Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

Abstract

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.

Membrane raft association is a determinant of plasma membrane localization

“…GPMVs were isolated from rat basophilic leukemia (RBL) cells as described previously (4,5). Constructs based on the trLAT backbone (30) were transfected into RBLs using Nucleofection (Lonza).…”

“…Thus, we produced 13 mutants (Table SI) of a model single-pass TM protein [the TMD of linker for activation of T-cells (LAT) fused to a C-terminal monomeric RFP (mRFP), LAT-TMD-mRFP, hereafter "trLAT" (30)] with various TMD lengths in an attempt to generate variants with varying raft affinities. Raft association was assayed by quantifying the relative partitioning of these constructs between coexisting raft/nonraft phases in GPMVs (4,5). Fig.…”

“…membrane domain | endocytosis | trafficking | phase separation | microdomains R ecent advances in superresolution microscopy (1), lipid analysis (2,3), and plasma membrane (PM) isolation (4,5) have confirmed the coexistence of lipid-driven, fluid domains in biological membranes. The relatively ordered domains, known as "membrane rafts," have been proposed to be involved in protein sorting (6), viral/pathogen trafficking (3,7), and PM signaling in a variety of contexts (8).…”

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

“…GPMVs were prepared as described previously ( 31 ). Briefl y, RBL-2H3 cells seeded out on a 60 mm petri dish ( ‫ف‬ 70% confl uent) were washed with GPMV buffer [150 mM NaCl, 10 mM HEPES, 2 mM CaCl 2 (pH 7.4)] twice.…”

Rituximab capping triggers intracellular reorganization of B cells

“…1F). To confirm the role of actin in rituximab-induced polarisation, we moved to giant plasma membrane vesicles (GPMVs), which are derived from living cells through vesiculation agents [6]. While GPMVs have similar membrane composition as their parent cells, they differ in lacking an intact actin cytoskeleton and cellular organelles.…”