Cadherin Mechanotransduction

这项资助是建立在我们的新发现之上的，即上皮细胞-细胞连接处的E-钙粘蛋白可以转导机械信号， 通过表皮生长因子受体（EGFR）激活激酶级联反应来传递信号。E-cadherin是一种 上皮细胞-细胞连接处的必需粘附蛋白，以及E-钙粘蛋白复合物也具有抑制作用， 调节细胞形状和上皮屏障完整性。这些新的发现表明，E-钙粘蛋白力- 转导还激活调节细胞增殖、形态发生和疾病的信号。广泛的目标 该计划的目的是确定E-钙粘蛋白机械激活EGFR的初始步骤，并建立 这一机制的更广泛的生理意义。我们的初步数据也表明， 力激活信号通路通过以下途径调节应激细胞-细胞连接的细胞骨架强化： E-钙粘蛋白复合物中的α-连环蛋白。这一出乎意料的发现支持了EGFR和E- 钙粘蛋白是上皮细胞连接处的核心力-转导机制中的基本组分。在这 程序，特异性目标1测试了机械刺激的E-钙粘蛋白激活EGFR的假设 磷酸化，通过触发假定的E-钙粘蛋白/EGFR复合物的破坏。具体目标2将使用 由合作者Hristova（约翰霍普金斯）开发的基于荧光的创新方法， 研究E-cadherin和EGFR在质膜上的直接相互作用。拟议的研究包括 基于大量的初步数据，揭示了直接的蛋白质-蛋白质关联。生物物理学研究将 建立这种关联的分子要求，使用一个子集的E-钙粘蛋白和EGFR突变体。 具体目标3将测试这些发现在三维，器官型的生理意义， 人乳腺上皮组织模型，与韦弗（加州大学旧金山分校）合作。研究将确定 E-钙粘蛋白/EGFR复合物是否确实是与整合素协调的中心力感应单位， 调节形态发生和恶性程度，以响应组织力学。乳腺上皮细胞的3D培养 工程化以表达E-钙粘蛋白突变体（Aim 2）将确定E-钙粘蛋白/EGFR复合物的影响， 破坏增殖、形态发生和侵袭，作为基质刚性的函数。整合素很好地 已知与EGFR协调调节乳腺组织发育和肿瘤进展。这些研究 将可能建立E-cadherin作为这个力敏感网络的重要组成部分。

This grant builds on our novel discovery that E-cadherin at epithelial cell-cell junctions transduces mechanical signals, by activating a kinase cascade via the epidermal growth factor receptor (EGFR). E-cadherin is an essential adhesion protein at epithelial cell-cell junctions, and E-cadherin complexes also transduce force, to regulate cell shape and epithelial barrier integrity. These new findings suggest that E-cadherin force- transduction also activates signals that regulate cell proliferation, morphogenesis, and disease. The broad goal of this program is to identify initial steps in the mechanical activation of EGFR by E-cadherin, and to establish the broader physiological implications of this mechanism. Our preliminary data also demonstrate that this force-activated signaling pathway regulates the cytoskeletal reinforcement of stressed cell-cell junctions by α−catenin in E-cadherin complexes. This unexpected finding supports the hypothesis that EGFR and E- cadherin are essential components in the core force-transduction machinery at epithelial cell junctions. In this program, Specific Aim 1 tests the hypothesis that mechanically stimulated E-cadherin activates EGFR phosphorylation, by triggering the disruption of putative E-cadherin/EGFR complexes. Specific Aim 2 will use innovative fluorescence-based methodology, developed by collaborator Hristova (Johns Hopkins) to investigate direct interactions between E-cadherin and EGFR at the plasma membrane. Proposed studies are based on substantial preliminary data, which reveal direct protein-protein association. Biophysical studies will establish the molecular requirements for this association, using a subset of E-cadherin and EGFR mutants. Specific Aim 3 will test the physiological implications of these findings in a three-dimensional, organotypic model of human mammary epithelial tissue, in collaboration with Weaver (UCSF). Studies will determine whether E-cadherin/EGFR complexes are indeed central force-sensing units that coordinate with integrins to tune morphogenesis and malignancy, in response to tissue mechanics. 3D cultures of breast epithelial cells engineered to express E-cadherin mutants (Aim 2) will determine the impact of E-cadherin/EGFR complex disruption on proliferation, morphogenesis, and invasion, as a function of matrix rigidity. Integrins are well known to coordinate with EGFR to regulate breast tissue development and tumor progression. These studies would potentially establish E-cadherin as an essential component in this force-sensitive network.