Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles

Abstract

Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured cells, were observed to reversibly phase segregate at temperatures significantly below 37 degrees C. In this contribution, we compare the temperature dependence of fluid phase segregation in HeLa and rat basophilic leukemia (RBL) cells. We find an essentially monotonic temperature dependence of the number of phase-separated vesicles in both cell types. We also observe a strikingly broad distribution of phase transition temperatures in both cell types. The binding of peripheral proteins, such as cholera toxin subunit B (CTB), as well as Annexin V, is observed to modulate phase transition temperatures, indicating that peripheral protein binding may be a regulator for lateral heterogeneity in vivo. The partitioning of numerous signal protein anchors and full length proteins is investigated. We find Lo phase partitioning for several proteins assumed in the literature to be membrane raft associated, but observe deviations from this expectation for other proteins, including caveolin-1.

Figure 1

Temperature dependence of phase coexistence in HeLa and RBL cell bleb membranes. (a) HeLa cells were labeled either with the lipid fluorophore TR, or with both TR and either CTB or annexin V. In all cases, the fraction of phase-separated membrane vesicles is observed to depend essentially monotonically on temperature, with a broad spread of transition temperatures. Both the addition of CTB and Annexin V raise transition temperatures relative to those GPMVs that were labeled with TR only. The effect of Annexin V on phase behavior appears to be slightly larger at low temperatures compared to CTB, although the error bars overlap. (b) RBL cell blebs were labeled with TR. Similar temperature-dependent phase behavior is found compared to HeLa cell blebs labeled with TR.

Figure 2

Variable partitioning of wild type Lck anchor eGFP construct. (a)–(c): Representative images of vesicles displaying preferential Ld phase partitioning (a), non-preferential partitioning (b), and preferentially ordered phase partitioning (c) wt Lck anchors (green channel, left) in HeLa cell giant plasma membrane vesicles labeled with the lipid dye TR. Scale bars: 2 µm. (d) Histogram of fluorescence intensity ratios of Lo phase versus Ld phase fluorescence, comparting protein and lipid dye. Fluorescence intensity ratios are shown as base-10 logarithmic values. The broad histogram reflects the variable partitioning behavior shown in the fluorescence images.

Figure 3

Partitioning of wt Fyn anchor eGFP construct. (a) Representative fluorescence images comparing protein (green, left) and lipid dye (red, right) fluorescence in a phase separated HeLa cell GPMV. Scale bars, 2 µm. (b) Fluorescence intensity ratio distribution for protein and lipid dye demonstrates primarily disordered phase partitioning of the wt Fyn protein anchor. Scale bars, 2 µm.

Figure 4

Partitioning of GPI anchor eGFP construct. (a) Representative fluorescence images comparing protein (green, left) and lipid dye (red, right) fluorescence in a phase separated HeLa cell GPMV. Scale bars, 2 µm. (b) Fluorescence intensity ratio distribution for protein and lipid dye demonstrates primarily ordered phase partitioning of the GPI anchor.