Chromokinesins NOD and KID use Alternative Nucleotide Mechanisms and One-Dimensional Diffusion to Target Microtubule Plus Ends

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Chromokinesins have been found to associate with chromosomes during cell division and play a variety of functions including chromosome positioning, chromosome condensation, spindle bipolarity, and cytokinesis. These motors are thought to generate the elusive polar ejection force that drives chromatin away from the spindle poles. Kinesin-10's are chromokinesins that include human KID (also known as KIF22) and Drosophila melanogaster NOD, which share an N-terminal kinesin-like catalytic domain, a central stalk domain, and a C-terminal DNA-binding domain. Due to their common in vivo physiologies, we propose that kinesin-10's work together in teams of motors to translocate/target to spindle microtubule plus-ends. Using steady-state kinetics, we have characterized the motor domain constructs of NOD and KID by measuring the basal, unpolymerized tubulin-stimulated, and MT-stimulated steady-state ATPase and GTPase kinetics under both low (∼21 mM) and physiologically relevant (∼111 mM) ionic strength conditions. These results suggest both purine nucleotides may be utilized by kinesin-10's in vivo and set the foundation for comparing the ATPase and GTPase mechanisms of these two unconventional kinesins. Using GFP-tagged kinesin-10's and total internal reflection fluorescence (TIRF) microscopy, we observed the microtubule-bound NOD either remaining stationary or engaging in a one-dimensional random walk across the microtubule lattice. These two modes of microtubule binding are interchangeable and movement along the microtubule is rapid (similar to kinesin-13 and other microtubule end-binding proteins) and without directionality preference. Under these conditions, we observe significantly higher incidence of microtubule end-binding events, which is increased after removal of the negatively-charge C-terminal tails of tubulin using subtilisin. Our continued studies reveal mechanistic and functional details of kinesin-10 force generation and therefore provide new insights into the molecular motion that drives the polar ejection force during cell division.