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The formation of focal contacts by fibroblast-like 3T3 and 9390 cells and endothelial XTH-2 cells was examined with the use of reflection interference contrast microscopy at different cell densities. It was found that cells density-inhibited in growth loose all focal contacts with the substratum. This observation explains the disappearance of stress fibers in density-inhibited cells, and also offers an explanation for the mechanism of density-dependent inhibition of proliferation of anchorage-dependent cells.

A pressure device has been used to obtain information about the forces involved in the maintenance of the aggregated state of melanophores of the angelfish, Pterophyllum scalare. Single aggregated melanophores of isolated scales were submitted to mechanical compression with forces ranging from 50-320 mup. As a function of the gradually increasing force melanophores disperse their pigment, the degree of dispersion being proportional to the intensity of the force. When microtubules are destroyed by treatment with 0.3 mM vinblastine in KCl solution, pigment dispersion in response to the external force is similar to that observed in KCl alone. After changing the medium to NaCl solution, melanin granules remain concentrated in the cell center; the force-induced melanosome dispersion, however, is significantly enhanced. Distinctly lower forces are required to produce an expansion equivalent to that observed in KCl solution. When the medium is changed to vinblastine-KCl again, the dispersion in response to the external force resembles that obtained before NaCl treatment. Removal of Ca++ and Mg++ ions by treatment with 2 mM EDTA or EGTA in Ringer's solution containing 0.1 mM adrenalin produces a remarkable enhancement of melanosome dispersion in response to increasing external force. This effect of EDTA or EGTA is completely reversible. When the medium is changed to normal Ca++-Ringer's, the force/dispersion curve resembles that obtained before EDTA or EGTA treatment. It is concluded that a state of equilibrium exists between the external force and an opposing force produced by the melanophore. The differences in the opposing force under different experimental conditions may be due to a "contractile component". This component seems to be independent of microtubules, as indicated by vinblastine experiments. It "contracts" under aggregating stimulus and "relaxes" under dispersing stimulus. From the data presented in this paper, the order of magnitude of the pressure developed by the contractile component in the completely aggregated state was calculated as between 5-7 p/cm2 in the relaxed state and about 20 p/cm2 during contraction. These values are comparable to those observed in other nonmuscular cells.

A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.

The scanning of fluorescence of a cell culture along a path several milimeters long, gives a series of signals, which allow calculation of the fluorescence emitted per cell. The emission at 450 +/- 10 nm, excited at 360 nm, provides a measure of NADH (and NADPH) per cell. Combination of this method with modifications in energy metabolism, allows determination in the same sample of total NAD content, of NAD redox state, content of NAD in the mitochondria and content of NAD plus NADP in the cytoplasm. The method can be extended to measure other cellular constituents by labelling with fluorescent markers, e.g. antibodies.

A method is described for the determination of cellular F-actin content using fluorometry of TRITC-phalloidin at very high dilution. In this case no saturation of all binding sites available is reached, however the staining is highly specific and the specificity is not affected by the preparative procedure as may be the case at high concentrations of TRITC-phalloidin. The method is based on calculation of fluorescence intensity at equilibrium conditions from measurements at two different numbers of exchange of the staining solution by using Lineweaver-Burk plotting. The relative content of F-actin has been determined for three established cell lines, an amphibian cell line XTH-2 of endothelial origin, 3T3 cells and SV 40 transformed 3T3 cells. In the two non transformed lines F-actin decreases with increasing cell density starting with the onset of confluency of the culture. SV 40 transformed 3T3 cells generally contain less F-actin and do not show any significant point of change. The decrease in F-actin with increasing cell density is accompanied by a disappearance of stress fibres. SV 40 3T3 cells generally are devoid of stress fibres. The observations are discussed considering a possible involvement of F-actin in growth control.