Journal: Molecular Pharmacology
Article Title: Detergent-Resistant Microdomains Determine the Localization of ?-1 Receptors to the Endoplasmic Reticulum-Mitochondria Junction S⃞
Figure Lengend Snippet: MAM accumulates ceramides. A, accumulation of NBD-Cer at subcellular membranes. CHO cells were incubated with NBD-ceramide (1.25 μM) at 4°C for 30 min. Confocal microscope (left). Arrowhead, ER cisternae; small arrow, mitochondria with a spaghetti-like shape; large arrow, juxta-mitochondria structures accumulating NBD-Cer; N, nucleus. Bar, 10 μm. HPTLC (right), where lipid extracts from NBD-Cer-labeled CHO cells were visualized under UV. 1, the lipid extract from CHO cells incubated with NBD-Cer at 4°C for 30 min; 2, the lipid extract from CHO cells incubated with NBD-Cer at 4°C for 30 min, followed by washings and incubation at 37°C for 30 min. Note the few conversions of NBD-Cer to NBD-GlcCer in lane 1. B, accumulation of NBD-Cer at intracellular domains apposing mitochondria. NBD-Cer (top) and mitochondria expressing Mito-DsRed (bottom) were visualized in the same cell. The two panels were superimposed in the inset (NBD-Cer in green, MitoDsRed in red). Bar, 2.5 μm. C, MAM-specific distribution of Sig-1R (top; in green inset). Shown at the bottom is mitochondria in the same sample. Bar, 2.5 μm. Inset, two panels superimposed. D, colocalization between Sig-1R-EYFP and fluorescent ceramides. CHO cells expressing Sig-1R-EYFP (top) were labeled with fluorescence ceramide BODIPY-Cer TR (1.25 μM; bottom) at 4°C for 30 min. Inset, two panels are superimposed (Sig-1R-EYFP in green, BODIPY-Cer TR in red). Bar, 2.5 μm. Inset, colocalization is represented by yellow. The same results were obtained from four independent samples ( > 5 fields/sample). E, MAM accumulates NBD-Cer. CHO cells labeled with NBD-Cer at 4°C for 30 min were subjected to membrane fractionations. The level of NBD-Cer in each fraction was measured by a fluorescence microplate reader. Fluorescence intensities were normalized to protein concentrations in each sample. F, retention of NBD-Cer at MAM. CHO cells were labeled with NBD-ceramides at 4°C for 30 min (left image). After washings, cells were incubated in normal medium at 37°C for 30 min (right image). Thin arrows, mitochondria; thick arrows, MAM; N, nucleus; G, Golgi apparatus; Bar, 10 μm.
Article Snippet: After separation from other lipids, the ceramides were extracted from an HPTLC plate (Merck) and spotted on a Hybond-C membrane (GE Healthcare).
Techniques: Incubation, Microscopy, High Performance Thin Layer Chromatography, Labeling, Expressing, Fluorescence