Endothelial cell adhesion in real time. Measurements in vitro by tandem scanning confocal image analysis.

Abstract

Real time measurements of cell-substratum adhesion in endothelial cells were obtained by tandem scanning confocal microscopy of sites of focal contact (focal adhesions) at the abluminal cell surface. Focal contact sites were sharply defined (low radiance 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-substratum 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 calculating separation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center of each focal contact, separation distances throughout the adhesion regions were always < 50 nm.Subtraction ofconsecutive images revealed continuous spontaneous remodeling of individual focal adhesions in unperturbed cells during periods of < 1 min. Despite extensive remodeling 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 adhesions. 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 of new 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 approach should allow more precise quantitative analyses to be A preliminary report ofparts ofthis work has appeared in abstract form (1991. FASEB (Fed. Am. Soc. Exp. Biol.) J. 5:1603A).

Model of cellular mechanotransduction via actin stress fibers

“…f 0 is a reference frequency taken to be 1 Hz pN. The adhesion density at the cell surface is typically d = 0.15 adhesions/µm 2 (Davies et al, 1993(Davies et al, , 1994. If 20 to 30% of the bead is embedded in the membrane, the bead surface area available for binding to membrane proteins is 0.8 − 1.2πr 2 , where r is the bead radius, and the number of cell-bead adhesions would be ∼ 0.8−1.2πr 2 d. This yields a typical force per adhesion of F ext = F bead /πr 2 d ≈ 600 pN.…”

Microtopography of metal surfaces influence fibroblast growth by modifying cell shape, cytoskeleton, and adhesion

“…The focal adhesion sizes had a wide size distribution (between 0.3-15 mm), which can be interpreted as being representative of the constant dynamic remodeling of all adhesion sites produced. 16 For the rougher surface of TS, the cell morphology indicated that the topography impinged significantly upon cell spreading with the cell periphery often aligned to the rough ''basketweave'' topography, producing a jagged polygonal cell periphery. Fluorescence indicated considerable interaction; however, quantitatively the cell area was significantly different to that of cells on SS, but not to TE or NE.…”

Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification

“…An intensity threshold was determined to define the contact/adhesion area. The threshold intensity I thresh was derived from equation 1 by setting the threshold membrane-to-substrate distance at 40 nm [25][26][27][28] for a wavelength of λ 1 = 593 nm or λ 2 = 546 nm and using the minimum and maximum intensities I m and I M on each cell image [29]. This is a semi-quantitative method [25,29] which distinguishes between adherent and nonadherent parts of the cell membrane.…”

“…To identify adhesive areas we used a semi-quantitative analysis [25] to distinguish between adherent and non-adherent areas within the projected cell area. To this end, we applied a threshold intensity corresponding to a cell-to-substrate distance of 40 nm [26]. Figures 3A & B show RICM images and respective threshold images of a representative PatuT and PatuS cell at different stages of cell spreading, namely 16, 30 and 60 min post plating.…”