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，涉及物理粘附力学和细胞内信号传导，两者相互依存。细胞迁移的机制已经在体外培养的单细胞中进行了深入的研究，但我们对体内细胞迁移的理解仍然有限。细胞在体内的迁移通常发生在多细胞阵列中，产生对形态发生、正常成人生理和各种病理至关重要的大块组织运动。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。我将验证完整迁移组织中由钙粘蛋白介导的细胞-细胞粘附启动的机械信号传导在协调细胞极化和单个细胞定向迁移以指导完整组织运动方面发挥核心作用的假设。细胞迁移的机制在不同的物种、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，穿越胚顶纤维连接蛋白基质的迁移中胚层将被用作这些研究的模型系统。该模型为研究正常的体内迁移过程提供了一个理想的系统，因为在体外培养时，完整组织的运动行为被保留，因此，该系统可用于各种实验操作。在调节中胚层细胞极性和迁移的机械信号的传播中，提出了四个特定的目的来解决细胞-细胞粘附的重要性。在具体目标1中，将使用中胚层外植体和磁珠“拉力”试验来确定生物力学张力对c -钙粘蛋白依赖性粘附的作用，作为该组织中极化和定向迁移的重要调节因子。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。在特定目的2中，GFP融合蛋白将用于跟踪由钙粘蛋白粘附引发的机械信号传导的细胞骨架动力学。参与迁移的肌动蛋白细胞骨架动力学是通过小Rho家族gtpase的时空激活来信号化的。在特定目标3中，FRET分析将用于检查Rho家族gtpase的局部亚细胞激活，这是c -钙粘蛋白和a5（31）整合素依赖的机械敏感细胞信号传导的结果。最后，在特定目的4中，将使用牵引力显微镜和反射对比显微镜方法来确定迁移中胚层中细胞-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.