Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Abstract

Giant Plasma Membrane Vesicles (GPMVs) are a widely used model system for biochemical and biophysical analysis of the isolated mammalian plasma membrane (PM). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the plasma membrane while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes, and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed; i.e. that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic giant unilamellar vesicles (GUVs). We attribute this leakiness to relatively large and heterogeneous pores formed by rupture of vesicles from cells. These pores are stable and persist throughout experimentally relevant time scales. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration are of critical importance for interpreting experiments utilizing GPMVs as plasma membrane models, particularly for drug permeation and membrane asymmetry. STATEMENT OF SIGNIFICANCE:A critical assumption in using Giant Plasma Membrane Vesicles to study membrane penetration and interactions is that these vesicles maintain the permeability barrier of the native membrane from which they form. Using large fluorescently-labeled hydrophilic probes, we demonstrate that this assumption is often incorrect and conclude that macromolecular solutes permeate GPMVs through stable pores formed during shear-induced rupture of vesicles from cells. Using these insights into the mechanisms of poration, we demonstrate an approach to isolate sealed GPMVs.

CHO/LY-B cell growth under limiting sphingolipid supply: correlation between lipid composition and biophysical properties of sphingolipid-restricted cell membranes+

“…S12B, C). Both Cer and HexCer are known to increase membrane lipid order [72][73][74] GPMV have been shown to be permeable to hydrophilic macromolecules 71 , and this could again be related to the increased Cer, and partly HexCer, since these SL happen to increase membrane permeability 72,73 . The observed increases in GP and breakthrough forces could be secondary to the use of dithiothreitol in GPMV formation.…”

“…GPMV constitute a frequently used PM preparation 37,40,41,71 . However, the lipidomic data show that their lipid composition departs from those of the whole cells and from other PM preparations (patches).…”

The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

“…This indicates that additional proteolytic processing of the protein evidently occurs, although we did not pursue the identity(ies) of the putative additional proteases. In light of recent reports that GPMVs contain pores that are permeable to some hydrophilic solutes (135), further studies will be required to determine whether these soluble cleavage products are quantitatively retained in the GPMV lumen. It is also currently unclear if proteolytic processing of C99-mEGFP only occurs in unperturbed cells or continues as GPMVs are being prepared and imaged.…”

Topical review: Shedding light on molecular and cellular consequences of NCX1 palmitoylation

“…Indeed, it is likely that numerous mechanisms, including cholesterol binding motifs, transmembrane domain physical properties and tertiary structure, and the presence of lipid modifications control microdomain affinity [29][30][31][32]. Investigations in Giant Plasma Membrane Vesicle (GPMV); a model system that is cell-detached structures (Figure 2A) [33][34][35][36], have elucidated an important role for palmitoylation to enhance the affinity of proteins for the raft (ordered) phase of isolated membranes which is rich in saturated phospholipids, cholesterol, glycolipids and sphingolipids [31,37]. For example, the transmembrane linker for activation of T cells (LAT) clustering in the lipid rafts is markedly dependent on its palmitoylation status at two juxtamembrane cysteines [31].…”