Mechanosensitive signaling and cell adhesion during migration

描述(申请人提供)：细胞迁移是一个复杂的过程，涉及物理黏附机制和细胞内信号传递，这两个过程彼此之间有着深刻的相互依存关系。利用体外培养的单个细胞对细胞迁移机制进行了深入的研究，但我们对体内细胞迁移的了解仍然有限。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-细胞黏附中对张力的机械敏感反应以及这些黏附接触对细胞迁移的后续影响尚未阐明。我将测试这样一种假设：在完整的迁移组织中，由钙粘附素介导的细胞-细胞黏附启动的机械信号在协调细胞极化和单个细胞定向迁移以指导完整的组织运动方面发挥着核心作用。细胞迁移机制在不同物种、组织和细胞类型之间广泛保守。在非洲爪哇原肠发育过程中穿过胚腔顶纤维连接蛋白基质的迁移性中胚层将被用作这些研究的模型系统。这个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了下来，因此，该系统可以进行各种实验操作。在调节中胚层细胞极性和迁移的机械信号的传播中，细胞-细胞黏附的重要性被提出了四个具体的目标。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-钙粘附素依赖的粘连的作用，作为该组织中极化和定向迁移的重要调节因素。钙粘附素和整合素在细胞外配体和内部细胞骨架元素之间提供了物理联系。GFP融合蛋白将被用来跟踪细胞骨架动力学，这是由于钙粘附素粘连在特定的目的2启动的机械信号的结果。参与迁移的肌动蛋白细胞骨架动力学信号是通过小的Rho家族GTP酶的时空激活来传递的。在具体目标3中，FRET分析将被用来检测C-钙粘素和a5(31整合素依赖的机械敏感细胞信号)导致的Rho家族GTP酶的局部亚细胞激活。最后，将在特定目标4中使用牵引力显微镜和反射对比显微镜方法来确定迁移中的中胚层中细胞-基质粘连和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.