Increased assembly of clathrin occurs in response to mitogenic activation of murine lymphocytes

Abstract

The unassembled (soluble) and assembled (particulate) pools of clathrin in murine lymphocytes have been separated by centrifugation, and specifically quantified by immunoblotting of cellular extracts with an anticlathrin heavy chain monoclonal antibody. In resting spleen lymphocytes only 25-30% of the total cellular clathrin was found to be present in an assembled form. Upon activation of lymphocytes with B or T cell mitogens (lipopolysaccharide or concanavalin A), the levels of assembled clathrin increased to 60% of the total. These changes in the levels of assembled clathrin were not due to an increase in total cellular clathrin concentration following lymphocyte activation, but rather to changes in the steady state ratio of assembled to unassembled clathrin. The increase in assembled clathrin preceded the expression of transferrin receptors, as measured by the cell surface binding of an antitransferrin receptor monoclonal antibody, and maximal DNA synthesis, indicating that clathrin assembly occurs early after lymphocyte activation and precedes cell division. Immunofluorescence analysis of activated lymphocytes with an anti-clathrin heavy chain monoclonal antibody revealed a punctuate staining pattern characteristic of coated pits and vesicles. Activated B lymphocytes displayed particularly prominent staining in the perinuclear region compared to T cells, suggesting that clathrin assembly may be important for B cell functions such as immunoglobulin synthesis or secretion. These results suggest that in lymphocytes, clathrin assembly is a dynamic process that is triggered by mitogenic stimuli.