Early phorbol ester induced release of cell surface fibronectin: direct observation by photoelectron microscopy

One of the early effects of the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) on cultured normal fibroblasts is the release of fibronectin into the culture medium. Immunophotoelectron microscopy was used to follow the loss of fibronectin from the upper cell surface of normal human foreskin fibroblasts exposed to TPA. Fibronectin labeled with silver-enhanced colloidal gold-antibody conjugates appears in photoelectron images as streak- and network-like patterns of bright dots against the less photoemissive uncoated cell surface. Labeled fibronectin present beneath the culture is not detected due to the surface-specificity of this technique. Ten to 30 min of exposure to 100 ng/ml TPA in culture medium results in a readily visible decrease in upper cell surface fibronectin. In these experiments, 60 min of exposure to TPA releases nearly all upper cell surface fibronectin, leaving only occasional short streaks of label. In contrast, extracellular matrix fibronectin between cells is apparently more resistant to release and can still be seen in photoelectron images even when the upper cell surface appears to be fibronectin-free. This immunophotoelectron study shows the distribution of fibronectin on fibroblasts at high resolution and demonstrates that the initial fibronectin release resulting from TPA exposure is at the expense of preexisting cell-surface fibronectin. These results also illustrate the application of photoelectron microscopy as a useful technique in cell biology.