The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux

Sedgwick, Alanna; Balmert, M. Olivia; D’Souza‐Schorey, Crislyn

doi:10.1016/j.yexcr.2018.03.001

“…These GPMVs fill a unique niche, maintaining the approximate lipid and protein content of living membranes while offering the experimental tractability of synthetic models (3). This combination has allowed investigation of PM composition (4), physical properties (5-7), dynamics (8,9), and potentially signaling and transport (8,(10)(11)(12)(13) in isolation from the complexity of living cells.…”

Section: Introductionmentioning

confidence: 99%

Cell-Derived Plasma Membrane Vesicles Are Permeable to Hydrophilic Macromolecules

Skinkle

¹

,

Levental

²

,

Levental

³

2020

Biophysical Journal

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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.

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“…These GPMVs fill a unique niche, maintaining the approximate lipid and protein content of living membranes while offering the experimental tractability of synthetic models (3). This combination has allowed investigation of PM composition (4), physical properties (5)(6)(7), dynamics (8,9), and potentially signaling and transport (8,(10)(11)(12)(13) in isolation from the complexity of living cells.…”

Section: Introductionmentioning

confidence: 99%

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Skinkle

¹

,

Levental

²

2019

Preprint

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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.

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“…Previous studies on NPC1 mutant cells proposed that cyclodextrin induces the secretion of cholesterol in vitro (Chen et al, 2010;Feltes et al, 2020;Ilnytska et al, 2021;Juhl et al, 2021;Sedgwick et al, 2018;Vacca et al, 2019) probably by distinct modes depending on cell type and experimental conditions. Our observations on primary cultures suggested that CD .…”

Section: Discussionmentioning

confidence: 97%

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neuronsin vivo

Barthélémy

¹

,

Demais²,

Stancu

³

et al. 2021

Preprint

0

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Niemann-Pick type C (NPC) disease is a rare and fatal lysosomal storage disorder presenting severe neurovisceral symptoms. Disease-causing mutations in genes encoding either NPC1 or NPC2 protein provoke accumulation of cholesterol and other lipids in specific structures of the endosomal-lysosomal system and degeneration of specific cells, notably neurons in the central nervous system (CNS). 2-hydroxypropyl-beta-cyclodextrin (CD) emerged as potential therapeutic approach based on animal studies and clinical data, but the mechanism of action on neurons has remained unclear. To address this topic in vivo, we took advantage of the retina as highly accessible part of the (CNS) and intravitreal injections as mode of drug administration. We find that CD enters the endosomal-lysosomal system of neurons and enables the release of lipid-laden lamellar inclusions, which are then removed from the extracellular space by specific types of glial cells. Thus, CD triggers a concerted action of neurons and glial cells to restore lipid homeostasis in the central nervous system.

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The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux

Cited by 12 publications

References 93 publications

Cell-Derived Plasma Membrane Vesicles Are Permeable to Hydrophilic Macromolecules

Cell-Derived Plasma Membrane Vesicles Are Permeable to Hydrophilic Macromolecules

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neuronsin vivo

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