proteins. We find that purified centralspindlin transports RhoGEF along single microtubules as cargo. Following transport, centralspindlin then retains RhoGEF at the plus-end tips for extended periods of time, leading to local RhoGEF accumulation. Transport and retention are inherent behaviors of centralspindlin. Additionally, we find that RhoGEF binding to centralspindlin may stimulate centralspindlin motor activity, suggesting a possible positive feedback loop. Collectively, these results demonstrate centralspindlin’s motor activity and strong association with microtubules rapidly generate a high concentration of RhoGEF at areas where plus-end tips are abundant, such as at overlapping aster microtubules at the cell equator, to specify the position of cleavage furrow formation.