Molecular diversity of cell-matrix adhesions

In this study we have examined for molecular heterogeneity of cell-matrix a dhesions and the involvement of actomyosin contractility in the selective r ecruitment of different plaque proteins. For this purpose, we have develope d a novel microscopic approach for molecular morphometry, based on automati c identification of matrix adhesions, followed by quantitative immunofluore scence and morphometric analysis. Particularly informative was fluorescence ratio imaging, comparing the local labeling intensities of different plaqu e molecules, including vinculin, paxillin, tensin and phosphotyrosine-conta ining proteins. Ratio imaging revealed considerable molecular heterogeneity between and within adhesion sites. Most striking were the differences betw een focal contacts, which are vinculin- and paxillin-rich and contain high levels of phosphotyrosine, and fibrillar adhesions, which are tensin-rich a nd contain little or no phosphotyrosine, Ratio imaging also revealed consid erable variability in the molecular substructure of individual focal contac ts, pointing to a non-uniform distribution of phosphotyrosine and the diffe rent plaque constituents. Studying the quantitative relationships between t he various components of the submembrane plaque indicated that the levels o f vinculin, paxillin and phosphotyrosine in adhesion sites are positively c orrelated with each other and negatively correlated with the levels of tens in, Tyrosine phosphorylation of focal contacts was highly sensitive to cell ular contractility, and was diminished within 5 minutes after treatment wit h the kinase inhibitor H-7, an inhibitor of actomyosin contractility. This was followed by the loss of paxillin and vinculin from the focal adhesions. Tensin-rich fibrillar adhesions were relatively insensitive to H-7 treatme nt. These findings suggest a role for contractility in the generation of ma trix adhesion diversity.