Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

Abstract

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and loss of reversible FM 4–64 binding confirm that 40–75% of the cell surface is internalized. Ongoing MEND stops in 2–4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and β-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces.

Biophysics of cell membrane lipids in cancer drug resistance: Implications for drug transport and drug delivery with nanoparticles

“…This assumption is further supported by recent studies showing that perturbation of the outer monolayer of the cell membrane with common detergents induces massive endocytosis in cell-membrane lipids with order domains [130, 131]. An interesting observation was that this endocytic process appears to be independent of the usual endocytic machinery such as dynamins and functional membrane cytoskeleton and other G proteins [130, 131]. Therefore, these studies clearly suggest that nanocarriers modified with cationic surfactants or cell-penetrating peptides might induce endocytosis due to interactions with the cell membrane and thereby enhance intracellular drug uptake.…”

“…It is possible that upon interaction between cell-penetrating peptides or cationic surfactant-modified nanocarriers and resistant cell membranes, the outer monolayer phospholipids of the cell membrane can develop lateral inhomogeneities and promote vesiculation of the plasma membrane into the cells. This assumption is further supported by recent studies showing that perturbation of the outer monolayer of the cell membrane with common detergents induces massive endocytosis in cell-membrane lipids with order domains [130, 131]. An interesting observation was that this endocytic process appears to be independent of the usual endocytic machinery such as dynamins and functional membrane cytoskeleton and other G proteins [130, 131].…”

The biophysical and molecular basis of intracellular pH sensing by Na+/H+exchanger‐3

“…This increase in NHE3 due to the perfusion of NHE3 478–500 ‐WT was not caused by the exocytosis of NHE3 at the plasma membrane, given that the C m (an index of cell surface; refs. 33, 44) did not change (Fig. 3 A , inset).…”

Human TRPML1 channel structures in open and closed conformations

“…Patch-clamp recordings of cell electrical parameters were performed as described previously 12,35,43 , using MATLAB-based Capmeter v7.2 44 with a National Instruments digital acquisition board and an Axopatch 200B patch-clamp amplifier. Square-wave voltage perturbation (20 mV; 0.2–0.5 kHz) was used for capacitance measurement.…”