COVISUALIZATION BY COMPUTATIONAL OPTICAL-SECTIONING MICROSCOPY OF INTEGRIN AND ASSOCIATED PROTEINS AT THE CELL-MEMBRANE OF LIVING ONION PROTOPLASTS

Using higher-resolution wide-field computational optical-sectioning fl uorescence microscopy, the distribution of antigens recognized by anti bodies against animal beta(1) integrin, fibronectin, and vitronectin h as been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spo ts, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta(1) integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specificity of antibody recognitio n. Affinity purification of onion cell extract with the same anti-inte grin used for visualization has yielded protein that separates in SDS- PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised again st the extracellular domain of chicken beta(1) integrin, and are also recognized by an antibody against the intracellular domain of chicken beta(1) integrin. Because beta(1) integrin is a key protein in numerou s animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the a dhesion sites long known to occur in cells of this tissue. Because vit ronectin and fibronectin are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins bot h in the wall (matrix) and on enzymatically prepared protoplasts reinf orces the concept that onion cells have adhesion sites with some simil arity to certain kinds of adhesion sites in animals.