Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

Abstract

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.

Figure 1

Partitioning of headgroup-labeled phosphoglycerolipids in phase separated GPMVs. GPMVs were labeled subsequent to isolation from unlabeled cells. Equatorial (A, C) or tangential (B) sections of GPMVs colabeled with fluorescent lipid analogues NBD-DPPE, NBD-DOPE or FITC-DPPE, together with Rh-DOPE were imaged at 20°C. (A) Headgroup-labeled NBD-DPPE and Rh-DOPE exhibit complementary partitioning in GPMVs, while, (B) headgroup-labeled NBD-DOPE partitions similarly to Rh-DOPE. (C) FITC-DPPE, unlike NBD-DPPE, preferentially partitions into the same phase as Rh-DOPE. (Scale bars, 5 μm.)

Figure 2

Partitioning of acyl chain-labeled lipid analogues in coexisting fluid phases in GPMVs. Equatorial (A–C) or tangential (D) sections of GPMVs, colabeled after formation, with Rh-DOPE and 12-NBD-SM, 12-NBD-PC, 12-NBD-Ceramide, or 6-NBD-SM and imaged at 20°C. (A–B) Acyl-chain labeled 12-NBD-SM and 12-NBD-PC exhibit differential partitioning in GPMVs: NBD-SM partitions equally between Lo-like and Ld-like phases (A), whereas, NBD-PC shows similar partitioning as Rh-DOPE (Ld-phase) (B). (C) Acyl chain labeled 12-NBD-Ceramide exhibits equal partitioning between Lo-like and Ld-like phases. (D) Acyl chain labeled 6-NBD-SM prefers the Lo-like phase and exhibits partitioning complementary to Rh-DOPE. (Scale bars, 5 μm.)

Figure 3

Partitioning of glycosphingolipids between coexisting fluid phases in GPMVs. GPMVs were generated from cells pre-labeled with Rh-DOPE and either A488-CTB (A), A488-AA4 (B) or bodipy-C5-GM1 (C). A488-CTB bound to GM1 (A), A488-AA4 bound to acetylated GD1b (B), and acyl chain-labeled Bodipy-C5-GM1 (C) prefer the Lo-like phase and shows partitioning complementary to Rh-DOPE in GPMVs. (Scale bars, 5 μm.)

Figure 4

Fluorescence images depicting equatorial confocal sections of GPMVs labeled with Rh-DOPE and the cholesterol analogues FITC-Cholesterol or 25-NBD-Cholesterol after isolation from cells. Both (A) headgroup labeled FITC-Cholesterol, and (B) alkyl chain labeled 25-NBD-Cholesterol, exhibit similar partitioning into Ld-like phase similar to Rh-DOPE in phase separated GPMVs. (Scale bars, 5 μm.)

Figure 5

Partitioning of GPI-anchored proteins between coexisting fluid phases in GPMVs. GPMVs were generated from cells transiently expressing YFP-GPI or mYFP-GL-GPI and subsequently labeled with Rh-DOPE. Confocal images of equatorial (A) or tangential sections (B) through GPMVs comparing the partitioning preference of GPI-anchored proteins with the Ld-marker Rh-DOPE. (A) YFP-GL-GPI is strongly enriched in the Lo-like phase in GPMVs while Rh-DOPE labels the Ld-like phase, and (B) mYFP-GL-GPI partitions preferentially into the Lo-like phase, complementary to the Rh-DOPE labeled Ld-like phase. (Scale bars, 5 μm.)

Figure 6

Partitioning of fluorescently labeled inner leaflet-associated proteins in phase-separated GPMVs. GPMVs were labeled either with A488-CTB or Rh-DOPE after isolation from cells transiently expressing PH-dsRed or flotillin-2-EGFP. (A) PH-dsRed shows partitioning complementary to Lo marker A488-CTB, and, (B) flotillin-2-EGFP cosegregates with the Ld marker Rh-DOPE. (Scale bars, 5 μm.)

Figure 7

Fluorescence images of tangential (A) or equatorial sections (B–C) through GPMVs comparing the partitioning of transmembrane proteins with the Ld-preferring lipid probe Rh-DOPE in the presence of coexisting fluid phases in GPMVs. GPMVs were isolated from Rh-DOPE-labeled RBL cells, transiently expressing either VSVG-EGFP, LAT-EGFP, or LAT-mEGFP. (A) VSVG-EGFP partitions strongly into the Ld-like phase colabeled with Rh-DOPE, whereas (B) LAT-EGFP shows equal partitioning between coexisting fluid phases, and (C) LAT-mEGFP preferentially partitions into the Ld-like phase colabeled with Rh-DOPE. (Scale bars, 5 μm.).

Figure 8

Distributions of antibody-labeled proteins and lipid probes associated with GPMVs, following lysis of GPMVs with 0.04% TritonX-100 and sucrose gradient fractionation. In this representative experiment, percentages of total fluorescence are shown for A488-CTB-GM1 (filled circles), Cy3-OX7-Thy1 (open triangles), 6-NBD-SM (filled squares), Rh-DOPE (open diamonds), and A488-IgE-FcεRI (filled triangles) in different fractions. Fractions 2–3 (10–20 % sucrose) include DRMs, whereas fractions 4–8 (~40% sucrose) contain solubilized proteins and lipids.