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Fig. 5 | BMC Biology

Fig. 5

From: Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy

Fig. 5

Accumulation of DAG at the ER in TAG production defective cells. a Starvation triggers intracellular DAG accumulation in dga1Δ lro1Δ cells. Cells of the indicated genotype expressing DAG probes (GFP-PKCδ and GFP-PKCβ) were grown to mid-log phase and then starved for 1 h. Images presented as in Fig. 1a. Yellow arrows, concentration of DAG at the buds in normal cells. Purple arrows, accumulation of DAG at intracellular bulbs. Scale bar, 2 μm. b Starvation triggers DAG accumulation at the ER in dga1Δ lro1Δ cells. Cells treated as in a, except that additional organelle markers (ER, vacuole, late Golgi, and late endosome) were co-expressed. Image presented as in Fig. 2a. Arrows, incidences of GFP-PKCδ colocalization with organelle markers. Scale bar, 2 μm. c, d Total cellular DAG. Cells of the indicated genotype were grown to mid-log phase and then starved for 1 h. Lipids were extracted and analyzed by mass spectrometry-assisted quantification (c) or thin layer chromatography (TLC) (d). c Error bar, standard deviation, n = 3. d Representative image from three independent repeats. e Manipulation of glycerolipid synthesis pathway alters DAG localization. Phospholipid production was upregulated by (1) knocking out OPI1 or (2) supplying key reactants (inositol, choline, and ethanolamine/ICE). Precursor influx was constrained by (1) 100-fold reduction of glucose supply (0.02% glucose), (2) chemical inhibition of fatty acid synthase (cerulenin), or (3) elimination of a key lysoPA acyltransferase (slc1Δ). Cells were starved for 1 h. Images presented as in a. Scale bar, 2 μm. f Exogenous DAG induces endomembrane defects. 1,2-Dioctanoyl-sn-glycerol of the indicated concentrations was added to starvation medium containing 0.02% glucose. Cells were starved for 1 h. Images presented as in Fig. 1a. Scale bar, 2 μm

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