Genetic strategies for the quantitative control of cell-matrix mechanobiology

描述(申请人提供)：现在广泛承认细胞行为对与细胞外基质(ECM)的机械串扰高度敏感。虽然已经开发了许多强大的方法来通过操纵ECM来控制这种通信，但几乎没有可用的工具来直接、细胞内在地控制细胞机械转导信号。在这个建议中，我们提出并应用了我们最近开发的一种遗传策略，在该策略中，我们通过机械转导基因的诱导表达来控制细胞-ECM机械信号。我们已经证明，这种方法能够对皮质硬度、牵引力产生、细胞迁移速度和细胞外基质重塑进行分级和动态控制。我们还表明，这种方法在剂量-反应关系、靶标可用性、毒性和安全性方面远远优于传统的药物策略。 诉讼的持续时间。我们现在建议开发这一策略的第二代，并利用它来解决细胞机械生物学领域中两个尚未得到满足的需求：第一，我们将把两个基因置于启动子的控制下，这两个基因可以被两个不同的小分子诱导或抑制，从而实现对两个机械转导基因的正交控制。我们将利用这一能力来构建细胞机械特性的“相图”，定量地映射肌球蛋白激活剂Rho相关激酶和肌球蛋白轻链激酶如何对机械生物学表型做出贡献。其次，我们将应用这一策略来定量控制ECM的机械特性如何调节两个重要的细胞行为：细胞运动速度和神经干细胞神经发生。如果成功，这将使我们能够将机械触发的细胞行为与输入本身分离，从而潜在地提供了一种方法，可以重新连接细胞矩阵串扰，以在任意指定的微环境中实现所需的表型端点。这可能为在体外和体内设计细胞-材料界面上的细胞行为提供一种新的、非常有效的方法。总而言之，这些研究将提供关键证据 作为定量细胞生物学发现和细胞与组织工程/再生医学应用的工具，这种方法的原则。

DESCRIPTION (provided by applicant): It is now widely acknowledged that cell behavior is highly sensitive to mechanical crosstalk with the extracellular matrix (ECM). While many powerful methods have been developed to control this communication through manipulation of the ECM, there are few tools available for the direct, cell-intrinsic control of cellular mechanotransductive signaling. In this proposal we advance and apply a genetic strategy we recently developed in which we control cell-ECM mechanical signaling through inducible expression of mechanotransductive genes. We have shown that this method enables graded and dynamic control of cortical stiffness, traction force generation, cell migration speed, and ECM remodeling. We have also shown that this approach vastly outperforms traditional pharmacologic strategies in terms of dose-response relationship, target availability, toxicity, and duration of action. We now propose to develop a second generation of this strategy and leverage it to address two unmet needs in the field of cell mechanobiology: First, we will place two genes under the control of promoters that can be induced or suppressed by two different small molecules, thereby enabling orthogonal control over two mechanotransductive genes. We will use this capability to construct a "phase diagram" of cell mechanical properties that quantitatively maps how the myosin activators Rho- associated kinase and myosin light chain kinase contribute to mechanobiological phenotype. Second, we will apply this strategy to quantitatively control how ECM mechanical properties regulate two important cell behaviors: cell motility speed and neural stem cell neurogenesis. In successful, this will enable us to decouple mechanically-triggered cell behaviors from the inputs themselves, thus potentially offering a way to "rewire" cell-matrix crosstalk to achieve desired phenotypic endpoints in arbitrarily specified microenvironments. This could offer a new and very powerful way to engineer cell behavior at cell-material interfaces in vitro and in vivo. Taken together, these studies will provide key proof of-principle for this approach as a tool for both quantitative cell biological discovery and cell ad tissue engineering/regenerative medicine applications.