Investigating Kinesin-based Mitotic Spindle Assembly Backup Mechanisms Using Single Molecule Methods

Abstract

Molecular motors are essential to sustain life, carrying out cellular processes that require directed motion over diffusion. Kinesins are motors that use ATP hydrolysis to perform mechanical work. Mitotic kinesins generate force to move cytoskeletal filaments to facilitate accurate cell division. Single molecule methods, such as optical trapping, are well-suited to study kinesin mechanics, having the ability to obtain measurements with nanometer and piconewton resolution. Here, optical tweezers and fluorescence microscopy were used to investigate kinesin-12 Kif15 and kinesin-14 HSET. These motors act as different back-up mechanisms for mitosis. Kif15 ensures that centrosomes become separated by sliding microtubules apart to form the bipolar mitotic spindle when the main microtubule slider, Eg5, has been inhibited. HSET aids in focusing the spindle into a bipolar array when cancer cells have too many centrosomes, allowing for proper chromosome alignment. To elucidate how these unique kinesins are acting redundantly, their biophysical properties were investigated using a hierarchal approach.

Measurements of motor subdomains, single motors, and motor-microtubule bundles paired with stochastic simulations unveiled a mechanism for how Kif15 can rescue Eg5 function under chemotherapeutic conditions. Results also revealed the location-specific roles that Kif15 plays in the spindle: static MT crosslinker/force regulator under physiological conditions (parallel kinetochore MTs) and active MT slider under drugged conditions (anti-parallel interpolar MTs). These studies illuminate the therapeutic importance of Kif15, whose inhibition in tandem with an Eg5 poison would be more effective at halting cancer in the clinic.

HSET was also investigated using optical trapping to give insight about how a previously defined non-processive motor could accomplish macroscopic force generation during mitosis. We found that HSET acts as a force brake against Eg5 in vitro. The motor's ability to crosslink and slide MTs, as well as its processive nature, suggest that HSET can discriminate between different cellular environments. This reflects its conditional ability to be physiologically relevant when centrosome number is not two, making it an attractive cancer therapy target.