Massive Endocytosis Activated by Perturbing the Outer Plasmalemmal Monolayer

Abstract

The roles played by lipids in endocytic processes are the subject of much ongoing debate. Using electrophysiological, optical, and ultrastructural methods, we describe massive endocytosis (MEND) of >50% of the plasmalemma in response to perturbing the outer plasmalemma monolayer of fibroblasts and cardiac myocytes by multiple means. Extracellular application of a bacterial sphingomyelinase causes MEND within seconds, and similar responses occur with the nonionic detergents, Triton X-100 and NP-40, proapoptotic drugs (e.g. edelfosine and tamoxifen), and an amphipathic phospholipase inhibitor, U73122. At the concentrations employed, the effective agents do not cause membrane permeability changes, and they are inactive from the cytoplasmic side. Ca transients that do not cause MEND decrease markedly the threshold concentrations of amphipaths that cause MEND, perhaps by generating a lipid catalyst of MEND. Noise analysis of NP-40 records suggests that the average vesicle size is initially small (<100nm). However, internalized vesicles evidently fuse rapidly, as horseradish peroxidase is found within seconds in large vacuoles and multi-lamellar bodies. These MEND responses do not require cytoplasmic ATP, Ca, or dynamins, and they can be repeated multiple times with reversal taking place over several minutes in the presence of ATP. For nonionic detergents, ongoing MEND stops within 2 to 4 seconds when detergent is removed. For dodecylsulfate and dodecylglucoside, MEND occurs only after detergent removal. These results suggest that endocytosis can be driven primarily by lipidic forces, possibly by lipid and protein partitioning into domains that pinch off to the cytoplasm as a result of line tension to their surround.