Single-molecule biophysics of kinesin family motor proteins [electronic resource]
Keywords: Physics.

Kinesin family proteins are nanoscale motors involved in many essential biological processes, such as intracellular transport and cell division. The biological function of most kinesin motors is to use the energy from ATP hydrolysis to move cargo through a crowded cellular environment, quickly taking 8-nm steps along cytoskeletal microtubules. By maintaining its two motor domains (heads) out of phase, kinesin can complete hundreds of steps per encounter with the microtubule, and can do so against pN-scale loads. The physiological role of kinesin is directly related to its movement and in this dissertation I present several single-molecule studies where the force-dependent motion of individual kinesin motors was studied using optical trapping techniques. In humans, the kinesin superfamily includes over forty genes encoding different kinesin proteins, classified into 15 families, and motors from several families were studied in this work. Optical traps use lasers to detect the position of biological molecules, at nm-scale resolution, and to directly manipulate them by applying pN-scale forces. In this dissertation, I present two novel optical traps. The first uses highly linear electro-optic deflection of the laser light to create an instrument with fast feedback that is optimized for work with kinesin motors. The second instrument, an "Optical Torque Wrench", is a trap that can apply both forces and torques on birefringent particles. By controlling the light polarization in the sample plane, the rotation of nanofabricated quartz cylinders can be controlled in real time while the applied torque is measured directly. The functionalized particles can be used to twist DNA or other biological molecules. The kinesin motor domains are coordinated during stepping and the inter-head communication is believed to be conferred by the neck linker, a 14-amino acid structural element connecting the head to the common coiled-coil stalk. By extending this segment, we could examine its role in gating the mechanochemical cycle. A six-amino acid insert in the neck linker of a cysteine-light human kinesin construct led to unexpected ATP-dependent backstepping under load. These observations could be explained by a branched pathway where both ATP unbinding and hydrolysis were gated by the direction of the neck linker. Lengthening the neck linker also led to futile hydrolysis. Further experiments on the effects of neck linker length were done with a series of Drosophila Kinesin-1 mutants, with one to six extra residues in the neck linker. The rate of force-dependent rear head release and the internal strain developed during stepping was determined from force-dependent velocities and we also found that the mechanism of detachment from the microtubule depends on the direction of load. The heterotrimeric Kinesin-2 motors are unique in that they are the only kinesin family motors that consist of two different catalytic domains. Here, the mammalian Kinesin-2, KIF3A/B, was studied in detail by performing optical trapping experiments with both the wild-type dimer and with homodimers (KIF3A/A and KIF3B/B). A pathway that incorporates the individual catalytic cycles for KIF3A and KIF3B could explain all force- and ATP-dependent kinetics and surprisingly we found that the run lengths for KIF3A/B were significantly shorter than for Kinesin-1. Furthermore, motors with the weakly force-dependent KIF3A head "slipped" and exhibited short run lengths that were rescued under no load, indicating that KIF3A/B combines a Kinesin-1-like motor domain (KIF3B) with a unique and "weak" one (KIF3A). Finally, I present motility experiments where force-dependent kinetics were explored for several other kinesin family motors. KIF17 (Kinesin-2) and CENP-E (Kinesin-7) are robust, processive motors whereas KIF4A, a Kinesin-4 motor, is fast but unable to sustain significant loads. These results, together with those for Kinesin-1, KIF3A/B (Kinesin-2), and other motors, show that forces are needed fully reveal the motor characteristics and differences between various kinesin proteins. They also illustrate the remarkable diversity within the kinesin superfamily.