Structure and assembly of cytoskeletal filaments

描述（由申请人提供）：细胞、器官和生物体的形态学是由细胞发展和感知机械力的能力来定义的。癌症、遗传畸形和其他疾病涉及力产生、力感应或刚性感应的改变。我们的长期目标是对细胞粘附、迁移和力产生过程的生化和生物物理功能进行定量的分子理解，包括对底物硬度的感知。最近的研究结果为刚性感知和不同肌球蛋白II亚型在周期性收缩中的作用提供了见解。在刚性感应的情况下，它在许多癌细胞中明显改变。在物理方面，刚度由单位力的位移来定义，我们建议在主动板足的前缘检测这两个参数。我们的研究已经确定了几种传感纤维连接蛋白基质刚性所需的蛋白质，如avB3整合素、RPTPa、Fyn和p130Cas。在活性板足中，这些蛋白质集中在边缘附近，并通过酶连接。我们的工作假设是，p130Cas的力依赖性展开导致刚性表面上的Fyn磷酸化，但在软表面上，Fyn和p130Cas被机械移位，从而抑制磷酸化。我们建议确定Fyn是否以及如何在刚性传感期间固定在前缘。我们将通过FRET实验检查p130Cas是否在前缘被拉伸，并将研究其在刚性感应过程中的结合机制。在某些情况下，细胞利用周期性收缩来感知刚性。在许多类型的扩散性和迁移性细胞中也观察到类似的收缩。然而，肌凝蛋白II-A和II-B的作用有很大的不同。收缩的周期性性质使得在荧光显微镜下更容易将肌球蛋白丝的组装与垂直和侧向力产生的定量联系起来。板基在正常收缩过程中的垂直弯曲对三维运动和基质重塑具有重要意义。收缩的控制取决于肌凝蛋白轻链激酶（MLCK）及其在肌动蛋白上的转运。使用GFP-myosin II-A或II-B、抑制剂和myosin- depletion细胞系，我们将分析局部组装和磷酸化是否与力产生相关。我们将确定定位和肌凝蛋白激活需要哪个MLCK结构域。这些定量分析将在分子水平上提供对刚性传感和周期性收缩的生化和物理方面的理解，然后可用于伤口愈合，转移，组织畸形和功能性组织工程的新疗法的建模和设计。

DESCRIPTION (provided by applicant): The morphologies of cells, organs and organisms are defined by the ability of cells to develop and sense mechanical forces. Cancers, genetic malformations, and other diseases involve alteration of either force production, force sensing or rigidity sensing. Our long-range goal is to develop a quantitative, molecular understanding of the biochemical and biophysical functions underlying the processes of cell adhesion, migration and force generation, including the sensing of substrate rigidity. Recent findings have provided insight into rigidity sensing and the roles of different myosin II isoforms in periodic contractions. In the case of rigidity sensing, it is notably altered in many cancerous cells. In physical terms, rigidity is defined by the displacement per unit force and we suggest that both parameters are sensed at the leading edges of active lamellipodia. Our studies have identified several proteins that are required for sensing the rigidity of fibronectin matrices, avB3 integrin, RPTPa , Fyn, and p130Cas. In active lamellipodia, these proteins are concentrated near the leading edge and are linked enzymatically. Our working hypothesis is that force-dependent unfolding of p130Cas results in Fyn phosphorylation on rigid surfaces but on soft surfaces Fyn and p130Cas are mechanically displaced thereby inhibiting phosphorylation. We propose to determine if and how Fyn is immobilized at the leading edge during rigidity sensing. We will examine if p130Cas is stretched at the leading edge by a FRET assay and will study the j mechanism of its binding during rigidity sensing. In some cases, cells use periodic contractions to sense rigidity. Similar contractions are observed in many types of spreading and migrating cells. However, the roles of myosin II-A and II-B are dramatically different. The periodic nature of the contractions makes it easier to correlate assembly of the myosin filaments with the quantification of both vertical and lateral force generation in the fluorescent microscope. Vertical bending of the lamellipodium during normal contractions has important implications for 3-D motility and matrix remodeling. Control of the contractions depends upon myosin light chain kinase (MLCK) and possibly its transport on actin. Using GFP-myosin II-A or II-B, inhibitors and myosin-depleted cell lines, we will analyze whether localized assembly and phosphorylation correlate with force production. We will determine which MLCK domain is needed for localization and myosin activation. These quantitative analyses will provide an understanding of the biochemical and physical aspects of rigidity sensing and periodic contractions at a molecular level that can then be used for modeling and design of novel therapies for wound healing, metastasis, tissue malformation and functional tissue engineering.