Dynamic Interactions of the Cytoskeleton

描述（由申请人提供）：微管和肌动蛋白细胞骨架之间的特异性和动态相互作用涉及关键的细胞过程，包括细胞运动、细胞粘附和细胞分裂。假设微管运动细胞质动力蛋白介导这些相互作用中的一些。动力蛋白，锚定在肌动蛋白丰富的细胞皮质，可以特异性地捕获和拴系微管加端。在细胞分裂过程中，皮质锚定的动力蛋白被认为与星状微管相互作用，使S。酿酒酵母，并在高等真核生物极化纺锤体方向。我们最近本地化的细胞质动力蛋白的哺乳动物上皮细胞的皮质在间期，在那里它是特别丰富的细胞-细胞接触的网站。我们假设，我们观察到的动力蛋白与皮质蛋白β-连环蛋白和PLAC-24之间的相互作用，起到了将动力蛋白锚在粘附连接处的作用，在粘附连接处，马达可以与微管加端蛋白如EB 1、CLIP-115和BPAG-1（游击手）相互作用，并捕获和束缚微管加端。这种特定微管的皮层捕获将允许微管-皮层相互作用的空间和时间调节，并且可能提供细胞-细胞接触细胞骨架的位点之间的通信机制。为了研究这样的假设，即在细胞-细胞相互作用位点的微管的皮质捕获对细胞粘附和正常发育至关重要，动力蛋白定位于皮质，在细胞-细胞连接位点束缚微管，并且这种皮质捕获在介导细胞-细胞连接处的动态细胞骨架相互作用中起关键作用，我们将追求以下具体目标：（1）位于细胞-细胞接触部位的细胞质动力蛋白是否捕获并瞬时束缚特定的微管？(2)是否有一个蛋白质网络，允许微管和肌动蛋白细胞骨架之间的动态和特定的相互作用，在细胞间的相互作用网站？以及（3）在体内和发育过程中，皮质-微管相互作用是否促进了细胞-细胞相互作用位点与细胞骨架之间的通讯？活细胞测定（目的1），抑制和生物化学方法（目的2），并在哺乳动物和后生动物模型系统（小鼠和果蝇，目的3）的相互作用蛋白的分析相结合，应使我们能够评估的机制作用，为动力蛋白介导的粘附连接和上皮细胞的细胞骨架之间的相互作用。

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.