Raft domains of variable properties and compositions in plasma membrane vesicles

Abstract

Biological membranes are compartmentalized for functional diversity by a variety of specific protein-protein, protein-lipid, and lipid-lipid interactions. A subset of these are the preferential interactions between sterols, sphingolipids, and saturated aliphatic lipid tails responsible for liquid-liquid domain coexistence in eukaryotic membranes, which give rise to dynamic, nanoscopic assemblies whose coalescence is regulated by specific biochemical cues. Microscopic phase separation recently observed in isolated plasma membranes (giant plasma membrane vesicles and plasma membrane spheres) (i) confirms the capacity of compositionally complex membranes to phase separate, (ii) reflects the nanoscopic organization of live cell membranes, and (iii) provides a versatile platform for the investigation of the compositions and properties of the phases. Here, we show that the properties of coexisting phases in giant plasma membrane vesicles are dependent on isolation conditions--namely, the chemicals used to induce membrane blebbing. We observe strong correlations between the relative compositions and orders of the coexisting phases, and their resulting miscibility. Chemically unperturbed plasma membranes reflect these properties and validate the observations in chemically induced vesicles. Most importantly, we observe domains with a continuum of varying stabilities, orders, and compositions induced by relatively small differences in isolation conditions. These results show that, based on the principle of preferential association of raft lipids, domains of various properties can be produced in a membrane environment whose complexity is reflective of biological membranes.

Fig. 1.

GPMVs derived with a combination of PFA and DTT remain phase separated at higher temperatures than other sets of isolation conditions. (A and B) Representative images and quantification of GPMVs derived with either 2 mM NEM or a combination of 25 mM PFA and 2 mM DTT (curves are sigmoidal fits to data). Microscopically large round coalesced domains persist in PFA + DTT GPMVs to approximately 20 °C. In contrast, domains in NEM-derived vesicles are smaller, more dispersed, and not observable above 10 °C. (C) Miscibility transition temperature (T_misc, defined as the temperature at which 50% of vesicles are phase separated) in GPMVs as a function of isolation agent(s). Isolating GPMVs with 2 mM NEM alone, with a combination of 2 mM NEM and 25 mM PFA, or with PFA + NEM supplemented with either a nonsulfhydryl reducing agent (TCEP) or a palmitoylation inhibitor (2BP) does not recapitulate the PFA + DTT phenotype.

Fig. 2.

Postisolation treatment reproduces PFA/DTT phenotype only when both PFA and DTT are present. (A–F) Representative images (all taken at 10 °C) of GPMVs isolated with the conditions shown on the left of the images, then dialyzed to remove isolation chemicals, then treated with the chemicals indicated above the arrows. Phase separation at this temperature is observed only when PFA and DTT are present, either as the isolation condition or the postisolation treatment. (G) Percentage of phase-separated GPMVs as a function of temperature for different isolation and treatment conditions. Each point represents 50–100 vesicles; curves are sigmoidal fits to data. Results representative of three independent experiments.

Fig. 3.

Phase separation in PMS. (A) PMS isolated by cell swelling without additional chemicals show clear liquid–liquid phase separation below 5 °C (staining with 1 μg/mL FAST-DiO, disordered phase marker). (B) Treatment of PMS with PFA + DTT reproduces the PFA + DTT phenotype in GPMVs, whereas treatment with PFA or DTT alone does not have a significant effect on phase behavior.

Fig. 4.

[DTT]-dependent phase separation. (A) Phase separation could be induced at progressively higher temperatures by increasing [DTT] in the GPMV isolation buffer at constant [PFA] (25 mM). Each point is representative of 75–100 vesicles per condition; curves are sigmoidal fits to data. Results representative of three independent experiments. (B) T_misc increases from below 5 °C to above 15 °C between [DTT] = 0.2 and 2 mM. Data at 0 mM DTT includes vesicles produced with NEM alone, NEM + PFA, and PFA alone. (C) No variation in T_misc observed with varying [PFA] at constant [DTT] (2 mM) through the range that yields GPMVs. Points in B and C are average ± SD from three to seven independent trials.

Fig. 5.

Order difference between coexisting phases is dependent on [DTT]. (A) Exemplary GP images of coexisting domains in GPMVs (all imaged at 5 °C). GP is a relative indicator of the membrane order (higher GP equals more ordered membranes). (B and C) Although the inclusion of PFA did not have a major effect on order (comparing NEM versus 25 mM PFA + 0.2 mM DTT), increasing [DTT] reduced the overall order and increased the relative order difference between the coexisting phases. Average ± SD from 10 vesicles per condition and representative of three experiments.

Fig. 6.

Relative protein concentration in coexisting phases is dependent on [DTT]. (A) Relative protein concentration in coexisting phases of GPMVs can be quantified by fluorescent imaging of antibiotin Fab’ after nonspecific protein labeling with Sulfo-NHS-biotin. (B) K_p,raft (ratio of intensity in the raft versus nonraft phase) of extracellularly accessible proteins decreases with [DTT] from a high concentration of raft proteins in NEM GPMVs to barely observable raft protein signal in 2 mM DTT. Points are average ± SD of > 15vesicles per condition, representative of three independent experiments.

Fig. 7.

GPMV isolation with PFA and DTT leads to reduction of extracted PE. (A) TLC of lipids extracted from GPMVs as a function of isolation conditions. The PE band decreases in intensity relative to PC with increased [DTT]. (B) Average ± SD of background subtracted peak intensities of PE relative to PC. Results representative of three independent experiments. (C) TLC of lipids extracted from 80/20 PC/PE liposomes. PFA + DTT treatment does not reduce extractable PE unless either a TM peptide (3 mol % LW peptide) or a soluble protein (3 mol % γ-globulin) is included in the preparation.

Fig. 8.

Domains of continuously varying orders and compositions observable in PM vesicles isolated with PFA and varying [DTT].