Dynamic Interactions of the Cytoskeleton

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DESCRIPTION (provided by applicant): Specific and dynamic interactions between microtubules and the actin cytoskeleton are involved in critical cellular processes, including cell motility, cell adhesion, and cell division. The microtubule motor cytoplasmic dynein is hypothesized to mediate some of these interactions. Dynein, anchored at the actin-rich cell cortex, may specifically capture and tether microtubule plus ends. During cell division, cortically anchored dynein is thought to interact with astral microtubules, allowing for nuclear migration in S. cerevisiae, and for polarized spindle orientation in higher eukaryotes. We have recently localized cytoplasmic dynein to the cortex of mammalian epithelial cells during interphase, where it is specifically enriched at sites of cell-cell contact. We hypothesize that the ineractions we have observed between dynein and the cortical proteins beta-catenin and PLAC-24 serve to anchor dynein to adherens junctions, where the motor can can interact with microtubule-plus end proteins such as EB1, CLIP-115, and BPAG-1 (shortstop), and capture and tether microtubule plus ends. This cortical capture of specific microtubules would allow for spatial and temporal regulation of microtubule-cortical interactions, and potentially may provide a mechanism for the communication between sites of cell-cell contact the cellular cytoskeleton. To investigate the hypothesis that the cortical capture of microtubules at sites of cell-cell interactions is critical to cell adhesion and normal development, that dynein localized to the cortex tethers microtubules at cell-cell junction sites, and that this cortical capture has a key role in mediating dynamic cytoskeletal interactions at cell-cell junctions, we will pursue the following specific aims: (1) Does cytoplasmic dynein localized at the sites of cell-cell contact capture and transiently tether specific microtubules? (2) Is there a network of proteins that allows for dynamic and specific interactions between the microtubule and actin cytoskeletons at sites of cell-cell interactions? And (3) Do cortical-microtubule interactions facilitate communication between cell-cell interaction sites and the cellular cytoskeleton in vivo and during development? The combination of live cell assays (Aim 1), inhibitory and biochemical approaches (Aim 2), and analysis of interacting proteins in both mammalian and metazoan model systems (mouse and Drosophila, Aim 3), should allow us to assess the mechanistic role for dynein in mediating interactions between adherens junctions and the cellular cytoskeleton in epithelial cells.