Single-molecule characterization of Cytoplasmic Dynein

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DESCRIPTION (provided by applicant): Cytoplasmic dynein is poorly understood in vitro, which makes it hard to understand its function and regulation in vivo. For instance, because its single-molecule properties are not known, it is unclear what roles various accessory proteins might need to play in order to achieve correct in vivo function. Further, although some in vivo evidence suggests that multiple dynein motors work together (and with dynactin), the functional significance of employing multiple motors is unknown. We address these questions by studying dynein function in a controlled environment, first at the single-molecule level and then in increasingly complex situations. We will employ an in vitro bead assay that uses an optical trap to quantify how single---or multiple--dynein molecules move along microtubules, both in the presence or absence of proteins that might alter dynein function such as dynactin and MAPs. Initial results published in a recent paper showed that Dynein functions in a fundamentally different manner than kinesin, and appears to have a gear mechanism enabling it to adjust force production to meet external applied load. Further, our unpublished preliminary results suggest that unlike kinesin, at the single molecule level dynein is not a very good efficient cargo transporter. The research will fully investigate dynein's single-molecule function (aim 1), clarify how this function can be altered (aims 2 and 3), and further study how the proposed gear might function (aim 4). We proposed the following specific aims: Aim 1: Elucidate the single-molecule functions of dynein, combining measurements including force-velocity curve, processivity vs load, dependence of function on ATP, etc. with modeling. Aim 2: Determine how multiple dyneins function together Aim 3: Determination of the role of some accessory proteins in regulating altering dynein function. Investigate the functional ramifications of the presence of the dynactin complex and or MAPs. Is a single dynein-dynactin pair an effective transport system ? Do MAPs impair motor-driven transport? Aim 4: Test our hypothesis explaining the gear's function Understanding dynein is directly related to public health: correct dynein function and regulation is essential for development, alteration of dynein function likely occurs in many cancers, and dynein and dynactin mutations cause neuronal degeneration.