Mechanisms regulating movement and force generation by cytoplasmic dynein. (360G-Wellcome-092436_Z_10_Z)

The goal of this proposal is to gain a mechanistic understanding of how cytoplasmic dynein, the large microtubule-based motor protein, is regulated to produce essential movement of cargo towards the cell interior (towards the microtubule minus-end). An initial step in many dynein transport events is its targeting to the plus-ends of microtubules, which grow and shrink near the cell periphery. Here, dynein is thought to search-and-capture cargos before transporting them towards the minus-end. T he recent advance of a dynein expression system in yeast makes in vitro reconstitution and detailed dissection of these critical regulatory processes possible. I will establish the minimal proteins required to target dynein to the microtubule plus-end, and analyse their behaviour using single-molecule fluorescence microscopy. The structural basis for control of dynein s motility will be elucidated by single-particle electron microscopy and chemical crosslinking, using non-natural amino acids to precisely insert reactive probes within dynein s large structure. I will also isolate a dynein cargo protein and use optical tweezers to test if it forms a load-bearing interaction with dynein. This combination of biochemical, biophysical and structural approaches will shed new light on the regulation of motor proteins inside living cells, which I will develop into a molecular movie.