The Effect of Tubulin Acetylation and Detyrosination on the in vitro Motility of Kinesin-1.

Abstract

The purpose of this study was to investigate possible molecular mechanisms by which a wide variety of cargoes are transported to specific locations in cells. Long-range transport is carried out mainly by motor proteins moving cargo along microtubules. Tubulin, the protein that assembles into microtubules, undergoes a number of dynamic, post-translational modifications (PTMs) in cells, including α-tubulin acetylation and detyrosination, that generate microtubule heterogeneity. In vivo, motility of kinesin-1, a well-characterized plus-end directed motor, translocates along subsets of microtubules marked by α-tubulin acetylation and detyrosination. The goal of this dissertation work was to examine if microtubule PTMs directly modulate the motility of motors and serve as signals for regulating intra-cellular traffic. This hypothesis was examined by using total internal reflection (TIRF)-based in vitro motility assays to observe the motility of kinesin-1 along microtubules polymerized from purified tubulin in which acetylation and deacetylation was controlled through in vitro enzyme treatment. A comparison of the velocity, run length and microtubule binding of kinesin-1 from experiments on differentially modified microtubules was used to evaluate the effect of PTMs. The velocity of kinesin-1 on acetylated and deacetylated microtubules was indistinguishable at 0.64 μm/s. The run length on acetylated microtubules was found to be 0.64 μm, while it was virtually identical (0.65 μm) on deacetylated microtubules. The landing rate on acetylated and deacetylated microtubules was also indistinguishable at 4.0 events per μm/min. Further investigation of the physiologically-relevant and commonly occurring combination of α-tubulin acetylation and detyrosination yielded similar results with regard to the measured velocities, run lengths and landing rates for kinesin-1 as compared to deacetylated and tyrosinated microtubules. From these results I conclude that neither acetylation nor detyrosination, independently or in combination, directly affect kinesin-1 motility. Control assays with kinesin-1 motors extracted from a cell lysate revealed an approximately two-fold increased landing rate on tyrosinated versus detyrosinated microtubules. Taken together with published in vivo observations, these findings suggest that additional factors must play a role in the interaction between kinesin-1 and microtubule PTMs. I suggest that future work should investigate the role of α- and β-tubulin polyglutamylation and MAPs on kinesin-1 motility.