Regulation of Cytoplasmic Dynein Based Vesicle Transport

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DESCRIPTION (provided by applicant): Dynactin is a multisubunit protein complex that works in conjunction with the microtubule-based motor, cytoplasmic dynein, to support a range of subcellular motile functions. Dynactin enhances motor processivity and serves as an adaptor protein that allows dynein to bind membrane and protein cargoes. Dynactin can also function independently of dynein, to tether protein complexes and regulatory molecules to microtubules. Dynactin has even been shown to interact with motors other than dynein. To perform these diverse functions, dynactin relies on multiple binding activities that are distributed across its structure. Motor binding uses one part of the molecule, microtubule binding and processivity enhancement another, and cargo binding yet another. Dynactin's structural integrity is maintained by its dynamitin (p50) subunit, which forms a flexible bridge that anchors the motor and microtubule-binding subunit to the cargo-binding domain. Specific Aim 1 is focused on understanding dynamitin's interactions with other dynactin subunits and with itself. Specific Aim 2 describes experiments to clearly define the molecular and ultrastructural basis of the dynein/dynactin interaction. The nucleus is one of many organelles that interacts with dynactin and dynein. Motor activity can power nuclear translocation and also the nuclear envelope rupture that occurs at the onset of mitosis. Work from our lab suggests that dynactin binds nuclei using subunits that are located at the very end of the cargo-binding domain. The goal of Specific Aim 3 is to elucidate the mechanism of cell cycle regulation and identify components of the nuclear envelope that bind dynactin. We recently discovered that dynactin is required for the normal metabolism and microtubule-dependent cytoplasmic tethering of signal transduction molecules. The link to dynactin is a pair of subunits, p25 and p27, that are loosely associated with the dynactin "core" structure, p25 and p27 may be exchangeable subunits that allow different cytosolic components to be targeted to dynactin and microtubules. The dynamic interactions between p25/p27, the dynactin core and microtubules are evaluated in experiments described in Specific Aim 4.