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. 2013 Jul 8;202(1):35-44.
doi: 10.1083/jcb.201301039.

Direct imaging reveals stable, micrometer-scale lipid domains that segregate proteins in live cells

Alexandre Toulmay  1 , William A Prinz
Affiliations

Direct imaging reveals stable, micrometer-scale lipid domains that segregate proteins in live cells

Alexandre Toulmay et al. J Cell Biol. .

Abstract

It has been proposed that membrane rafts, which are sterol- and sphingolipid-enriched liquid-ordered (Lo) domains, segregate proteins in membranes and play critical roles in numerous processes in cells. However, rafts remain controversial because they are difficult to observe in cells without invasive methods and seem to be very small (nanoscale) and short lived, leading many to question whether they exist or are physiologically relevant. In this paper, we show that micrometer-scale, stable lipid domains formed in the yeast vacuole membrane in response to nutrient deprivation, changes in the pH of the growth medium, and other stresses. All vacuolar membrane proteins tested segregated to one of two domains. These domains formed quasi-symmetrical patterns strikingly similar to those found in liposomes containing coexisting Lo and liquid-disordered regions. Indeed, we found that one of these domains is probably sterol enriched and Lo. Domain formation was shown to be regulated by the pH-responsive Rim101 signaling pathway and may also require vesicular trafficking to vacuoles.

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Figures

Figure 1.
Figure 1.
Vacuole membrane proteins segregate into domains in Stat-phase. (A) Cells expressing Vph1-GFP, in Stat-phase, were visualized live at room temperature by fluorescence microscopy focusing on either the top or middle of the vacuole. Three types of microdomains are shown: partial, quasi-symmetrical, and coalesced. (B) Cells expressing Vph1-GFP were grown in SC medium. The percentage of cells with the indicated vacuolar domain patterns and the OD600nm (black line) were determined (n = 100–300 cells/time). (C) Cells expressing Vph1-GFP and Ivy1-mCherry, in Stat-phase, were visualized by fluorescent microscopy. Bars, 5 µm.
Figure 2.
Figure 2.
Role of sterols in vacuolar domain formation. (A) WT cells were grown in medium with FM4-64 to Stat-phase and visualized live by fluorescence microscopy. (B) Intact vacuoles from Stat-phase cells expressing Vph1-GFP were incubated with filipin for 30 min and imaged by fluorescence microscopy. Fluorescence intensities of filipin and GFP around the vacuole (arrows) were plotted. (C) 5 µM fenpropimorph or vehicle (DMSO) was added to growing cells expressing Vph1-GFP, and the cells were visualized after 16 h when they had reached Stat-phase. (D) Intact vacuoles from cells expressing Vph1-GFP were incubated with 10 mM MβCD for 30 min and imaged by fluorescence microscopy. Stat, Stat-phase; log, logarithmic growth phase. (E) Percentage of erg1Δ cells expressing Vph1-GFP that contained vacuoles with membrane domains when grown in YPD with ergosterol or cholesterol. The percentage of domains was calculated from 100–300 cells or vacuoles from two independent experiments. Error bars show means ± SD. Bars: (A, C, and D) 5 µm; (B) 1 µm.
Figure 3.
Figure 3.
Membrane ordering in the vacuolar domains. (A) Intact vacuoles from cells expressing Vph1-GFP were incubated with Fast DiI for 30 min and imaged by fluorescence microscopy. (B) Images of live cells expressing Vph1-GFP and Ivy1-mCherry (shown in Fig. 1 C). (C) Images of isolated vacuoles from cells expressing Vph1-GFP and strained with filipin. In B and C, arrows indicate inward bending of sterol-enriched domains. (D) Generalized polarization (GP) of liposomes (left) and cellular membranes (right) incubated with Laurdan for 1 h at 30°C (means ± SD, n = 4–5 independent experiments). chol, cholesterol; DOPC, dioleoylphosphatidylcholine; SM, sphingomyelin; vac, vacuole. Where indicated, a two-tailed t test was used to calculate p-value. Bars: (A) 2 µm; (B and C) 1 µm.
Figure 4.
Figure 4.
Stresses that induce domain formation in vacuolar membranes. (A) Growing cultures of cells expressing Vph1-GFP were washed and resuspended in SC, H2O, SC lacking nitrogen (SD-N), or SC lacking glucose (SC-glc), and the indicated compounds were added (Rapa, rapamycin; 2DG, 2-deoxyglucose; CHX, cycloheximide). The percentage of cells with vacuolar domains was measured after 3 h. (B–D) The cultures in A were grown to Stat-phase (18 h), and the percentage of cells with vacuolar domains (B), the final OD600nm (C), and pH (D) of the cultures were determined (n = 100–300 cells from at least two independent experiments; means ± SD). (E) pH-dependent dissipation of vacuolar domains; cells expressing Vph1-GFP were grown to Stat-phase, and the percentage of cells with vacuolar domains was determined. A portion of the cultures was pelleted, and the medium was replaced with 100 mM Tris, pH 9.3. After 75 min of incubation at 30°C, the half of the culture in Tris was pelleted and resuspended in used SC medium. Means ± SD; two independent experiments.
Figure 5.
Figure 5.
Mutants that affect vacuolar domain formation or dissipation. (A) The Rim101 pathway. (B) WT or rim21Δ cells expressing Vph1-GFP were grown to Stat-phase, and the percentage of cells with vacuolar domains was determined. Portions of the cultures were pelleted, and the media were replaced with 100 mM Tris, pH 9.3. (C) The indicated strains were grown as in B, and the percentage of cells with vacuolar domains was determined. (D) Percentages of vacuolar domain types in cells expressing Vph1-GFP and with the indicated genotypes were determined in Stat-phase. (E) Midlogarithmic growth–phase cultures were untreated (SC) or treated with 10 µg/ml CHX or 60 mM NaOAc, pH 5.2, for 3 h, and the percentages of cells with vacuolar domains were determined. (F) WT or sec18-1 cells were grown at 24°C to midlogarithmic growth phase, and a portion of the cultures was shifted to 37°C for 3 h. The percentage of cells with vacuolar domains was determined. Graphs show means of 100–300 cells ± SD from two independent experiments. CHX, cycloheximide; MVB, multivesicular body.
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