Cytoplasmic dynein is a microtubule-based motor with critical and diverse cellular functions, ranging from vesicle transport to mitotic spindle assembly. Genetic and biochemical studies have shown that dynactin is a required co-factor for most of these functions of dynein. Both dynein and dynactin are large protein complexes with multiple subunits. Recent research has also identified a number of interacting proteins that mediate dynein/dynactin function in vivo, including spectrin, Huntingtin, Lis1 and Nudel. However, we still lack a mechanistic understanding of how these proteins interact during organelle and vesicle transport in the cell. Here, we propose to use a battery of in vitro and in vivo approaches to establish a unified model for dynein/dynactin function in intracellular transport. We will examine the specific steps of cargo binding and motor recruitment, initiation of motility, and regulation of minus end-directed as well as bi-directional organelle transport. To develop this model, we will address the following questions: (1) What are the mechanisms of cargo attachment and recruitment for the cytoplasmic dynein/dynactin motor complex? We hypothesize that dynein binds cargo through multiple distinct mechanisms, allowing for precise and specific targeting in the cell. (2) How is cytoplasmic dynein motor function activated and how is dynein regulated during transport? We hypothesize that dynein-mediated transport is regulated at multiple levels, through interactions with proteins affecting recruitment, activation, and motor function. (3) How are multiple motors coordinated during bidirectional transport? We hypothesize that microtubule plus- and minus-end directed motors are coordinated during bidirectional transport via direct interactions between the active motor complexes. Recent progress has provided new insight into dynein/dynactin function, leading to the identification of interacting proteins that may function as modulators of the transport pathway. Using a combination of biochemical and cellular approaches will allow us to test specific hypotheses, and to develop a unified model for dynein's role in vesicular and organelle transport. Our recent work has demonstrated a direct link between defects in dynein/dynactin function and human motor neuron disease, so it is clear that a more thorough and mechanistically rigorous model of dynein/dynactin function in intracellular transport is required. ? ? ?
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