The structure and mechanism of cytoplasmic dynein andelectron microscopy. (360G-Wellcome-080709_Z_06_Z)

The structure and mechanism of cytoplasmic dynein by X-ray crystallography and electron microscopy Dynein is a microtubule-associated motor protein involved in numerous fundamental processes in eukaryotic cells including the movement of cilia and flagella by axonemal dyneins and intracellular transport of organelles by cytoplasmic dynein. Dynein-driven mRNA translocation is critical for embryogenesis and during mitosis dynein powers spindle assembly, chromosome positioning and sister chromatid separation. In neurones, cytoplasmic dynein drives retrograde axonal transport and mutations are associated with neurodegenerative diseases. Yet despite its necessity for eukaryotic life, the structure and mechanism of dynein remain unknown. The aim of this project is to determine the structure of the cytoplasmic dynein motor. This will be achieved by the following specific objectives: 1) Expressing and purifying yeast dynein motor domain for cryo-electron microscopy and crystallographic studies. 2) Obtaining 3D cryo-EM structure of Dictyostelium discoideum dynein motor domain with a yeast-like truncation for comparison to (1). 3) Expressing and purifying AAA4-stalk-AAA5 fragment of a yeast motor domain for cryo-EM and crystallographic studies.