Dynamic Interactions of the Cytoskeleton

描述(申请人提供)：微管和肌动蛋白细胞骨架之间特定和动态的相互作用涉及关键的细胞过程，包括细胞运动、细胞黏附和细胞分裂。微管运动胞浆动力蛋白被假设为介导其中的一些相互作用。动力蛋白定位于富含肌动蛋白的细胞皮质，可以特异性地捕获和拴住微管和末端。在细胞分裂期间，皮质锚定的动力蛋白被认为与星形微管相互作用，允许酿酒酵母的核迁移，以及高等真核生物的极化纺锤体方向。我们最近在间期将细胞质动力蛋白定位于哺乳动物上皮细胞的皮质，在那里细胞与细胞接触的部位特异地富含动力蛋白。我们假设，我们观察到的dynein与皮质蛋白β-catenin和plac-24之间的惰性作用有助于将dynein锚定到粘连连接上，在那里马达可以与微管+末端蛋白如EB1、CLIP-115和BPAG-1(游击键)相互作用，并捕获和拴住微管+末端。这种对特定微管的皮质捕获将允许微管-皮质相互作用的空间和时间调节，并可能为细胞-细胞接触部位与细胞骨架之间的通信提供一种机制。为了研究这样一种假设，即细胞-细胞相互作用部位的皮质微管捕获对细胞黏附和正常发育至关重要，定位于皮质的动力蛋白束缚细胞-细胞连接部位的微管，并且这种皮质捕获在调节细胞-细胞连接处的动态细胞骨架相互作用中具有关键作用，我们将追求以下具体目标：(1)细胞质动力蛋白是否定位于细胞-细胞接触捕获部位，并瞬时拴住特定的微管？(2)是否存在允许细胞-细胞相互作用部位的微管和肌动蛋白细胞骨架之间动态和特定相互作用的蛋白质网络？以及(3)在体内和发育过程中，皮质-微管相互作用是否促进细胞-细胞相互作用部位和细胞骨架之间的沟通？结合活细胞分析(目标1)、抑制和生化方法(目标2)以及对哺乳动物和后生动物模型系统(小鼠和果蝇，目标3)相互作用蛋白的分析，我们应该能够评估动力蛋白在调节黏附连接和上皮细胞细胞骨架之间的相互作用中的机制作用。

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.