Clathrin‐Dependent and Clathrin‐Independent Endocytosis are Differentially Sensitive to Insertion of Poly (Ethylene Glycol)‐Derivatized Cholesterol in the Plasma Membrane

Abstract

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036-3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.

Azithromycin, a Lysosomotropic Antibiotic, Has Distinct Effects on Fluid-Phase and Receptor-Mediated Endocytosis, but Does Not Impair Phagocytosis in J774 Macrophages

“…Bulkflow endocytosis may be affected not only by local lipid asymmetry across the bilayer, but also by physical forces such as tension and fluidity [84,85]. That clathrin-independent endocytosis is particularly sensitive to such biophysical parameters is supported by its selective inhibition by low concentrations of PEG-cholesterol, whereas impairment of incurvation of clathrincoated pits requires higher concentrations [86,87]. In this respect, interaction of azithromycin with membrane phospholipids is supported by direct in vitro evidence based on equilibrium dialysis with liposomes [88].…”

Physics of the Chemical Asymmetry of the Cell Membrane: Implications in Gene Regulation and Pharmacology

“…Finally it was also demonstrated that reversing lipid asymmetry by adding lipids to the outer leaflet that could not be transported to the inner leaflet opposed fluid phase and clathrin endocytosis triggering the exocytosis of caveolae vesicles from the cytoplasm [12]. This highlighted how the physical properties of the cell membrane are able to oppose specific and active biological processes.…”

“…This highlighted how the physical properties of the cell membrane are able to oppose specific and active biological processes. However, the energy required to impact on specific processes-e.g., pinocytosis versus clathrin-coated pit formation-may differ as it was also demonstrated that the energy required to inhibit clathrin-mediated endocytosis is about 100 times higher than that required to stop pinocytosis [12]. So the same conceptual physics is used but the energy differs.…”

Membranes, molecules and biophysics: enhancing monocyte derived dendritic cell (MDDC) immunogenicity for improved anti-cancer therapy

“…Let us assume that the lipid asymmetry is annihilated, endocytosis will stop. Now, if we reverse the lipid asymmetry -i.e., compact more the outer leaflet via the addition of exogenous lipid species compared to the inner leaflet; endocytosis will stop and exocytosis should be promoted [79][80][81]. In cells, not all receptors are displayed on the membrane, some stay within cells in endosomes or vesicles some may even recycle constantly between the membrane and the intracellular vesicles.…”