Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation - PubMed (original) (raw)
Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation
Ariadna P Velázquez et al. J Cell Biol. 2016.
Abstract
Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases.
© 2016 Velázquez et al.
Figures
Figure 1.
LD deficiency conditionally impairs autophagy. (A) Autophagy flux of wt, Δ_atg7_, ΔTG, ΔSE, and ΔLD cells expressing 2xGFP-ATG8 during starvation or rapamycin treatment. Data are means ± SD (n = 4). (B) Cells were treated as in A and imaged by fluorescence microscopy after 1 h. Mean number of Atg8 puncta (left panel) and APs (right panel) per cell are shown as mean ± SD (≥150 cells; n = 3). (C) wt and ΔLD cells coexpressing _2xGFP_-ATG8 and genomically tagged _SEC13_-mCherry were shifted to starvation for 1 h before imaging. Arrows indicate Atg8 puncta associated with ERES (Sec13-mCherry). Data are means ± SD (≥150 cells; n = 3). Dashed lines indicate cell boundaries. (D) Survival of indicated strains during starvation. Bars, 1 µm. t test in A and B: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2.
Inhibition of de novo FA synthesis improves autophagy, ER morphology, and cell survival of LD-deficient cells during starvation. (A) Number of BODIPY493/503-stained LDs in wt, Δ_atg7_, ΔTG, ΔSE, and ΔLD cells during log phase and starvation ± glucose (Glu) after 6 h (150 cells per strain) analyzed by fluorescence microscopy as shown in
Fig. S1 C
. (B) wt, Δ_atg7_, ΔTG, ΔSE, and ΔLD cells expressing 4xUPRE-GFP grown to log phase were analyzed by whole-cell extraction and Western blot analysis using α-GFP and α-Pgk1 antibodies. GFP signals were normalized to Pgk1 and expressed relative (rel.) to wt cells in log phase (set as one). Data are means ± SD (n = 4). (C) wt and ΔLD cells expressing GFP-HDEL (ER-targeted GFP) were imaged in log phase or after 1 h of starvation ± cerulenin (10 µg/ml). Images show single cortical and mid sections of the same Z-stack. Quantifications of cells with a collapsed ER network of dilated tubules (arrows) as seen for ΔLD cells during starvation are mean ± SD (≥150 cells; n = 3). Bar, 5 µm. (D) Autophagy flux of wt and ΔLD cells expressing 2xGFP-ATG8 during starvation ± cerulenin (10 µg/ml). Data are means ± SD (n = 5). (E) Cells were treated as described in D and imaged and analyzed by fluorescence microscopy as described in Fig. 1 B. Data are means ± SD (≥150 cells; n = 3). Dashed lines indicate cell boundaries. Bar, 1 µm. (F) Survival of indicated strains treated as in D during starvation. t test in B, D, and E: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3.
Restoring altered PL composition in LD-deficient cells improves autophagy and cell survival during starvation. (A) wt, ΔLD, Δ_atg7_, Δ_opi1_, and Δ_opi1_ΔLD cells were grown to log phase ± inositol (2 mg/l), and PLs were analyzed by mass spectrometry as described in the Materials and methods. Relative (rel.) distribution of PC, PE, PI, PS, PA, and PG is shown as mean ± SD (n ≥ 3). (B) Autophagy flux of wt and ΔLD cells expressing 2xGFP-ATG8 during starvation (starv.) in the presence of indicated inositol concentrations. Data are means ± SD (n = 3). (C) wt and ΔLD cells expressing 2xGFP-ATG8 were grown to log phase and shifted to starvation ± inositol (2 mg/l). Cells were imaged and analyzed as described in Fig. 1 B. Dashed lines indicate cell boundaries. Bar, 1 µm. (D) Survival of indicated strains treated as in C during starvation. (E) Growth of wt, ΔLD, or Δ_atg7_ strains on plates containing oleate (O) or palmitoleate (P) at indicated concentrations ± inositol (2 mg/l). t test in A, B, and C: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 4.
Deletion of OPI1 buffers FA and improves ER stress and morphology in LD-deficient cells during starvation. (A) Localization of Opi1-3xGFP in wt and ΔLD cells in log phase ± inositol (2 mg/l) analyzed by fluorescence microscopy. Single focal planes of representative cells during growth in +inositol media are shown. Quantifications of cells with nuclear ER (nER) or diffuse nuclear (nucl.) Opi1 localization (≥100 cells; n = 3). (B) Growth of indicated strains on plates containing oleate (O) or palmitoleate (P) at indicated concentrations ± inositol (2 mg/l). (C) wt, Δ_ire1_, ΔLD, Δ_opi1_, and Δ_opi1_ΔLD cells expressing 4xUPRE-GFP in log phase. Cells were analyzed as described in Fig. 2 B. (D) wt, ΔLD, Δ_opi1_, and Δ_opi1_ΔLD cells expressing GFP-HDEL (ER-GFP) were grown to log phase and starved ± inositol (2 mg/l) for 1 h and analyzed as in Fig. 2 C. Arrowheads and arrows mark ER extensions or dilated tubules, respectively. Bar, 5 µm. t test in A and C: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Figure 5.
Increased FA resistance and restored PL composition cures AP biogenesis and autophagy flux in LD-deficient cells. (A and B) Autophagy flux in wt, ΔLD, Δ_opi1_, and Δ_opi1_ΔLD cells expressing 2xGFP-ATG8 during starvation (starv.) in the presence (A) or absence (B) of inositol (2 mg/l). Data are mean ± SD (n = 4). (C) Survival of indicated strains during starvation ± inositol (2 mg/l). (D) Autophagy flux in wt, ΔLD, Δ_opi1_, and Δ_opi1_ΔLD cells grown in inositol-free media and starved in the presence of cerulenin (10 µg/ml). Data are mean ± SD (n = 4). (E–G) wt, ΔLD, Δ_opi1_, and Δ_opi1_ΔLD cells expressing 2xGFP-ATG8 were treated as described in A, B, and D and imaged and analyzed as described in Fig. 1 B. Bar, 1 µm. Data are means ± SD (150 cells/condition, n = 3). t test in A, B, D, F, and G: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
References
- Axe E.L., Walker S.A., Manifava M., Chandra P., Roderick H.L., Habermann A., Griffiths G., and Ktistakis N.T.. 2008. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182:685–701. 10.1083/jcb.200803137 - DOI - PMC - PubMed
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