Biochemical and single-molecule characterization of the human cytoplasmic dynein motor complex

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Human cytoplasmic dynein is an essential molecular motor involved in multiple cellular functions such as cargo transport along microtubules, cell division and cell migration. Dynein and its cofactors are implicated in neurological diseases including lissencephaly and Perry syndrome, and contribute to pathologic processes such as viral transport and cancer cell division. Developing drugs for these conditions, and manipulating dynein function in the treatment of other diseases, requires a detailed knowledge of the human motor. However, the biochemical and biophysical properties of human dynein remain uncharacterized in vitro. Mechanisms regulating dyneins multiple cellular functions have not been correlated sufficiently with its single-molecule characteristics, yet are likely responsible for ambiguous results on yeast and other eukaryotic dyneins. In this research project, I will purify dynein from human cell cultures and analyze the biochemical composition of the motor complex by Western blot and mass-spectrometry. Using single-molecule fluorescence and optical trapping microscopy, I will then scrutinize dyneins motive and force-generating properties. In combination with the biochemical analysis, these single-molecule studies may reveal potential points of dynein regulation in vivo, such as variable phosphorylation states or subunit composition. In addition to clarifying controversies in the dynein field, an in-depth characterization of human dynein is likely to have cross-disciplinary implications for dynein-dependent signal transduction pathways, as well as cancer and cell biology.