Figure 5.

C6-SM prevents the formation of liquid-ordered domains in GUVs and reduces order in the Golgi membranes. (A) GUVs made of DOPC (dioleoylphosphatidylcholine):C16-SM:Chol (2:1:1) (top panels), and DOPC:C6-SM:Chol (2:1:1) (bottom panels) were heated to 37°C, and visualized by fluorescence microscopy using the dyes naphthopyrene (green) that preferentially stains liquid-ordered membrane regions, and Lissamine-Rh-PE (red) that preferentially stains liquid-disordered regions. Scale bar is 10 μm. (B) Generalized polarization (GP) measured in DOPC, C16-SM, or C6-SM containing LUVs in a 1:1 molar ratio to cholesterol at 20 or 37°C. Average values±s.d. (N=3) are represented. (C) HeLa-MannII-RFP cells were treated with 20 μM L- or D-ceramide-C6 for 30 min and then washed and incubated in laurdan-containing FCS-free medium for 90 min before fixation. Cells were observed by two-photon fluorescence microscopy and the laurdan GP was measured and plotted in the whole cell (left) and also in the MannII-RFP-positive area (right). Colour code represents the measured GP value. (D) Laurdan GP from different cells in (C) was measured. Average values±s.d. (N=17) are represented.