Endothelial Cell Adhesion in Real Time

Abstract

Real time measurements of cell-substratum adhesion in endo- thelial cells were obtained by tandem scanning confocal micros- copy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radi- ance levels) in the living cell such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal determinant of cell-sub- stratum adhesion. Measurements of (a) the focal contact area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time. The topography of focal contacts was estimated by calculat- ing separation distances from radiance values using a calibra- tion technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and sub- stratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm. Subtraction ofconsecutive images revealed continuous spon- taneous remodeling of individual focal adhesions in unper- turbed cells during periods of< 1 min. Despite extensive remod- eling of focal contact sites, however, cell adhesion calculated for an entire cell over extended periods varied by < 10%. When cytoskeletal stability was impaired by exposure to cytochalasin or when cells were exposed to proteolytic enzyme, endothelial adhesion declined rapidly. Such changes were recorded at the level of single cells, groups of cells, and at single focal adhe- sions. In both unperturbed and manipulated cells, the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance of existing sites and appearance ofnew ones often occurred within minutes while adjacent sites underwent minimal remodelling. Tandem scanning confocal microscopy image analysis of living cells in real time provides repetitive spatial, temporal, and quantitative information about cell adhesion. Such an ap- proach should allow more precise quantitative analyses to be made of the interactions between extracellular matrix, adhe- sion proteins, integrins, and the cytoskeleton in the living cell.