Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells

Abstract

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Fig. 1.

Chemical structures. A: Photoactivable sphingosine derivate, Sph-N₃. B: Photoactivable sphingomyelin, SM-N₃. C: Reduced form of SM-N₃, SM-NH₂.

Fig. 2.

Experimental model scheme. Sph-N₃ and [methyl-³H]choline were administered to human fibroblasts in culture with the aim of providing cells with photoactivable and radioactive precursors able to enter the sphingolipid metabolic pathways, allowing the production of radioactive and photoactivable sphingomyelin able to interact with neighboring proteins and to form, after cell illumination, stable radioactive SM-protein complexes.

Fig. 3.

Effect of normal and photoactivable sphingosine on proliferation and viability of cultured fibroblasts. Six hours after seeding, NPA and control fibroblasts were treated with normal (gray dotted line) or photoactivable (black dotted line) sphingosine solubilized in cell culture medium, both at the final concentration of 38 nM for up to 72 h. The proliferation and viability were compared with untreated cells (gray line). After 24, 48, and 72 h, the number of total cells (A1, B1) and of dead cells (A2 and B2, left) was evaluated by Trypan blue exclusion assay as described in Materials and Methods. Data are the means ± SD of three different experiments.

Fig. 4.

Radioactive lipids analysis of normal (lanes 1, 2, 3, 4) or NPA (lanes 5, 6, 7, 8) cells treated (lanes 1, 3, 5, 7) or not (lanes 2, 4, 6, 8) with Sph-N₃ and fed with [3-³H(Sph)]GM1(lanes 1, 2, 5, 6) or [3-³H(Sph)]SM (lanes 3, 4, 7, 8). One thousand dpm of the total lipids extracted from normal and NPA fibroblasts fed with Sph-N₃ followed by radioactive GM1 or SM administration were separated by HPTLC using CHCl₃/CH₃OH/(CH₃)₂CHOH/0.2% aqueous CaCl₂, 20:60:20:4 by vol. as solvent system. Radioactive lipids were detected by digital autoradiography performed with a Biospace β-imager instrument for 48 h.

Fig. 5.

Radioactive lipids analysis. TLC separation of the total lipids extracted from normal (lane A) and NPA (lane B) fibroblasts fed with Sph-N₃ and [methyl-³H]choline. One thousand dpm were applied on a 4 mm line for each sample. TLC was run in CHCl₃/CH₃OH/0.2% aqueous CaCl₂, 50:40:8 by vol. Digital autoradiography was performed with a Biospace β-imager instrument for 48 h.

Fig. 6.

Gradient fractions analysis. Gradient fractions distribution (abscissa axis) of total radioactivity (A1, B1), radioactive sphingomyelin (A2, B2), and cell proteins (A3, B3) in normal and NPA cells. Data are the means ± SD of three different experiments.

Fig. 7.

Radioactive lipids distributions within gradient fractions. TLC separation of radioactive total lipid extracts obtained from sucrose gradient fractions 4, 5, 6, 10, and 11 prepared from normal (A) and NPA (B) fibroblasts. One thousand dpm were applied on a 4 mm line for each sample. TLC was run in CHCl₃/CH₃OH/0.2% aqueous CaCl₂, 50:40:8 by vol. Digital autoradiography was performed with a Biospace β-imager instrument for 48 h.

Fig. 8.

[³H]SM-N₃ analysis. TLC separation of radioactive total lipid extracts obtained from DRM fractions prepared from normal (A) and NPA (B) fibroblasts. A total of 1000 dpm of control lipid extracts (lane 2) and 1000 dpm of lipid extracts previously subjected to chemical reduction with dithiothreitol (lane 1) were applied on a 4 mm line. TLC was performed with two sequential runs in CHCl₃/CH₃OH/H₂O, 50:40:8 by vol. The radioactivity was detected by digital autoradiography using a Biospace β-imager instrument for 48 h.

Fig. 9.

Proteins cross-linked with [³H]SM-N₃ from cell homogenates (lane 1), HD fraction (lane 2) and DRM fraction (lane 3) prepared from normal (A) and NPA (B) fibroblasts were separated by 10% SDS-PAGE, blotted on a polyvinyldifluoride (PVDF) membrane, and visualized by digital autoradiography for 120 h. Proteins recovered in the immunoprecipitation experiments performed in domain preserving conditions starting from aliquots of DRM fractions prepared from normal (A) and NPA (B) fibroblasts were separated by 10% SDS-PAGE. The proteins were directly detected by silver staining (lane 4) or blotted on a PVDF membrane and then visualized by Western blot using HRP-streptavidin (lane 5) or by digital autoradiography for 120 h (lane 6). Data are the means of three different experiments.

Fig. 10.

Topology of SM-protein complexes belong to detergent resistant membrane (DRM) of normal cells. The IP sample obtained in domain preserving conditions from DRM fraction of L40 fibroblasts was reimmunoprecipitated in domain disrupting conditions. Proteins recovered in the IP100 (lane 2) and in supernatant remaining after immunoprecipitation 100 (lane 1) were separated by 10% SDS-PAGE and blotted on a PVDF membrane, which was stained with HRP-streptavidin (A) and then visualized by digital autoradiography to detect the radioactive SM-protein complexes (B). Data are the means of three different experiments.