How Kinesin-8 Alternates Between Motility and Microtubule Depolymerization to Control Mitotic Spindle Dynamics

During mitosis, microtubules undergo dynamic reorganization to form the mitotic spindle that aligns chromosomes at the cell’s equator and then equally distributes this genetic information to daughter cells. Kinesin-8 motor proteins regulate the lengths of microtubules during these events by walking processively to microtubule ends where they influence the addition or removal of tubulin subunits. This form of bimodal operation has not been observed in any other kinesin family, and thus the molecular mechanisms of kinesin-8s have drawn significant attention. Using X-ray crystallography, cryo-electron microscopy, and biochemical analyses of recombinant kinesin-8 motors from Candida albicans (CaKip3), we obtained evidence that this ability to switch between walking and microtubule-shortening activities stems from an extended and flexible loop-2 region, which is unique to the kinesin-8 motor domain. We show that kinesin-8s use loop-2 to form interactions with tubulin that sense the shape of tubulin protofilaments in the microtubule, and then activate either their motile or microtubule shortening activities accordingly. On straight tubulin protofilaments in the middle of the microtubule, loop-2-tubulin contacts restrict conformational changes of the motor domain in a way that couples ATP hydrolysis energy to walking. On curved tubulin protofilaments, which are abundant at microtubule plus ends, the loop-2 region morphs to accommodate the different shape of tubulin. This causes the kinesin to couple ATP binding to conformational changes in the motor domain that further bend and destabilize tubulin-tubulin contacts. When these kinesin-8 activities are eliminated from C. albicans cells by knockout of both copies of the KIP3 gene, the astral microtubules and mitotic spindles of these cells become abnormally long and less dynamic. This loss of microtubule length regulation subsequently prevents the fungus from forming invasive hyphal filaments. Based on these observations, and the unique structural properties of CaKip3, we probed the effects of CaKip3 inhibition with the kinesin-8 inhibitor sovilnesib to assess its potential application for the treatment of C. albicans infections. Together, our structure-function work on CaKip3 and our live-cell analysis of C. albicans provide valuable insight into the mechanism of the bifunctional kinesin-8 family and how these motors enable the segregation of genetic material.
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