Supplementary Figures to the Manuscript by Aresta-Branco et al. “Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, sphingolipid-enriched lipid rafts.”
Figure S1- Identification of a long lifetime component in the fluorescence intensity decay of t-PnA due to the presence of gel domains. A) Global analysis of fluorescence intensity decay of wt (BY4741) cells in mid-exponential phase at 24ºC labeled with t-PnA (blue) or unlabeled (red). B) Individual analysis of fluorescence intensity decays of t-PnA at 24ºC incorporated into MLVs of POPC (blue) and POPC/phytoceramide with molar ratios of 90:10 (green) and 70:30 (grey). Top panels: experimental decays and best fitting function with a sum of exponentials (white lines). Middle panels: random distribution of weighted residuals of the fitting. Bottom panels: autocorrelation of the residuals.
Figure S2 – t-PnA incorporates stably into gel domains of the plasma membrane of S. cerevisiae wt cells. The cells were labeled with t-PnA as described under Experimental Procedures, except that different incubation times were used, as indicated. The fluorescence intensity decay was measured, and the mean fluorescence lifetime (A) and long component lifetime (C) obtained thereof are shown. The values are the mean ± standard deviation of at least four independent experiments. The kinetics of incorporation was studied by following the time evolution (1 s resolution) of the steady-state fluorescence intensity of the cell suspension labeled with t-PnA immediately after probe addition (B).
The mean fluorescence lifetime of t-PnA is practically identical between 2 and 15 min of probe incorporation. There is, at the most, a slight decrease between 2 and 5 min, during which the labeling is still proceeding, as can be seen from the incorporation kinetics showed in panel B. After 2 min of incubation with the probe, the gel phase (long component of 41 ns) is already detected (panel C), and it does not change with longer incorporation times. Since the incorporation into gel domains is slightly slower than into fluid domains, due to their rigidity, but the disincorporation rate is much slower, due to the partition preference of t-PnA for gel domains, the fact that the long component is detected at short times means that the gel domains should be localized at the plasma membrane. After 5 min, the total fluorescence intensity practically stabilizes (panel B), and there are no alterations in the fluorescence intensity decay (panels A and C), which means that even if the probe is still incorporating, the distribution between ordered and disordered domains, and between the plasma and intracellular membranes, is maintained.
To further confirm that the cellular distribution of t-PnA after 5 min incorporation is stable and quantitative comparison between experiments is possible, the cells were washed once, twice, or not washed after probe addition. The washing does not change the results (not shown). This means that a fast equilibrium between the several cellular membranes and domains is established, even before the final equilibrium with the aqueous phase has been reached. This allows quantitative comparison of the fluorescence decay parameters between different experiments.
Figure S3 – The plasma membrane S. cerevisiae cells in mid-exponential phase contains gel domains at both 24ºC and 30ºC. The long component lifetime (A) and normalized amplitude (B), and the mean fluorescence lifetime (C) of t-PnA were obtained from the fluorescence intensity decay of the probe as described in Experimental Procedures for wt (BY4147) cells in mid-exponential phase at 24ºC (black) or 30ºC (white). The values are the mean ± standard deviation of at least four independent experiments. * P < 0.001 vs 24ºC
Figure S4 – Both phytoceramide, and ergosterol increase the membrane order when mixed with POPC. Steady-state fluorescence anisotropy of DPH incorporated into MLVs composed of POPC/phytoceramide (grey) and POPC/ergosterol (black) mixtures at 24ºC. X stands for mole fraction. The data is the average of 3 independent experiments. The standard deviation is enclosed in the data points or shown by the error bars. The lines are merely to guide the eye.
Figure S5 – The highly ordered domains in S. cerevisiae cells that accumulate zymosterol, a non-raft promoting sterol, are gel domains. The steady-state fluorescence anisotropy of t-PnA (black) versus DPH (white) was obtained as described in Experimental Procedures for wt (RH3435) and erg2Derg6D cells in mid-exponential phase. The values are the mean ± standard deviation of at least four independent experiments, except for DPH in erg2Derg6D (two independent experiments). *** P<0.05 vs wt cells
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