Functional regulation of cytoplasmic dynein in vivo

Abstract

In the crowded confines of the eukaryotic cell, where large cargo (gt 100 nm) are diffusionaly constrained, intracellular transport by motor proteins plays a crucial role in the exchange of material between various compartments of the cell and maintaining cellular homeostasis. Aberrant intracellular transport has been implicated in several disease states, including neurodegenerative disorders like Alzheimer s and Parkinson s disease. Cytoplasmic dynein and the kinesin family of motor proteins drive cargo movement on microtubules. Eukaryotes contain ~ 40 different types of kinesins with most carrying specific cargo towards the plus ends of the microtubules. While there are minus end directed kinesin motors (like kinesin-14 involved in mitosis), in many cell types cytoplasmic dynein is the only minus end directed motor protein and thus understanding how a single motor can transport various kinds of cargo remains an interesting question. Recent studies have revealed that dynein is inactive by default and is activated upon the formation of a tripartite complex containing dynein, the dynein regulator dynactin, and the cargo (via a cargo adaptor such as BicD2). However, how processive complexes come together in the complex in vivo milieu is unknown. In this thesis, I discuss my research over the past five years to understand how single molecules of dynein are activated to transport intracellular cargo. We first improved HILO microscopy to visualize fluorescently-labeled single dynein molecules in HeLa cells and observed that only ~30 % of the dyneins binding to the microtubule moved in a processive fashion towards the minus ends. Moreover, the dynein molecules detached from the microtubule after a residence time of ~ 0.7s. We hypothesized that the processive dynein molecules represented dyneins that were activated upon binding to cargo-dynactin complexes that were anchored to microtubules and that the cargo-dynactin-dynein complex moves in short bursts. To test this hypothesis, we first focused on understanding how d...