STRUCTURE AND ASSEMBLY OF CYTOSKELETAL FILAMENTS

描述：（申请人摘要中的逐字记录）单元格生成定义 整合素-基质接触的力，并对这些力作出反应， 接触对他们产生影响。传感矩阵刚度并生成正确的 力是细胞发育、伤口愈合和 疾病，如癌症。转化细胞的一个主要标志是它们的能力 在软琼脂上生长，即在基质上不产生力的情况下 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-细胞骨架的联系，并测量细胞 在亚微米区域产生。我们观察到， 整合素-细胞骨架连接与所施加的力成比例。 令人惊讶的是，基质配体的纤连蛋白受体-细胞骨架连接是 增强的但未配体化的整联蛋白不是。连锁形成和动力学是 酪氨酸磷酸酶/激酶依赖性。酪氨酸磷酸酶抑制剂阻断 酪氨酸激酶，Src，抑制增强，但只有 玻连蛋白-AVB 3-细胞骨架连接。我们现在建议测量增强 在缺少酪氨酸磷酸酶（Shp 2-/-、RPTP α-/-和PTEN-/-）的细胞中， 细胞）在纤连蛋白或玻连蛋白上的运动性改变。第一 两种磷酸酶与纤连蛋白密切相关， 玻连蛋白依赖的运动，而PTEN的功能主要是 作为脂质磷酸酶，通常抑制运动。此外，我们在 研究原发性Pyk 2-/-、FAK-/+/Pyk 2-/-、Src-/-/Pyk 2-/-、Fyn-/-、Fyn-/-/Src-/- 和相关的控制细胞，以检查酪氨酸激酶如何调节动力学 依赖力的联系。为了了解细胞力是如何产生的 和控制，我们已经开发了硅芯片中的力传感器， 改进了等长牵引力的激光镊子测量。我们 研究表明，牵引力在所有测试电池的前部都是向后的 并在核区域切换到正向。此外， 在背侧和腹侧表面上产生相同的水平，这验证了 测量来自背表面的牵引力。我们现在将 测量当肌动蛋白动力学改变以及细胞 刺激以改变收缩性或肌球蛋白磷酸化。这些研究 将使我们能够定义力感测和力的分子基础 代，这与更深入地了解转移和 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.