STRUCTURE AND ASSEMBLY OF CYTOSKELETAL FILAMENTS

描述：(逐字摘自申请者摘要)生成定义的单元格 对整合素-矩阵接触的力，并对这些力做出响应 人脉会对他们施加压力。检测矩阵刚性并生成正确的 力是细胞在发育、伤口愈合和修复过程中的关键功能 癌症等疾病。转化细胞的一个主要标志是它们的能力 在软琼脂上生长，即在没有对基质产生力的情况下 联系人。我们已经开发了新的方法来测量力依赖的变化 基质-整合素-细胞骨架的连接和测量细胞 在亚微米区域产生。我们观察到依赖于力的增强 整合素-细胞骨架连接与施加的力成比例。 令人惊讶的是，基质连接的纤维连接蛋白受体-细胞骨架连接 增强的但未连接的整合素不是。连杆形成和动力学是 依赖酪氨酸磷酸酶/激酶。酪氨酸磷酸酶抑制剂阻断 强化和酪氨酸激酶，Src，抑制强化，但仅 Vitronectin-avb3-细胞骨架连接。我们现在建议测量加固 缺失酪氨酸磷酸酶(Shp2-/-、RPTPα-/-和PTEN-/-)的细胞 细胞)，这些细胞在纤维连接蛋白或玻璃体连接蛋白上的运动性改变。第一 两种磷酸酶与纤维连接蛋白和 分别依赖于Vitronectin的运动，而PTEN主要起作用 作为一种脂质磷酸酶，通常会抑制运动性。此外，我们正在 研究主要的PYK2-/-，FAK-/+/PYK2-/-，Src-/-/PYK2-/-，FYN-/-，FYN-/-/Src-/- 和相关的控制细胞来研究酪氨酸激酶是如何调节动力学的 与力相关的联系。要了解细胞力是如何产生的 我们已经开发了硅芯片中的力传感器，并拥有 改进了激光镊子测量等距牵引力的方法。我们的 研究表明，所有被测试电池的牵引力都在前面。 并在核区切换到前进方向。此外，军队的力量 在背侧和腹侧表面生成相同的水平，这验证了 从背部表面测量牵引力。我们现在就会 测量力在肌动蛋白动力学改变时以及细胞改变时的变化 刺激以改变收缩能力或肌球蛋白磷酸化。这些研究 将使我们能够定义力感知和力的分子基础 这与更深层次地理解转移和 发展。

DESCRIPTION: (Verbatim from the applicant's abstract) Cells generate defined forces on integrin-matrix contacts and respond to the forces that those contacts exert on them. Sensing matrix rigidity and generating the correct force are critical functions for cells in development, wound healing and diseases such as cancer. A major hallmark of transformed cells is their ability to grow on soft agar, i.e. in the absence of force generated on matrix contacts. We have developed new methods to measure force-dependent changes in matrix-integrin-cytoskeleton links and to measure the forces that cells generate on submicron areas. We observed force-dependent reinforcement of integrin-cytoskeleton linkages in proportion to the force applied. Surprisingly, matrix-liganded fibronectin receptor-cytoskeleton linkages are reinforced but unliganded integrins are not. Linkage formation and dynamics are tyrosine phosphatase/kinase dependent. Tyrosine phosphatase inhibitors block reinforcement and the tyrosine kinase, Src, inhibits reinforcement but only of vitronectin-avb3-cytoskeleton linkages. We now propose to measure reinforcement in cells missing tyrosine phosphatases (Shp2-/-, RPTP alpha-/-, and PTEN-/- cells) that are altered in motility on fibronectin or vitronectin. The first two phosphatases are strongly implicated in fibronectin and vitronectin-dependent motility, respectively, whereas PTEN functions primarily as a lipid phosphatase and normally suppresses motility. In addition, we are studying primary Pyk2-/-, FAK-/+/Pyk2-/-, Src-/-/Pyk2-/-, Fyn-/-, Fyn-/-/Src-/- and related control cells to examine how tyrosine kinases modulate the dynamics of force-dependent linkages. To understand how cellular forces are generated and controlled, we have developed force sensors in silicon chips and have improved the laser tweezers measurements of isometric traction forces. Our studies show that traction forces are rearward in the front of all cells tested and switch to forward direction in the nuclear region. Further, forces of the same level are generated on both dorsal and ventral surfaces, which validates the measurements of traction forces from the dorsal surface. We will now measure how force changes when actin dynamics are altered and when cells are stimulated to modify contractility or myosin phosphorylation. These studies will enable us to define the molecular bases of force sensing and force generation, which is relevant to a deeper understanding of metastasis and development.