Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the ␤1 and the ␣2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events.

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Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.