Gangliosides and cholesterol, two major components of the membrane lipid rafts, as new regulatory partners for stress granules assembly

Abstract

Stress granules are cytoplasmic inclusions with cyto-protective functions assembling in response to stress. They are now accepted to be part of the pathological mechanism in several diseases, from cancer to neurodegenerative disorders. However, the field is still struggling to find common regulators of their assembly and function. In this study, we describe a mechanism involving lipid rafts (gangliosides and cholesterol), in the regulation of stress granules formation. This study reports that membrane lipid composition is able to regulate the formation of stress granules potentially unraveling several disease mechanisms.

Keywords: Cholesterol; Gangliosides; Lipid rafts; Stress; Stress granules; Translation.

Fig. 1

Cells with lack of gangliosides and cholesterol at the membrane required higher dose of sodium arsenite (SA) to induce the assembly of stress granules. (a) MDA-MB-231 cells are treated with PPMP (5 μM, 48 h) or MβCD (5 mM, 24 h) before stress granule experimentation. On the day of the experimentation, cells were treated 1 h with SA at the indicated concentration and collected. (b) SH-SY5Y cells are treated with PPMP (10 μM, 48 h) or MβCD (1 mM, 24 h) before stress granule experimentation. The day of the experimentation cells were treated 1 h with SA at the indicated concentration and collected. (a) and (b) After collection cells are then fixed and stained with G3BP1 (green), Caprin-1 (red) for stress granule labeling and DAPI (bleu) for nuclei visualization before to by imaged using confocal microscopy. Left: representative pictures. Right: quantifications. N ≤ 3, *P < 0.05, ****P < 0.0001. Abbreviation used: MβCD, methyl-β-cyclodextrin.

Fig. 2

Lack of gangliosides and cholesterol at the membrane delay the formation of stress granules. (a) MDA-MB-231 cells are treated with PPMP (5 μM, 48 h) or MβCD (5 mM, 24 h) before stress granule experimentation. The day of the experimentation cells were treated with 200 μM sodium arsenite and collected at the indicated time. (b) SH-SY5Y cells are treated with PPMP (10 μM, 48 h) or MβCD (1 mM, 24 h) before stress granule experimentation. The day of the experimentation cells were treated with 100 μM sodium arsenite and collected at the indicated time. (a) and (b) After collection cells are then fixed and stained with G3BP1 (green), Caprin-1 (red) and DAPI (bleu) before to by imaged using confocal microscopy. Left: representative pictures. Right: quantifications. N ≤ 3, *P < 0.05. **P < 0.001. Abbreviation used: PPMP, D,L-threo-L-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol.

Fig. 3

Foci induced after lipid raft disruption are stress granules. (a) Mechanism of action on translation of cycloheximide and puromycin. CTR) Translation in basal condition: 1) tRNA binds in the A binding site (acceptor). 2) Peptide bond then forms between the new amino acid and the forming peptide chain in the P (Polypeptide) site. 3) The tRNAs translocate to the E (Exit) site, where the first tRNA can exit the ribosome. CHX) Inhibition of translation by cycloheximide: When cycloheximide is present, it binds to the E site of the ribosome. As a result, the translocation step cannot take place, and translation stops with the RNAs being translated blocked in the ribosome. Puro.) Inhibition of translation by puromycin: When cells are treated with puromycin, the latter integrates with nascent polypeptide chains, stopping translation and releasing mRNAs and polypeptide chains into the cytoplasm. (b-d) MDA-MB-231 are treated with PPMP (5 μM, 48 h) or MβCD (5 mM, 24 h) before stress granule experimentation. Stress granule experimentation is the indicated combination of cycloheximide (CHX, 50 μM) or puromycin (20 μM) and/or SA (50 μM or 100 μM as indicated) for 1 h.Ø: no cycloheximide, no puromycin. Cells are then fixed and stained with G3BP1 (green), Caprin-1 (red) and DAPI (bleu) before to by imaged using confocal microscopy. Left: representative pictures. Right: quantifications. N = 3, **P < 0.01, ****P < 0.001. (b) Cells are treated with cycloheximide, puromycin and compared to untreated samples (Ø) without any SA addition. (c) All samples are stressed using 100 μM of SA to induce a robust stress granule response (Ø). Cycloheximide is added to inhibit stress granule formation. (d) Cells are treated with suboptimal stress (50 μM) to induce minimum or no stress granule (Ø). Puromycin is added to enhance stress granule formation. Abbreviations used: CHX, cycloheximide; Puro., puromycin; SA, sodium arsenite.

Fig. 4

Modification of lipids raft decrease G3BP1 expression level and translation inhibition in response to stress. (a) Under stress exposure, polysome disassemble to allow mRNA to be recruited to stress granule with proteins.a- Keeping the translation active will prevent the assembly of SG. b- The downregulation of one of the two scaffolding protein G3BP1 or G3BP2 will also (partially) prevent the formation of stress granule. (b-d) MDA-MB-231 cells are treated with PPMP (5 μM, 48 h) or MβCD (5 mM, 24 h) before to be exposed to the indicated sodium arsenite (SA) concentration for 1 h. 5 min before cell lysis, cells were pulsed with puromycin 5 μg/ml. For all samples, 15 μg of total protein are loaded for each sample. N ≤ 3, *P < 0.05, **P < 0.01, ***P < 0.005. (b) Representative blots. (c) and (d) Quantifications. Signal intensity was measured using ImageJ software. Each band intensity was expression relatively to the GAPDH intensity of the same sample and then expression was plotted relatively to the untreated sample (CTR, 0 μM SA). (c) Puromycin level, representative of general protein expression. (d) G3BP1 expression. All the CTR, PPMP and MBCD treated samples were, respectively, pulled together for analysis. Abbreviations used: MβCD, methyl-β-cyclodextrin; PPMP, D,L-threo-L-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol.