High Resolution Measurements Of Kinesin Driven Microtubule Motility PDF Download

"Motor proteins kinesins and dynein are mechanoenzymes that catalyze ATP hydrolysis to take successive steps on microtubule filaments, while transporting organelles to their destinations. How do motor proteins efficiently navigate specific microtubule tracks to sort cargoes? The microtubule cytoskeleton regulates the trafficking and sorting of vesicles through interactions with microtubule-associated proteins, tubulin post-translational modifications and network organization. The impairment of transport causes neurodegenerative disease. The interplay of the microtubule-based regulatory factors poses challenges in understanding the regulation of intracellular transport and contributes to apparent discrepancies between studies in live cells and reconstituted in vitro systems. In my thesis, we address such regulatory mechanisms that modulate the efficient transport of cargoes on the microtubule network. Acetylated microtubules, abundant in neuronal axons and central regions of fibroblasts, constitute the preferred tracks for kinesin-1 motors. However, microtubule acetylation does not influence kinesin-1 motility in purified systems, suggesting that regulatory factors other than this post-translational modification contribute to the motor's track selection. We isolated intact microtubule networks from fibroblasts to conduct motility assays of purified kinesin-1. With this approach bridging live cell studies and reconstituted systems, we demonstrate that although tubulin acetylation does not directly impact kinesin-1 binding or motility, it is highly correlated with microtubule bundling, which leads to longer kinesin-1 run lengths. We expect that the higher local density of microtubules within bundles allows for rapid kinesin-1 re-attachment upon unbinding, extending the total displacement. We also investigated the role of neuronal microtubule-associated protein tau, which normally bundles acetylated axonal microtubules. Several studies show that tau strongly inhibits kinesin-1 in vitro, although paradoxically kinesin-1 driven transport is highly efficient on tau-decorated microtubules in neurons. We found that inhibition of kinesin-1 by tau is not affected by either microtubule acetylation or bundling on the isolated microtubule networks. This led us to further investigate how motor proteins can navigate tau-associated microtubules in fibroblasts, which do not endogenously express tau. As tau phosphorylation modulates tau's diffusivity on microtubules, we investigated the effects of wild-type (WT) tau (non-phosphorylated) and phosphomimetic tau (at Y18) on the motility of different organelles. We found that phosphomimetic tau does not inhibit the motility of centrally positioned lysosomes, whereas WT tau reduces the processivity of peripheral and central lysosomes similarly, as shown by mean squared displacement and radius of gyration measurements of their trajectories. Early endosomes are more sensitive to inhibition by both WT and phosphomimetic tau. Our model suggests that tau phosphorylation does not inhibit lysosomes that are driven by kinesin-1 on acetylated microtubule bundles, in contrast to the inhibition of kinesin-3 mediated transport in the periphery"--