Jha, R; (2017) Regulation of recombinant human dynein complex studied in vitro. Doctoral thesis , UCL (University College London).

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Abstract

In cells, cytoplasmic dynei n is important for cell division and retrograde transport of organelles and macromolecules. Although a microtubule minus end directed motor, dynein often localises to the plus ends of microtubules. The growing m icrotubule plus ends are thought to serve as important sites of cargo loading and initiation of dynein driven transport. During mitosis, dynein interacts with the plus ends of cortical and kinetochore microtubules where its activity appears essential for spindle positioning, chromosome alignment and checkpoint protein removal. In cells, to track plus ends, dynein interacts with its key regulators — dynactin and Lis1 — which, together with the cargo adaptor BicD2, are also involved in dynein driven motion away from the plus ends. How do these dynein regula tors control the plus end localisation versus minus end motion of dynein on dynamic microtubules? To address this, I study the regulation of the recombinant human dynein by dynactin, Lis1 and BicD2 on dynamic microtubules in the presence of the end binding protein EB1 using an in vitro reconstitution approach combined with TIRF microscopy. This allows simultaneous investigation of dyne in motility and microtubule end tracking in the same assay and sheds light on the mechanism of dynein regulation for the two dynein processes.

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