Regulation of microtubule bundle mechanics by prc1 in metaphase and anaphase

Abstract

May2025School of ScienceThe mitotic spindle is composed of distinct networks of microtubules, including interpolar bundles that can bridge sister kinetochore fibers and bundles that organize the spindle midzone in anaphase. The crosslinking protein PRC1 can mediate such interactions between antiparallel microtubules. PRC1 is a substrate of mitotic kinases including CDK/cyclin-B, suggesting that it can be phosphorylated in metaphase and dephosphorylated in anaphase. How these biochemical changes to specific residues regulate its function and ability to organize bundles is not known. Here, we perform biophysical analyses on microtubule networks crosslinked by two PRC1 constructs, one a wild-type reflecting a dephosphorylated state, and one phosphomimetic construct with two threonine to glutamic acid substitutions near PRC1’s microtubule binding domain. We find that the wild-type construct builds longer and larger bundles that form more rapidly and are much more resistant to mechanical disruption than the phosphomimetic PRC1. Interestingly, microtubule pairs organized by both constructs behave similarly within the same assays. Our results suggest that phosphorylation of PRC1 in metaphase would tune the protein to stabilize smaller and more flexible bundles, while removal of these PTMs in anaphase would favor the assembly of larger more mechanically robust bundles to resist chromosome and pole separation forces at the spindle midzone.In addition to these findings on PRC1’s biochemical regulation during mitosis via phosphorylation, we have begun characterizing the biophysical properties of PRC1 binding using a combination of in vitro experiments and computational simulations to theoretically model protein-protein interactions in the spindle. To achieve this, we are collaborating with mathematicians and physicists to focus on creating models that predict the formation of the mitotic spindle via relevant motor and non-motor crosslinking proteins. A computational model that reflects braking and coasting behaviors exhibited by crosslinked microtubule pairs based on previously published data from our lab has been developed. We find that braking occurs with smaller microtubule separation compared to coasting; the reduced separation between microtubule pairs results in increased resistive forces exerted by PRC1 and thus a reduced sliding speed. The model also shows that higher initial sliding speeds lead to a transition to braking. The results give insight on the relationship between microtubule separation and forces in the spindle exerted by crosslinkers and other MAPs. Furthermore, our collaborative project is currently exploring the possibility of PRC1 cooperativity. Thus far, data on the rate of PRC1 recruitment to single microtubules and overlaps from experiments suggest that a simplified binding model does not sufficiently explain PRC1’s binding behavior, as occupancy effects do not account for the experimental results. We plan on pursuing these findings further, as they may give insight into why PRC1 preferentially binds to antiparallel overlaps compared to single microtubules. The works presented here characterize the behaviors and regulatory mechanisms of the essential human mitotic crosslinker PRC1 via biochemical and biophysical approaches, as well as with structural and computational modeling.Ph