Structure and assembly of cytoskeletal filaments

描述(申请人提供)：细胞、器官和生物体的形态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到力产生、力感觉或僵硬感觉的改变。我们的长期目标是发展对细胞黏附、迁移和力产生过程中潜在的生化和生物物理功能的定量的、分子的理解，包括底物刚性的感知。最近的发现为僵硬感觉和不同肌球蛋白II亚型在周期性收缩中的作用提供了洞察力。在刚性感应的情况下，它在许多癌细胞中发生了显著的变化。在物理方面，刚性是由单位力的位移来定义的，我们认为这两个参数都是在活动板脂的前沿检测到的。我们的研究已经确定了几种检测纤维连接蛋白基质、avB3整合素、RPTPA、Fyn和p130Cas刚性所需的蛋白质。在活性片状脂体中，这些蛋白质集中在前缘附近，并通过酶连接。我们的工作假设是，依赖于力的p130Cas的展开导致Fyn在刚性表面的磷酸化，但在软表面，Fyn和p130Cas被机械置换，从而抑制磷酸化。我们建议在刚性检测过程中确定Fyn是否以及如何固定在前沿。我们将通过FRET实验检查p130Cas是否在前沿被拉伸，并将研究其在刚性传感过程中的结合机制。在某些情况下，细胞使用周期性收缩来感觉僵硬。在许多类型的扩张和迁移细胞中也可以观察到类似的收缩。然而，肌球蛋白II-A和II-B的作用截然不同。收缩的周期性使得在荧光显微镜下将肌球蛋白细丝的组装与垂直和侧向力产生的量化相关联变得更容易。在正常收缩过程中，板层的垂直弯曲对三维运动和基质重塑有重要意义。收缩的控制依赖于肌球蛋白轻链激酶(MLCK)及其对肌动蛋白的运输。使用GFP-肌球蛋白II-A或II-B、抑制剂和肌球蛋白缺失的细胞系，我们将分析局部组装和磷酸化是否与FORCE产生相关。我们将确定定位和肌球蛋白激活所需的MLCK结构域。这些定量分析将在分子水平上提供对僵硬感觉和周期性收缩的生化和物理方面的了解，然后可用于创伤愈合、转移、组织畸形和功能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.