EGF induces recycling membrane to form ruffles

KB cells are know to respond to epidermal growth factor (EGF) by producing prodigious ruffles in the plasma membrane within minutes. The signal transduction pathway underlying this effect in fibroblasts is mediated through Rac, a member of the Ras-like family of GTPases. As ruffles are rich in components of the cytoskeleton--particularly in actin and ezrin--it has been suggested that ruffles arise when activated Rac modulates the actin cytoskeleton to push out a membrane protrusion. We set out to see whether the surface of new ruffles arises from neighbouring membrane, or whether it comes from an intracellular pool of endocytosed membrane. If it arose by exocytosis from endosomes, it would be expected to be enriched in those recycling receptors that are concentrated in coated pits in the endocytic side of the cycle. On the other hand, if it arose passively from the adjacent plasma membrane, a uniform distribution of these receptors would be expected. Here, we show that as soon as ruffles appear on KB cells in response to EGF, their membrane surfaces are enriched in both transferrin and low density lipoprotein (LDL) receptors. Both these proteins are known to be selectively concentrated into endosomal membranes by clathrin-mediated endocytosis. Our results reveal that the surfaces of ruffles arise by exocytosis of internal membrane from the endocytic cycle and, therefore, that a primary action of Rac is to redirect the exocytosis of recycling membrane into just those specific sites where ruffles form.