Quantitation of actin polymerization in two human fibroblast sub-types responding to mechanical stretching

Abstract

To study early reorganization of the cytoskeleton in response to physical forces, human gingival and periodontal ligament fibroblasts were cultured on flexible plastic substrata and stretched by mechanical deformation of the substratum. F-actin was measured by quantitative spectrofluorimetry of FITC-phalloidin-stained cells. Fluorescence due to FITC-phalloidin was reduced stoichiometrically by co-incubation with unlabeled phalloidin. Increases in stretch-induced fluorescence were blocked by preincubation of cells with cytochalasin D. Compared to baseline values, F-actin in gingival fibroblasts was reduced by 50% at 10 s after stretching but was increased more than 100% at 50 s after stretching. Increases of F-actin were also dependent on the amount of deformation of membrane: 3.3% stretching produced the largest increase and 9.2% stretching produced the least. Stretch-activated actin polymerization was blocked by pre-incubation of cells with 500 ng ml-1 pertussis toxin or 10 mM EGTA. Compared to gingival cells, periodontal ligament fibroblasts exhibited twice the total actin per cell and three times the baseline F-actin per mm2, however, there was no increase in fluorescence after stretching. In both cell types, stretching caused increased ruffling activity and no detectable shape changes at 60 s, but at 180 s the cells shortened and pseudopods retracted. The microfilament system of fibroblasts exhibits rapid dynamic responses to mechanical deformation prior to cell shape changes and appears to be dependent on the cell type.