Antiparallel microtubule bundling supports KIF15-driven mitotic spindle assembly

The spindle is a bipolar microtubule-based machine that is crucial for accurate chromosome segregation. Spindle bipolarity is generated by Eg5 (a kinesin-5), a conserved motor that drives spindle assembly by localizing to and sliding apart antiparallel microtubules. In the presence of Eg5 inhibitors (K5Is), KIF15 (a kinesin-12) can promote spindle assembly, resulting in K5I-resistant cells (KIRCs). However, KIF15 is a less potent motor than Eg5, suggesting that other factors may contribute to spindle formation in KIRCs. Protein Regulator of Cytokinesis 1 (PRC1) preferentially bundles antiparallel microtubules, and we previously showed that PRC1 promotes KIF15-microtubule binding, leading us to hypothesize that PRC1 may enhance KIF15 activity in KIRCs. Here, we demonstrate that 1) loss of PRC1 in KIRCs decreases spindle bipolarity, 2) overexpression of PRC1 increases spindle formation efficiency in KIRCs, 3) overexpression of PRC1 protects K5I naïve cells against the Eg5 inhibitor STLC, and 4) PRC1 overexpression promotes the establishment of K5I resistance. These effects are not fully reproduced by a TPX2, a microtubule bundler with no known preference for microtubule orientation. These results suggest a model wherein PRC1-mediated bundling of microtubules creates a more favorable microtubule architecture for KIF15-driven mitotic spindle assembly in the context of Eg5 inhibition.

Movie S1: HeLa KIRCEB3‐GFP by DIC and GFP. HeLa KIRCEB3‐GFP cells were imaged every 5 minutes for 190 minutes. Video shows DIC images taken at a single focal plane and a maximum z‐projection of images taken in the FITC (GFP) channel. Scale bar, 10μm.Download Original Video (.5 MB) Movie S2: HeLa KIRCEB3‐GFP with PRC1‐mCherry overexpression by DIC, GFP, and mCherry. HeLa KIRCEB3‐GFP cells transfected with PRC1‐mCherry were imaged every 5 minutes for 190 minutes. Video shows DIC images taken at a single focal plane and a maximum z‐projection of images taken in the FITC (GFP) and mCherry channels. Scale bar, 10μm.Download Original Video (.3 MB)Movie S3: HeLa KIRCEB3‐GFP mCherry‐TPX2 overexpression by DIC, GFP, mCherry. HeLa KIRCEB3‐GFP cells transfected with mCherry‐TPX2 were imaged every 5 minutes for 190 minutes. Video shows DIC images taken at a single focal plane and a maximum z‐projection of images taken in the FITC (GFP) and mCherry channels. Scale bar, 10μm.Download Original Video (.3 MB)