Mechanics of Curved Plasma Membrane Vesicles: Resting Shapes, Membrane Curvature, and In-Plane Shear Elasticity

Highly curved cell membrane structures, such as plasmalemmal vesicles (caveolae) and clathrin-coated pits, facilitate many cell functions, including the clustering of membrane receptors and transport of specific extracellular macromolecules by endothelial cells. These structures are subject to large mechanical deformations when the plasma membrane is stretched and subject to a change of its curvature. To enhance our understanding of plasmalemmal vesicles we need to improve the understanding of the mechanics in regions of high membrane curvatures. We examine here, theoretically, the shapes of plasmalemmal vesicles assuming that they consist of three membrane domains: an inner domain with high curvature, an outer domain with moderate curvature, and an outermost flat domain, all in the unstressed state. We assume the membrane properties are the same in these domains with membrane bending elasticity as well as in-plane shear elasticity. Special emphasis is placed on the effects of membrane curvature and in-plane shear elasticity on the mechanics of vesicle during unfolding by application of membrane tension. The vesicle shapes were computed by minimization of bending and in-plane shear strain energy. Mechanically stable vesicles were identified with characteristic membrane necks. Upon stretch of the membrane, the vesicle necks disappeared relatively abruptly leading to membrane shapes that consist of curved indentations. While the resting shape of vesicles is predominantly affected by the membrane spontaneous curvatures, the membrane shear elasticity (for a range of values recorded in the red cell membrane) makes a significant contribution as the vesicle is subject to stretch and unfolding. The membrane tension required to unfold the vesicle is sensitive with respect to its shape, especially as the vesicle becomes fully unfolded and approaches a relative flat shape.

Role of Plasmalemmal Vesicles on the HDL-Mediated Cholesterol Efflux in Aortic Endothelial Cells

It is well-known that high-density lipoprotein (HDL) mediates the transport of cholesterol from peripheral tissues to liver through “reverse cholesterol transport” for metabolism. However there is considerable debate about the mechanisms by which HDL removes excess cholesterol from cells. Two different pathways have been suggested: (i) a docking receptor promots cholesterol translocation, or (ii) a receptor mediates intracellular endosomal pathway termed “retroendocytosis“. Our previous studies have indicated that the removal of cholesterol from aortic endothelial and smooth muscle cells in the presence of HDL is facilitated by plasmalemmal vesicles. in this study, our emphasis is on the ultrastructural changes of cholesterol-loaded endothelial cells after incubated with HDL at different time intervals. Furthermore, the mechanism of HDL-mediated cholesterol efflux by cultured aortic endothelial cells is investigated by immunoelectrophoresis.Confluent monolayers of endothelial cells were incubated in cholesterol DMEM medium for 48 hr.

Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae).

Plasmalemmal vesicles (PVs) or caveolae are plasma membrane invaginations and associated vesicles of regular size and shape found in most mammalian cell types. They are particularly numerous in the continuous endothelium of certain microvascular beds (e.g., heart, lung, and muscles) in which they have been identified as transcytotic vesicular carriers. Their chemistry and function have been extensively studied in the last years by various means, including several attempts to isolate them by cell fractionation from different cell types. The methods so far used rely on nonspecific physical parameters of the caveolae and their membrane (e.g., size-specific gravity and solubility in detergents) which do not rule out contamination from other membrane sources, especially the plasmalemma proper. We report here a different method for the isolation of PVs from plasmalemmal fragments obtained by a silica-coating procedure from the rat lung vasculature. The method includes sonication and flotation of a mixed vesicle fraction, as the first step, followed by specific immunoisolation of PVs on anticaveolin-coated magnetic microspheres, as the second step. The mixed vesicle fraction, is thereby resolved into a bound subfraction (B), which consists primarily of PVs or caveolae, and a nonbound subfraction (NB) enriched in vesicles derived from the plasmalemma proper. The results so far obtained indicate that some specific endothelial membrane proteins (e.g., thrombomodulin, functional thrombin receptor) are distributed about evenly between the B and NB subfractions, whereas others are restricted to the NB subfraction (e.g., angiotensin converting enzyme, podocalyxin). Glycoproteins distribute unevenly between the two subfractions and antigens involved in signal transduction [e.g., annexin II, protein kinase C alpha, the G alpha subunits of heterotrimeric G proteins (alpha s, alpha q, alpha i2, alpha i3), small GTP-binding proteins, endothelial nitric oxide synthase, and nonreceptor protein kinase c-src] are concentrated in the NB (plasmalemma proper-enriched) subfraction rather than in the caveolae of the B subfraction. Additional work should show whether discrepancies between our findings and those already recorded in the literature represent inadequate fractionation techniques or are accounted for by chemical differentiation of caveolae from one cell type to another.