Ultrastructure and gold-immunolabelling of cell-substratum adhesions (podosomes) in RSV-transformed BHK cells

Abstract

Rous sarcoma virus-transformed BHK (RSV/B4-BHK) cells develop peculiar dot-like adhesions, that have been named podosomes, which, in the presence of serum, aggregate into ring- or crescent-shaped adhesion sites, the rosettes of podosomes. We have used the lysis-squirting technique and gold-immunolabelling to study the 3D-organisation of podosomes and the location of vinculin, gelsolin, phosphotyrosine-containing proteins and pp60src at an ultrastructural level. Podosomes appear to be conical bodies, 0.1-0.5 micron high, made by a dense aggregation of actin oligomers and several associated proteins, connected in the rosette by actin filaments. Gelsolin and some phosphotyrosine-containing proteins are found within the podosomes, often associated with the actin filaments, while vinculin is found predominantly at the podosome periphery, associated with microfilaments, and pp60src is located on the adjacent plasma membrane.