Figure 1.
GLTP readily inserts GM1 into the extracellular leaflet of HepG2 cells.
(A) HepG2 cells were incubated at 37°C for 10 min in a 20 min pre-incubated Ringer solution containing 0.2 mg/ml GM1-DOPC (5∶95 mol%)-liposomes with 0, 5, or 25 µM GLTP. Then cells were washed, stained in the cold with fluorescently labeled CTX, fixed and imaged. Pictures were recorded with a confocal laser scanning microscope in the differential interference contrast (DIC) and the fluorescence channel. Images in the lower panels are shown at the same scaling leading to the impression that in control cells staining is missing. However, also in control cells staining was present in the periphery (see Fig. S2A). (B) CTX staining intensity at the cell periphery was quantified by linescan analysis (three pixel width). Values are given as means ± SEM (n = 3 - 4 independent experiments; 19 - 35 cells were analyzed for each experiment).
Figure 2.
Insertion into the intra- and the extracellular plasmalemmal leaflet.
(A) For GM1 loading HepG2 cells were treated as in Fig. 1. Then membrane sheets were generated and CTX staining was performed at RT. Samples were fixed and basal plasma membranes were imaged in the TMA-DPH (general membrane staining) and CTX channel. (B) Quantitation of the mean CTX fluorescence intensities reflecting the GM1 increase in the extracellular leaflet. Values are given as means ± SEM (n = 3 independent experiments; 18 - 50 membrane sheets analyzed for each experiment). (C and D) For incorporation of GM1 into both membrane leaflets, membrane sheets were generated prior to the incubation with GLTP/liposomes, allowing biochemical access to the inner plasmalemmal leaflet. After GM1 insertion, GM1 staining, imaging and quantification of fluorescence was performed as in A and B. In this experiment fluorescence arises from both leaflets; fluorescence from the inner plasmalemmal leaflet has a more uniform appearance (Fig. 3). Values are given as means ± SEM (n = 4 - 5 independent experiments; 36 - 53 membrane sheets analyzed for each experiment).
Figure 3.
GM1 in the inner leaflet is more uniformly distributed.
The degree of clustering of the CTX staining was analyzed on membrane sheets from experiments shown in Fig. 2 by calculating the relative SD (SD/mean). For each membrane sheet, SD/mean was plotted against the mean fluorescence and a function (y = y0+ a/x) was fitted (with values for “outer leaflet” y0 = 0.2437; a = 62.0138 and for “both leaflets” y0 = 0.0454; a = 34.49). The higher the value of the relative mean, at a comparable GM1 concentration, the more clustered the signal [39]. Loading of the outer leaflet (black dots) results in overall higher values than loading of both leaflets (red dots). Arrows point to the values obtained for the membranes shown for illustration. For each condition, membrane sheets from all independent experiments were pooled (128 and 140 membrane sheets for loading of the extracellular and both leaflets, respectively).
Figure 4.
GM1 extraction from plasma membrane sheets.
(A) Membrane sheets were generated from HepG2 cells, incubated with GLTP at variable concentrations for 10 min (not shown) or 30 min at 37°C, washed, stained at RT with fluorescent CTX, fixed and imaged in the TMA-DPH and the CTX channel. (B) Quantitative analysis of mean CTX staining intensities, normalized to control values. GLTP decreases the GM1 staining in a time and concentration dependent manner though even under the harshest condition GM1 was reduced to only 45%. Values are given as means ± SEM (n = 4 - 5 independent experiments; 8 - 81 membrane sheets analyzed for each experiment).
Figure 5.
(A) HepG2 cell membrane sheets were loaded at 37°C for 10 min with 10 µM GLTP/liposomes (in the control GLTP was omitted) followed by a second incubation at 37°C for 30 min with 0, 50 or 100 µM GLTP. (B) Quantification of fluorescence in the CTX channel. Values are given as means ± SEM (n = 3 independent experiments; 36 - 68 cells were analyzed for each experiment).
Figure 6.
Extraction of GM1 from Jurkat cell membrane sheets.
Jurkat cell membrane sheets were incubated at 37°C for 30 min with variable concentrations of GLTP and fluorescence in the CTX channel was quantified. The bar chart includes also data shown in Fig. 8. Values are given as means ± SEM (n = 3 - 5 independent experiments; 142 - 316 cells were analyzed for each experiment).
Figure 7.
Micromolar concentrations of GLTP balance GM1 concentrations between Jurkat cell membrane sheets.
Jurkat cell membrane sheets were incubated at 37°C for 30 min with 0, 1, 5 or 25 µM GLTP, followed by staining with CTX and imaging. For each condition images from the TMA-DPH and the CTX channel are shown. CTX fluorescence intensity values from individual membrane sheets were normalized to the average intensity of the control and values were plotted as a histogram. At 1 and 5 µM a shift of the distribution peak to higher fluorescence values is observed. Fig. 6 shows the average intensity remaining after GLTP treatment. Values are given as means ± SEM (n = 3 - 5 independent experiments; 142 - 316 membrane sheets analyzed for each experiment).
Figure 8.
Transfer of Jurkat cell GM1 to membrane sheets from HepG2 cells.
GLTP solutions from Jurkat cell membrane treatments (Fig. 7; 30 min at 37°C) were collected and added to HepG2 membrane sheets. After 30 min at 37°C GM1 was visualized by CTX staining and imaged. The bar chart represents quantified CTX fluorescence of Jurkat cell membranes (from the experiment shown in Fig. 7) and HepG2 cell membranes. Jurkat cell membranes and HepG2 membranes were recorded with 100 ms and 1 s, respectively. To allow a direct comparison of the GM1 levels, the presented HepG2 values were divided by a factor of 10 assuming that the recorded intensity is linear to the exposure time. In controls, Jurkat cells had a 74-fold higher GM1 concentration than HepG2 cells. Values are given as means ± SEM (n = 3 independent experiments; 113 - 316 membrane sheets analyzed for each experiment).