Mechanosensitive signaling and cell adhesion during migration

描述（由申请人提供）：细胞迁移是一个复杂的过程，涉及物理粘附机制和细胞内信号传导，两者相互依赖。细胞迁移的机制已被深入研究，使用体外培养的单细胞，但我们的理解在体内的细胞迁移仍然有限。体内细胞迁移通常发生在多细胞阵列中，其产生对形态发生、正常成人生理学和各种病理学至关重要的大量组织运动。虽然通过细胞-基质牵引力产生的机械信号的功能经常是研究的主题，但细胞-细胞粘附中对张力的机械敏感性反应以及这些粘附接触对细胞迁移的后续影响尚未阐明。我将测试的假设，机械信号，启动钙粘蛋白介导的细胞间粘附在完整的迁移组织，有一个核心的作用，在协调细胞极化和定向迁移的个别细胞，以指导完整的组织运动。细胞迁移的机制在不同物种、组织和细胞类型之间广泛保守。在非洲爪蟾原肠胚形成过程中穿过囊胚腔顶部纤维连接蛋白基质的迁移中内胚层将被用作这些研究的模型系统。该模型提供了一个理想的系统，在其中研究正常的体内迁移过程，因为完整组织的运动行为保留在体外培养时，因此，该系统是可访问的各种实验操作。有四个具体的目标，提出了解决的意义，细胞间粘附的机械信号，调节中内胚层细胞的极性和迁移的传播。在具体目标1中，将使用中内胚层外植体和磁珠“牵拉”测定来确定生物力学张力对C-钙粘蛋白依赖性粘连的作用，作为该组织中极化和定向迁移的重要调节剂。钙粘蛋白和整合素提供了细胞外配体和内部细胞骨架元件之间的物理联系。GFP融合蛋白将用于跟踪细胞骨架动力学，作为由钙粘蛋白粘附启动的机械信号传导的结果，具体目标2。肌动蛋白的细胞骨架动力学参与迁移的信号通过小Rho家族GTP酶的时空激活。在具体目标3中，FRET分析将用于检查由于C-钙粘蛋白和α 5 β 1整联蛋白依赖性机械敏感性细胞信号传导导致的Rho家族GTP酶的局部亚细胞活化。最后，牵引力显微镜和反射对比显微镜方法将用于具体目标4中，以确定迁移中内胚层中细胞-基质粘附和C-钙粘蛋白细胞-细胞接触之间的比力关系。

DESCRIPTION (provided by applicant): Cell migration is a complex process that involves both physical adhesive mechanics and intracellular signaling, which are profoundly interdependent on one another. Mechanisms of cell migration have been studied intensively using single cells cultured in vitro, but our understanding of cell migration in vivo remains limited. Cell migration in vivo often occurs in multicellular arrays that produce bulk tissue movements critical to morphogenesis, normal adult physiology, and various pathologies. While the function of mechanical signaling generated through cell-matrix traction forces has frequently been the subject of investigation, mechanosensitive responses to tension in cell-cell adhesions and the subsequent effects of these adhesive contacts on cell migration have not been elucidated. I will test the hypothesis that mechanical signaling, initiated by cadherin-mediated cell-cell adhesions in intact migratory tissues, has a central role in coordinating cell polarization and directional migration of individual cells to direct intact tissue movements. Mechanisms of cell migration are widely conserved between different species, tissues, and cell types. The migratory mesendoderm that traverses the fibronectin matrix of the blastocoel roof during Xenopus gastrulation will be used as a model system for these studies. This model provides an ideal system in which to study normal in vivo migratory processes because the motile behaviors of the intact tissue are retained when cultured in vitro and thus, the system is accessible to a variety of experimental manipulations. There are four specific aims proposed to address the significance of cell-cell adhesion in the propagation of mechanical signals that regulate mesendoderm cell polarity and migration. In specific aim 1, mesendoderm explants and a magnetic bead "pull" assay will be used to establish the role of biomechanical tension on C-cadherin dependent adhesions as an important regulator of polarization and directional migration in this tissue. Cadherins and integrins provide a physical link between extracellular ligands and internal cytoskeletal elements. GFP fusion proteins will be used to track cytoskeletal dynamics as a consequence of mechanical signaling initiated by cadherin adhesions in specific aim 2. Actin cytoskeletal dynamics involved in migration is signaled by spatiotemporal activation of the small Rho family GTPases. In specific aim 3, FRET analysis will be used to examine the local subcellular activation of Rho family GTPases as a result of C-cadherin and a5 (31 integrin-dependent mechanosensitive cell signaling. Finally, traction force microscopy and reflection contrast microscopy approaches will be used in specific aim 4 to determine the specific force relationship between cell-matrix adhesions and C-cadherin cell-cell contacts in migrating mesendoderm.