Dissect the motility mechanism and regulation of the C-terminal kinesin KlpA using single-molecule fluorescence microscopy*

C-terminal kinesins such as HSET, Ncd and KlpA are naturally occurring microtubule-based motor proteins that play important roles in eukaryotic cell division by regulating spindle assembly. Microtubules are polar filaments with two distinct ends: the minus end and the plus end. Our previous work showed that while all other C-terminal kinesins exclusively exhibit directional preference for the microtubule minus end, KlpA uniquely exhibit directional preference for the microtubule plus end. In this talk, we will discuss our effort toward understanding the mechanisms underlying KlpA motility and its regulation. Specifically, we found that KlpA relies on a mechanical element called the central stalk (which connects the N-terminal tail and the C-terminal motor domains of KlpA) to achieve plus-end-directed motility on the microtubules. Building upon this finding, we have developed a method for engineering artificial C-terminal kinesins that uniquely exhibit KlpA-type plus-end-directed motility. Furthermore, we found that KlpA has a regulatory protein called TinA, which can directly interact with KlpA to reverse its directionality on the microtubule. Overall, our results are expected to significantly improve current understanding of C-terminal kinesin motors.