Lipid rafts are the new Stress Granules regulators
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Stress granules are cytoplasmic inclusions 1 with cyto-protective functions 2-6 assembling in response to stress. They are now accepted to be part of the pathological mechanism in several diseases, from cancer to neurodegenerative disorders 7-10 . However, the field is still struggling to find common regulators of their assembly and function 7,11 .
In this study, we describe an unraveled mechanism involving lipid raft, via gangliosides and cholesterol, in the regulation of SG formation. This is the first report about regulation of SG by the cell membrane composition. This discovery could have a significant impact on the understanding of several disease mechanism.
MATERIAL AND METHODES
Cell culture & cell treatment
MDA-MB-231 (ATCC) and SH-SY5Y (ATCC) cells were maintained at 37 °C with 5% CO 2 in Gibco Dulbecco’s Modified Eagle Medium: Nutrient Mixture F12 (DMEM-F12, GIBCO, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Eurobio, Les Ulis, France), 20 mM HEPES (GIBCO, Waltham, MA, USA), 1X Penicillin streptomycin (GIBCO, Waltham, MA, USA). Cells are treated with methyl-β-cyclodextrine (MβCD) (MDA-MD-231 5mM, SH-SY5Y 1mM) 48h before experimentation, or with d,l-threo-l-Phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) (MDA-MD-231 5μM, SH-SY5Y 10μM) for 24h.
Immunofluorescence
Cells were seeded on coverslips, treated 48h with PPMP or 24h with MβCD before the experiment. After stress treatment, cells are washed quickly with PBS before to be fixed for 15min with 4% Paraformaldehyde (Thermo Scientific, Waltham, MA, USA) in PBS. Cells were then permeabilized and blocked with IF buffer PBS-0.3% TX100 (Euromedex, Souffelweyersheim, France), 1% Glycine (Sigma, Saint-Louis, MO, USA), 5% Normal Horse Serum (Sigma, Saint-Louis, MO, USA), 5% Bovine Serum Albumine (Sigma, Saint-Louis, MO, USA) for 30 min at room temperature. Primary antibodies ( Table S1 ) were diluted in IF buffer and incubated 1 h at room temperature. Coverslips were washed three times for 5 min with 1X PBS between primary and secondary antibody incubations. Subsequently, secondary antibodies ( Table S1 ) were added along with DAPI for 1 h at room temperature in IF buffer. Cells were washed extensively 3 times with 1X PBS and mounted with ProLong Antifade reagent (Invitrogen, Carlsbad, CA, USA). Pictures were taken with confocal microscope LEICA LSM880
Western Blot
Following drug(s) treatment(s), cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer (150mM NaCl, 50mM Tris pH7.4, 1%TritonX100, 0.1% SDS, 1% Sodiun desoxycholate) with Halt phosphatase and protease inhibitors (Thermo Scientific). Laemmli’s sample buffer supplemented was added to samples to 1X final concentration. Samples were boiled, 5min 95°C before being loaded on a NuPAGE™ 4–12% Bis-Tris gel (Invitrogen) and transferred to nitrocellulose membrane (GE Healthcare). Membranes were blocked with Tris-buffered saline with 0.1% Tween-20 (TBS-T) with 5% BSA for at least 30 min at room temperature. Antibodies were diluted in 2.5% BSA in TBS-T. Primary antibodies were incubated overnight at 4°C and secondary antibodies for 1 h at room temperature; mouse anti G3BP1 antibody (Santa Cruz sc-365338), rabbit anti Caprin-1 antibody (ProteinTech Group 15112-1-AP), mouse anti puromycin antibody (Millipore MABE342), mouse anti GAPDH (abcam ab8245). Antibody detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Revelation of the blot was made using G:BOX machine (Syngene) via the GeneSys software. Blot analysis and quantification were done using ImageJ software.
Statistical Analysis
Statistical analyses were done on 3 independent experiments. Student T-TEST were performed to compare control to PPMP samples or control to MβCD samples.
