Mechanistic analysis of massive endocytosis in relation to functionally defined surface membrane domains

Abstract

A large fraction of endocytosis in eukaryotic cells occurs without adaptors or dynamins. Here, we present evidence for the involvement of lipid domains in massive endocytosis (MEND) activated by both large Ca transients and amphipathic compounds in baby hamster kidney and HEK293 cells. First, we demonstrate functional coupling of the two MEND types. Ca transients can strongly facilitate detergent-activated MEND. Conversely, an amphipath with dual alkyl chains, ditridecylphthalate, is without effect in the absence of Ca transients but induces MEND to occur within seconds during Ca transients. Ca transients, like amphipaths, enhance the extraction of lipids from cells by β-cyclodextrins. Second, we demonstrate that electrical and/or optical signals generated by selected membrane probes are nearly insensitive to MEND, suggesting that those probes segregate into membrane domains that are not taken up by MEND. Triphenylphosphoniums are increasingly excluded from domains that internalize as the carbon chain length increases from 4 to 12. The small cationic membrane dye, FM 4–64, binds well to domains that internalize, whereas a closely related dye with a larger hydrophobic moiety, di-4-ANEPPDHQ (ANEPPDHQ) is excluded. Multiple carrier-type ionophores and a small amphipathic anion, niflumic acid, are also excluded. Probes with modest MEND sensitivity include the hydrophobic anion, dipicrylamine, carbonyl cyanide m-chlorophenylhydrazone, and NBD-phosphatidylethanolamine. Third, we demonstrate that large Ca transients can strongly enhance the extracellular binding of several membrane probes, monitored electrically or optically, consistent with a more disordered membrane with more amphipath-binding sites. Fluorescence shifts of ANEPPDHQ report increased disorder of the extracellular monolayer after large Ca transients, consistent with an increased propensity of the membrane to phase separate and vesiculate. Collectively, the results indicate that >50% of the outer monolayer is ordered and can be selectively internalized during MEND responses initiated by two very different cell perturbations.

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].…”

Damage control: cellular mechanisms of plasma membrane repair

“…Conversely, cholesterol enrichment was reported to potentiate the MEND massive endocytic process triggered by Ca 2+ entry or exposure to sphingomyelinase 48 . The large fractions of plasma membrane internalized by MEND were found to bind amphipathic molecules less efficiently than membrane remaining at the cell surface, leading to the suggestion that this form of endocytosis involves more highly ordered lipid microdomains 52 . These observations support the involvement of ASM-generated ceramide microdomains, formed within ordered cholesterol/sphingolipid-enriched lipid rafts, in the Ca 2+ -dependent endocytic process that mediates plasma membrane repair.…”

Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs

“…Identifying cues and their signaling processes that can broadly enhance fluid-phase endocytosis, such as by enhancing flotillin-dependent endocytosis, would be very valuable from a cancer drug delivery perspective. Massive endocytosis (MEND) [25][26][27] may be a particularly attractive mechanism for targeted drug delivery. MEND occurs in response to large intracellular Ca 2+ transients (e.g.…”