Investigating the Kif15-TPX2 Interaction for Spindle Assembly

Abstract

Mitotic cell division relies on complicated interactions between motor proteins and spindle assembly factors (SAFs) that result in the rearrangement of the microtubule cytoskeleton. A majority of microtubules (MTs) for spindle assembly are generated by branching MT nucleation at branch sites marked by Targeting Protein for Xklp2 (TPX2) condensates. The resulting dense MTs are slid apart by molecular motors to form a bipolar spindle. A previous study observing binding between the motor protein Kif15 and TPX2 suggests that Kif15 may bind during branching MT nucleation, prompting investigation into the Kif15-TPX2 interaction. This project aimed to identify the site on Kif15 that mediates its binding with the SAF TPX2 and determine how this interaction influences recruitment to the MT. Using TIRF microscopy, I found that the presence of TPX2 enhances Kif15 tail localization to MTs. Since Kif15 has been previously shown to cooperate with the motor Eg5 to build and maintain the bipolar mitotic spindle, I performed additional pulldown assays with both Kif15 and Eg5 to provide insight into how multiple motor proteins interact with SAFs to build and maintain the bipolar mitotic spindle. I found that Eg5 motor binding to the C terminal portion of TPX2 decreases when in the presence of Kif15 motor and tail domains. This research furthers the model of Kif15 as a tethered MT crosslinker, providing a better understanding of how nucleation and motor molecules work together to orchestrate spindle assembly.