Cell-Cell and Cell-Matrix Interactions in Morphogenesis

细胞迁移是一种基本的生物学过程，目前对细胞外基质平面底物上单细胞运动的机制已有较多了解。较少被理解的是连续的细胞组(例如，片状、簇状和索状结构)的迁移，其中细胞-细胞相互作用涉及对迁移阵列内的细胞极性和突出活动的调节。在下一个进化期将检验的中心假设是，与中间纤维细胞骨架相连的钙粘附素复合体作为一种可能的机械感觉装置来指导和定向非洲爪哇胚胎中胚层中的集体细胞运动。先前项目期间的进展证实了钙粘附素依赖的细胞-细胞接触在体内指导中胚层细胞的极性、突起行为和对纤维连接蛋白(FN)的迁移的重要性。此外，FN上定向的细胞运动需要通过钙粘附素和完整的中间丝细胞骨架施加压力。钙粘附素和整合素在这一过程中的协调参与是这一提议的一个关键目标，该提议试图建立所涉及的机械/细胞骨架和信号联系。该提案有两个具体目标。首先，将建立钙粘附素黏附、施加力与中间丝和肌动蛋白细胞骨架之间的关系，以确定细胞极性的调节和细胞定向迁移的机制。牵引力显微镜(TFM)和顺磁珠拉实验将被用来确定维持FN上定向迁移所需的细胞-细胞粘附力。微光高分辨率成像也将被用来将细胞骨架动力学与施加在衬底上的力和牵引力联系起来。在第二个目标中，将使用功能丧失(例如，反义吗啉)和生化方法来研究参与钙粘素依赖的细胞极性和突起活动的机械敏感调节的蛋白质的作用。这些实验的最初焦点将是将钙粘附素连接到中间细丝细胞骨架(例如，蛋白球蛋白、桥粒蛋白和其他细胞-细胞连接成分)所涉及的蛋白质。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，这些状态包括胚胎发育、伤口愈合、癌症和转移、炎症和广泛的先天性出生缺陷。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.