Cell-Cell and Cell-Matrix Interactions in Morphogenesis

细胞迁移是一个基本的生物学过程，目前对细胞在细胞外基质平面基质上运动的机制已知之甚多。不太清楚的是相邻细胞组的迁移（例如，片、簇和索状结构），其中细胞-细胞相互作用涉及细胞极性的调节和迁移阵列内的迁移活动。在下一个进展期间要测试的中心假设是，一个钙粘蛋白复合物与中间丝细胞骨架的链接作为一个假定的mechanosensory设备的功能，以指导和定向集体细胞运动中的非洲爪蟾胚胎中内胚层。 在前一个项目期间的进展建立了钙粘蛋白依赖的细胞间接触在体内指导中内胚层细胞极性、迁移行为和迁移到纤连蛋白（FN）上的重要性。 此外，FN上的定向细胞运动需要通过钙粘蛋白和完整的中间丝细胞骨架施加力。 钙粘蛋白和整合素在这一过程中的协调参与是本提案的关键目标，该提案旨在建立所涉及的机械/细胞骨架和信号联系。 该提案有两个具体目标。 首先，钙粘蛋白粘附，力的应用和中间丝和肌动蛋白细胞骨架之间的关系将被建立，以确定调节细胞极性和定向细胞迁移的机制。 牵引力显微镜（TFM）和顺磁珠牵拉实验将用于确定维持FN上定向迁移所需的细胞-细胞粘附力。 低光高分辨率成像也将被用来关联细胞骨架动力学与力的应用和牵引应力的基板上。 在第二个目标中，将使用功能丧失（例如，反义吗啉代）和生物化学方法。 这些实验的最初焦点将是参与将钙粘蛋白连接到中间丝细胞骨架的蛋白质（例如，斑珠蛋白、桥粒斑蛋白和其它细胞-细胞连接组分）。集体细胞迁移对各种正常生理和病理状态的进展至关重要，包括胚胎发育、伤口愈合、癌症和转移、炎症以及各种先天性出生缺陷。这一建议将寻求扩大我们对这一重要的细胞迁移模式所涉及的分子机制的理解。

Cell migration is a fundamental biological process and much is now known about mechanisms involved in single cell motility on planar substrates of extracellular matrix. Less well understood is the migration of contiguous groups of cells (e.g., sheets, clusters, and cord-like structures) where cell-cell interactions are involved in the regulation of cell polarity and protrusive activities within the migratory array. The central hypothesis to be tested during the next progress period is that a cadherin complex with links to the intermediate filament cytoskeleton functions as a putative mechanosensory apparatus to direct and orient collective cell movements in the mesendoderm of Xenopus embryos. Progress during the previous project period established the importance of cadherin-dependent cell-cell contact in directing mesendoderm cell polarity, protrusive behaviors and migration on fibronectin (FN) in vivo. Moreover, directed cell movements on FN require force application through cadherins and an intact intermediate filament cytoskeleton. The coordinate involvement of both cadherins and integrins in this process is a key target of this proposal, which seeks to establish the mechanical/cytoskeletal and signaling linkages involved. The proposal has two Specific Aims. First, the relationship between cadherin adhesion, force application and the intermediate filament and actin cytoskeletons will be established in order to identify mechanisms of regulation of cell polarity and directed cell migration. Traction force microscopy (TFM) and paramagnetic bead-pull experiments will be used to define cell-cell adhesive forces required to maintain directional migration on FN. Low-light high-resolution imaging will also be used to correlate cytoskeletal dynamics with force application and traction stresses on the substrate. In the second aim, the roles of proteins involved in the cadherin-dependent mechanosensitive regulation of cell polarity and protrusive activity will be investigated using loss-of-function (e.g., antisense morpholinos) and biochemical approaches. The initial focus of these experiments will be proteins involved in linking cadherins to the intermediate filament cytoskeleton (e.g., plakoglobin, desmoplakin and other cell-cell junctional components). Collective cell migration is critical to the progression of a variety of normal physiological and pathological states that include embryonic development, wound healing, cancer and metastasis, inflammation, and a wide range of congenital birth defects. This proposal will seek to broaden our understanding of the molecular mechanisms involved in this important mode of cellular migration.