The use of the gene knockout technique for functional and mechanical studies of the roles of vinculin in cells

摘要

Vinculin is a cytoskeletal protein that forms a molecular bridge between integrins (receptors for extracellular matrix materials) in the cell membrane, and F actin. This link is not merely structural but has a mechanical or regulatory function.The functional characterization of vinculin was made possible by abrogating the vinculin gene in embryo stem cells, and hence making animals that lacked one vinculin gene. Lack of all vinculin was lethal during embryo development, since vinculin(-/-)embryos died on or before day E10 Cardiac development was particularly retarded and the reduced structure never achieved contractility. The cessation of development by the 10th day of gestation could be explained by the altered characteristics of cellsisolated from the mutant embryos. The cytoskeletal organization of vinculin(-/-) cells was aberrant. Vinculin null embryo cells moved at twice the rate of normal fibroblasts and adhered half as well. Vinculin null cells have a rounded, non-spreading shape and proliferate faster than wildtype cells. These defects must in part contribute to poor embryo development where cell shape, locomotion and adhesion must be finely controlled. Atomic force microscopy and magnetometry have demonstrated changes inviscosity and stiffness of cells correlated to their vinculin content.