Deciphering the Mechanics of Microtubule Bundles in Mitosis.

The proper organization of the mitotic spindle is required for successful chromosome segregation during cell division. This highly dynamic network consists of microtubules that are organized, bundled, and transported by motor and non-motor proteins that generate precisely balanced ‘active’ pushing and ‘passive’ frictional forces to give the spindle its mechanical robustness. How these mesoscale forces are produced and regulated by ensembles of mitotic proteins has been unclear. We are addressing this knowledge gap by directly measuring force production across reconstituted micron-scale microtubule bundles and simultaneously observing by single molecule fluorescence microscopy the localization of proteins that build these networks. Here we demonstrate that the essential microtubule crosslinking protein PRC1 performs distinct mechanical tasks in metaphase and anaphase to regulate motor protein activity. In anaphase, we have shown that PRC1 ensembles act like viscous dashpots, providing velocity-dependent resistance to microtubule separation. In metaphase, when CDK/cyclin-B activity is high, PRC1 is phosphorylated at two threonine residues near its microtubule-binding domain, while in anaphase these marks are removed. Surprisingly, we find that a phosphomimetic PRC1 construct organizes smaller bundles containing fewer filaments than the wildtype protein. In addition, this metaphase PRC1 analog produces significantly less mechanical resistance against motor-driven microtubule sliding than the anaphase analog. These changes are biochemically regulated by phosphorylation of microtubule-adjacent residues within the PRC1 protein and help explain the functional differences observed in cells between bridging fibers that connect sister kinetochore fibers in metaphase and the central spindle in anaphase that bridges the two separating spindle halves.