Cell-substrate adhesions are a mechanosensitive protein complex that regulates various cellular functions. Molecular mechanisms underlying the physical force-dependent regulation remain elusive partly because endogenous forces are distributed in a spatially heterogeneous manner within cells, thus complicating the interpretation on the effect of forces. Here we use a micropatterning technique to focus spatially distributed intracellular contractile forces onto a particular subcellular area, with which mechanical and pharmacological effects are separately analyzed. Single human osteosarcoma U2OS cells were plated within square micropatterns, and phosphorylation of an adhesion-associated adaptor protein paxillin was analyzed. Paxillin, visualized with immunostaining, was highly accumulated in the proximity of the corners of the square micropatterns where cellular forces are concentrated, but the huge paxillin-labeled adhesions were less phosphorylated compared to those present elsewhere as a small patch. Pharmacological inhibition of the endogenous forces resulted in disassembly of the huge dephoshorylated paxillin clusters
in contrast, the small, highly phosphorylated paxillin patches persisted. Similar negative regulation of paxillin phosphorylation is also induced upon loading of exogenous forces. Unexpectedly, on the other hand, immunoblot of cell lysates showed a tendency of a reduction in paxillin phosphorylation upon the same pharmacological inhibition of the endogenous forces. Thus, the response of paxillin phosphorylation to mechanical forces was the opposite between the immunostaining and immunoblot data
i.e., the phosphorylation is reduced and enhanced at subcellular level and whole-cell level, respectively, in response to loading of mechanical forces. To reconcile the contradictory results, we submit a simple model that is consistent with not only the present but also previous reports on the regulation of paxillin. The model implies that similar opposite response can generally emerge if the protein activation is negatively regulated at a local place while the activation trigger alters the assembly of the protein to the local place.