STRUCTURE AND ASSEMBLY OF CYTOSKELETAL FILAMENTS

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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.