Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

Abstract

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below approximately 25 degrees C. At 37 degrees C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcepsilonRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.

Fig. 1.

Cell-attached GPMVs can laterally segregate into coexisting fluid phases. (A, C, and D) RBL cells treated with 4% (vol/vol) ethanol. Cells containing fluorescent membrane probes show formation of large GMPVs attached to the cell bodies by superposition of CM or two-photon microscopy image z-stacks. (A) Cell expressing geranylgeranyl-EGFP (GG-GFP) with attached GPMV measured by CM-imaged stack at ≈23°C. Note the absence of internal membranes within the GPMV and significant partitioning of GG-GFP into the GPMV compared with the cell body structures. (B) CM images of an NIH 3T3 GPMV incubated at room temperature with 1% (vol/vol) DMSO and R-DOPE, showing large-scale fluid/fluid phase coexistence similar to the RBL cells. (C and D) Two-photon microscopy images of a cell-attached GPMV colabeled with the membrane markers Nap (C) and R-DOPE (D), imaged at 5°C. Note their contrasting wavelength-selected labeling of the separated phases. Cell bodies show large fluorescence (attenuated here) due to internalized membrane probes. (Scale bars, 5 μm.)

Fig. 2.

Plasma membrane lipids and lipid fluorophores in GPMVs demonstrating characteristic phase preferences resembling the L_o/L_d partitioning behavior in model membranes. GPMVs, all prepared by formaldehyde/DTT treatment of RBL cells, were colabeled by Nap, R-DOPE, or DiIC16 and by A488-CTB bound to GM₁ or A568-annexin V bound to phosphatidylserine. Images are equatorial CM sections obtained at ≈23°C. (A) GPMVs colabeled with Nap and R-DOPE show contrasting partitioning. (B) GPMVs colabeled with CTB and R-DOPE show contrasting partitioning. (C) GPMVs colabeled with CTB and DiI C16:0 show contrasting partitioning. (D) CTB/annexin V-colabeled GPMV shows contrasting partitioning, indicating that annexin V labels an L_d-like phase.

Fig. 3.

GPMVs reveal membrane protein phase preferences in detergent-free GPMVs. Fluorescence images of equatorial confocal sections through GPMVs comparing the fluid phase partitioning of membrane-associated proteins (left column) to the lipid probes R-DOPE and CTB bound to GM₁ (right column). All GPMVs were prepared by formaldehyde/DTT treatment of RBL cells and imaged at ≈23°C. (A) GPI-anchored protein Thy-1 labeled with A488-anti-Thy1 mAb is preferentially in L_o phase, showing fluorescence in regions contrasting to R-DOPE partitioning in the L_d-like phase. (B) Lyn-GFP partitions preferentially into the L_d-like phase, in contrast with the CTB-labeled L_o-like phase. (C) PM-GFP partitions strongly into the L_d-like phase, in contrast to the CTB-enriched phase, in a cell-attached GPMV. (D) GG-GFP partitions preferentially into the L_d-like phase, complementary to CTB. (E) FcεRI labeled with A488-IgE partitions into the L_d-like phase colabeled with R-DOPE. (F) FITC-Con A labels glycoproteins and glycolipids that preferentially cosegregate with the L_d phase marker R-DOPE. (G) A significant fraction of Con A receptors populate GPMVs, compared with those in the attached cell. (Scale bar, 5 μm.)