Biophysical control of cell migration by mechanical gradients in cell microenvironment

细胞迁移是一种基本的生物学现象，对单细胞和多细胞生物体的生命至关重要。简单的生物，如阿米巴，必须迁徙以寻找食物和交配。脊椎动物等多细胞生物的复杂性带来了个体细胞和细胞组在发育形态形成过程中定位的必要性，这在很大程度上是通过细胞的定向迁移实现的。在成人，定向迁移在广泛的生理和病理过程中发挥着重要作用，包括皮肤和肠道上皮的更新、免疫系统功能和伤口愈合，以及癌症转移和心血管疾病。在组织环境中，细胞的迁移受到许多指导信号的严格调控，这些信号调节信号网络的活性，进而控制细胞内的机制，如肌动蛋白细胞骨架和焦点粘连，从而推动细胞迁移。在过去的几十年里，人们的注意力主要集中在了解化学信号，如可溶性生长因子，如何引导细胞迁移。然而，最近的研究强调了机械信号在细胞微环境中的重要性，包括细胞外基质(ECM)的密度和硬度在调节细胞迁移中的作用。通过使用合成的ECM，已经表明许多类型的细胞显示出向更坚硬的ECM区域迁移的强烈倾向-这一过程被称为趋多性。多柔趋化被认为是上皮向间充质转化和发育形态发生的关键，但其异常调节与癌细胞的恶性性质有关。尽管趋多性具有明显的生物学和临床意义，但细胞感知机械提示并将其转化为细胞反应(如迁移)的确切机制在很大程度上尚不清楚。在这里，我们建议通过使用最先进的生物物理技术的组合来揭示这些机制：具有可调机械性能的合成水凝胶来模拟生物组织的僵硬景观，以及遗传编码的FRET生物传感器来可视化跨迁移细胞的信号活动。通过将这些方法与定量图像分析和细胞内信号通路的遗传/药物扰动相结合，我们将揭示将机械引导线索转化为定向细胞迁移的特定分子角色。这一建议的具体目标包括：(1)解开细胞内受细胞外基质硬度梯度引导的信号分子的时空动力学；(2)确定整合素为基础的焦点粘连的机械传感如何调节细胞内由细胞外基质硬度梯度引导的信号活动。这项拟议的研究将揭示基本的生物学机制，这些机制支持细胞如何感知微环境的机械特性，并将这些信息转化为特定的细胞响应定向迁移。

Cell migration is a fundamental biological phenomenon that is critical for life of unicellular and multicellular organisms. Simple organisms, such as amebae, have to migrate to find food and to mate. Complexity of multicellular organisms, such as vertebrates, brings the necessity for individual cells and groups of cells to position themselves during developmental morphogenesis that is largely achieved through directed cell migration. In the adult, directed migration plays an important role in a wide range of physiological and pathological processes, including renewal of skin and intestinal epithelium, immune system function, and wound healing, as well as cancer metastasis and cardiovascular diseases. In tissue environment, cell migration is tightly regulated by numerous guidance cues that modulate activity of signaling networks, which in turn control intracellular machineries, e.g. actin cytoskeleton and focal adhesions, driving cell migration. In the last few decades, much attention has focused on understanding how chemical signals, such as soluble growth factors, guide cell migration. However, more recent studies have highlighted the importance of the mechanical signals in cell microenvironment, including density and stiffness of the extracellular matrix (ECM) in regulating cell migration. By using synthetic ECMs it has been shown that many cell types show a strong propensity to migrate toward areas of stiffer ECM - a process known as durotaxis. Durotaxis is thought to be critical to epithelial--to--mesenchymal transition and developmental morphogenesis, but its abnormal regulation is associated with the malignant nature of cancer cells. Despite the apparent biological and clinical significance of durotaxis, the exact mechanisms by which the cells sense mechanical cues and transduce them into cellular responses, such as migration,  are largely unknown. Here, we propose to reveal these mechanisms by using a combination of state--of--the--art biophysical techniques: synthetic hydrogels with tunable mechanical properties to mimic stiffness landscape of the biological tissues and genetically encoded FRET biosensors to visualize signaling activity across migrating cells. By coupling these approaches with quantitative image analysis and genetic/pharmacological perturbations of intracellular signaling pathways we will reveal specific molecular players that transduce mechanical guidance cues into directed cell migration. The specific aims of this proposal include: (1) To unravel the spatiotemporal dynamics of signaling molecules in the cells guided by a gradient of ECM stiffness; (2) To determine how mechanosensing by integrin-based focal adhesions modulates signaling activities in the cells guided by a gradient of ECM stiffness. The proposed study will uncover fundamental biological mechanisms that underpin how cells sense mechanical properties of the microenvironment and convert this information into a specific cellular response - directed migration.