GLTP Mediated Non-Vesicular GM1 Transport between Native Membranes
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).