Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis. at the Ministry of Higher Education and Research) and all mouse work was approved by the Veterinary Services of Paris (Authorization number: 75-1073). Because VAMP7 gene is located around the X chromosome only male embryos were utilized for cell culture. Cells were cultured in Dulbecco’s altered Eagle’s medium with 4.5g/L glucose and pyruvate (GIBCO Invitrogen) supplemented with 10% fetal bovine serum 2 L-Glutamine 10 models/penicillin and 10?μg/ml streptomycin in a 5% CO2-humidified atmosphere at 37°C and split at least 3?occasions to enrich the fibroblast populace. Experiments were performed before passage 6. Cells were transfected with either JetPrime (Polyplus transfection) or Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Immunofluorescence studies and antibodies For classical immunolocalization studies cells were fixed with PFA 4% at room temperature then washed 5?occasions in PBS and incubated with NH4Cl 50?mM in PBS for 20?min. Cell were then permeabilized with 0 1 Triton-PBS extensively washed and blocked for 30?min with Fish Gelatin 0 25 in PBS. Main antibodies were incubated overnight at 4°C in PBS-gelatin 0 125 Main Ab used included mouse anti-GM130 (Transduction Lab) anti-STX6 (Transduction Lab) anti-PrPc (SAF32 kind gift of Jacques Grassi CEA VCH-916 Fontenay-aux-Roses France) anti-mCherry (Clontech) rabbit anti-GolginA4 (kind gift of Mickey Marks University or college of Pennsylvania School of Medicine Philadelphia USA 57) and goat anti-mCherry (Acris). Secondary antibodies coupled to Alexa488 (Invitrogen) CY3 or CY5 (Jackson Laboratories) were incubated for 1?h at room temperature. Confocal microscopy Fixed cells were imaged with LSM 710 (Zeiss) confocal microscope using a 405?nm diode laser collection exciting DAPI a 488?nm argon laser collection exciting Alexa Fluor 488 and a 561?nm diode laser collection exciting TNFRSF10B CY3. Emission was detected between 410 and 480?nm for DAPI 495 for Alexa Fluor 488 and 565-600?nm for CY3. BFA treatment and live cell imaging WT and KO MEFs were cultured on 30?mm diameter coverglass and transfected with NAGT-GFP construct. Live cell imaging was conducted 16 to 24?h post-transfection in modified Krebs-Ringer-HEPES buffer (135?mM NaCl 2.5 KCl 1.2 MgCl2 1 CaCl2 20 HEPES 11.1 glucose pH VCH-916 7.4) supplemented with 1% FBS. Cells were imaged at 37°C using an inverted Leica DMI6000B microscope (Leica Microsystems Manheimm VCH-916 Germany) equipped with a 63X/1.4-0.6 NA Plan-Apochromat oil immersion Leica objective an EMCCD digital camera (Cascade:512B; Roper Scientific Trenton NJ) and controlled by Metamorph software (Roper Scientific). For BFA experiments VCH-916 cells were treated with 5?μM BFA for 20?min and then extensively washed by imaging buffer perfusion for 10?min. Acquisition started with BFA addition and was performed every 10?min for 2?h. For quantification NAGT-GFP VCH-916 transmission was segmented using “Wavelet spot detector” module of Icy imaging software (http://www.bioimageanalysis.org/) 58 and particle analysis of resulting masks was processed in ImageJ (imagej.nih.gov/ij/). For dynamic study of VCH-916 mCherry-GPI RUSH and GFP-VAMP7 co-expressed in Hela cells acquisition started 40?min after biotin addition and images were taken every 5?min for 150?min. Binary masks of each channel were obtained using Icy “Wavelet spot detector” analysis. Channels merging and movie montage were performed with ImageJ. Secretion assays For secretion studies cells were transfected with mCherry-tagged GPI and VSVG constructs of the RUSH system generated by Boncompain et?al.29 24?h after transfection secretion was induced with 40?μM Biotin (Sigma Aldrich). After induced release cells were quickly washed at 4°C with DMEM buffered with 20?mM HEPES and then incubated in the same solution with main mouse anti-mCherry antibody for 20?min at 4°C to allow surface epitope labeling. Cells were then washed fixed with PFA 4% and incubated with secondary Alexa488 antibody. To uncover the total pool cells were than permeabilized with 0 1 Triton X-100 and re-incubated with the same main anti-mCherry antibody and a.