Ulloa G et al. (2019), Lipid species affect morphology of endoplasmic ...

ECB-ART-47456

J Lipid Res 2019 Nov 01;6011:1880-1891. doi: 10.1194/jlr.RA119000210.

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Lipid species affect morphology of endoplasmic reticulum: a sea urchin oocyte model of reversible manipulation.

Ulloa G , Hamati F , Dick A , Fitzgerald J , Mantell J , Verkade P , Collinson L , Arkill K , Larijani B , Poccia D .

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The ER is a large multifunctional organelle of eukaryotic cells. Malfunction of the ER in various disease states, such as atherosclerosis, diabetes, cancer, Alzheimer''s and Parkinson''s and amyotrophic lateral sclerosis, often correlates with alterations in its morphology. The ER exhibits regionally variable membrane morphology that includes, at the extremes, large relatively flat surfaces and interconnected tubular structures highly curved in cross-section. ER morphology is controlled by shaping proteins that associate with membrane lipids. To investigate the role of these lipids, we developed a sea urchin oocyte model, a relatively quiescent cell in which the ER consists mostly of tubules. We altered levels of endogenous diacylglycerol (DAG), phosphatidylethanolamine (PtdEth), and phosphatidylcholine by microinjection of enzymes or lipid delivery by liposomes and evaluated shape changes with 2D and 3D confocal imaging and 3D electron microscopy. Decreases and increases in the levels of lipids such as DAG or PtdEth characterized by negative spontaneous curvature correlated with conversion to sheet structures or tubules, respectively. The effects of endogenous alterations of DAG were reversible upon exogenous delivery of lipids of negative spontaneous curvature. These data suggest that proteins require threshold amounts of such lipids and that localized deficiencies of the lipids could contribute to alterations of ER morphology. The oocyte modeling system should be beneficial to studies directed at understanding requirements of lipid species in interactions leading to alterations of organelle shaping.

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Genes referenced: als2 LOC100887844 LOC100888919 LOC100893907 LOC115919910 LOC115925415

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	Fig. 1. ER of unfertilized egg. A: Confocal microscopy image 30 min after microinjection of oil containing diIC18. B: Five hours after diIC18 injection illustrating similar pattern of ER. C: 3D reconstruction of egg made from z-stack of six 0.1 Î¼m optical sections analyzed with Microscopy Image Browser. Total ER was selected with a Strel filter and labeled yellow, brighter sheet-like areas (selected by black/white intensity thresholding) were labeled turquoise and superimposed. Tubular density is apparent. D, E: Representative transmission electron micrographs of 300 nm sections showing the injected oil (D) and egg edge (E). F, G: Tomographic slices from the boxed areas in D and E. Yolk granules (yellow arrows) appear relatively uniformly distributed throughout the cytoplasm. In areas with fewer yolk granules, annulate lamellae (red arrows), mitochondria (green arrows), membrane vesicles, ER tubules, and rare ER sheets can be seen.
	Fig. 2. ER of DGK-treated eggs. A: Confocal images of an egg injected with DGK showing accumulation of sheet regions at 28, 36, and 52 min post-DGK injection. Green arrows indicate representative sheet regions. Red scale lines = 10 Î¼m. B: Confocal image of an unlabeled egg injected with DGK and 20 min later injected with diIC18 to test for ER continuity. Green arrows indicate representative sheet areas; yellow arrows indicate representative tubular areas. Sheets and tubules remain connected. C: Confocal 3D reconstruction of control and DGK-treated eggs made from z-stack of 18 overlapping 0.1 Î¼m sections (total depth 0.9 Î¼m) and analyzed with Microscopy Image Browser using a Strel filter to select for the tubular network (Strel 4, b/w threshold 0.04, size limit 70) and Fiji volumes showing depletion of tubules by DGK.
	Fig. 3. The ER displays a variety of structures in sheet regions. A: Confocal image 80 min after DGK injection shows bright sheet-like areas against background of more faint connecting tubules with extensive sheet formation illustrating many structures confirmed by electron microscopy. B: Enlargement of A. Arrows indicate edge views of sheets or thick stacks (yellow), thicker stacks (orange), partially closed stacks (pink), closed stacks/cylinders (red), bifurcations of stacks (tan), en face views of sheets possibly fenestrated (green), area of tubules (blue). C: EM section of DGK-treated egg. D, E: Transmission electron micrograph of 300 nm sections with planes from tomographic reconstructions of the two boxed areas shown in C. Note sheets and fenestrated sheets extending from annulate lamellae. E: Note prominent annulate lamellae and fenestrated sheet areas. F: A coiled stacked sheet from elsewhere in the same egg. Dâ€“F: Yolk granules (purple arrows), mitochondria (green arrows), fenestrated sheet/annulate lamellae (yellow arrows), nonfenestrated sheets (red arrows).
	Fig. 4. DGK-treated egg with forming coils. Egg contains many regions with varying degrees of sheet and coil formation (blue arrows indicate some coils and green arrow a flat stack of two membranes). One of the coil regions with multiple sheets was selected (yellow box) and images from the SBF-SEM stack are shown in the bottom insets at relative depths of âˆ’1.5, 0.0, +1.5, and +3.0 Î¼m showing the complexity of thickness, shape, and sheet number and demonstrating a minimum length in the z direction of 4.5 Î¼m for this part of the forming coil. The structures suggest that coils form progressively from stacks that feed into them (see supplemental Movie S3, which shows a complete z-series of the tilt series tomographic reconstruction of this region). Scale bar = 5.0 Î¼m.
	Fig. 5. Confocal 3D reconstruction of bifurcated sheet region. A: Six confocal sections of a bifurcated sheet area taken from selected z-sections. B: 3D reconstruction of this region from 32 sections spaced at 0.5 Î¼m intervals indicating a folding of the sheets into a partial coil. Arrow indicates region of bifurcation.
	Fig. 6. Effect of bacterial PI-PLC injection into unfertilized eggs. A: Decline in sheet regions of five eggs injected with PI-PLC. B: Successive images of an unfertilized egg injected with PI-PLC (200 Î¼g/ml pipette concentration) showing slight decrease in sheet regions over time.
	Fig. 7. Effect of DGK/PI-PLC co-injection at various ratios on sheet region formation. A: Ratio of area of sheet regions to total egg area normalized to time of co-injection of DGK and bacterial phospholipase C (PI-PLC). Numbers indicate pipette concentrations in micrograms per milliliter of the two enzymes. Sheet regions accumulated at high DGK/PI-PLC ratios but were prevented or slightly reversed at lower ratios. B: Four images of one such egg injected at a 50/5 ratio showing decline in sheet areas over time (0â€“40 min).
	Fig. 8. Ability of 1,3-DAG- or PE-containing SUVS to reverse sheet formation in DGK-injected eggs. A: 1,3-DAG (10 mol%)/PC (90 mol%), four eggs. B: 1,3-DAG (20 mol%)/PC (80 mol%), seven eggs. C: PE (20 mol%)/PC (80 mol%), five eggs. D: PE (45 mol%)/PC (55 mol%) PC. SUVs were added 15â€“20 min after DGK injection, five eggs. All four SUV preparations led to reversal of sheet formation. SUVs containing higher ratios of phospholipids with negative curvature were generally most effective.

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