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, 17 (6), 657-69

Lipid Droplets Form From Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces Pombe

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Lipid Droplets Form From Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces Pombe

Alex Meyers et al. Traffic.

Abstract

Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation around the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. The results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell.

Keywords: endoplasmic reticulum; fission yeast; lipid body; lipid droplet; sterol ester; triacylglycerol.

Figures

Figure 1
Figure 1
Lipid droplets that are enriched in SEs form exclusively at the tips of elongating fission yeast cdc25-22 cells. (A) Confocal microscopy images of cdc25-22 cells expressing the ER luminal marker mCherry-AHDL (red) where the lipid droplets have been stained with BODIPY 493/503 (green). Left panels are maximum intensities of all z-stacks (green), center panels are the middle z-stack (red), and right panels are the merger of the left and center panels. Arrows highlight the increase in the number of lipid droplets at the tips of the cells. Scale bars are 5 microns. (B) Plot of the number (left axis) and normalized intensity (right axis) of the lipid droplets in the cell in (A) as a function of time. (C) Plot of the number of lipid droplets as a function of their cellular position in the cell in (A) at t = 0 min (green) and t = 160 min (purple). The symbol, d, represents the distance from the center of the nucleus to the tip of the cell. The symbol, x, represents the position of the lipid droplet from the center of the nucleus. The gray vertical rectangles in the plots represent spaces occupied by the nucleus. (D) Thin layer chromatography results of the neutral lipid content of lipid droplets isolated from wild-type cells and elongated cdc25-22 cells at 36°C. (E) Plot of the positions of lipid droplets in five cdc25-22 cells before (t = 0 m) and after (t = 160 m) elongation at 36°C. The cell in (A) is not included. (F) Phase contrast (left panels) and maximum intensity of all z-stacks (right panels) for elongated cdc25-22 and are1Δare2Δ-cdc25-22 cells where the lipid droplets have been stained with BODIPY 493/503. Scale bars are 1 micron. (G) Plot of the number of lipid droplets as a function of their cellular position in the cells in (F). (H) Plot of the number of lipid droplets in five elongated cdc25-22 and five elongated are1Δare2Δ-cdc25-22 cells at 36°C. (I) Confocal microscopy images of cdc25-22 cells expressing Atb2p-mCherry (red) where the lipid droplets have been stained with BODIPY 493/503 (green). Left panels are maximum intensities of all z-stacks (green), center panels are maximum intensities of all z-stacks (red), and right panels are the merger of the left and center panels. The cell on the right was treated with thiabendazole to disrupt the microtubule network. Scale bars are 1 micron. (J) Plot of the distribution of BODIPY 493/503-stained lipid droplets in elongated cdc25-22 cells expressing Atb2p-mCherry whose microtubule networks were intact (green) versus disrupted by thiabendazole (purple). (K) Maximum intensity confocal microscopy images of cdc25-22 cells lacking Myo51p (left panel) or Myo52p (right panel) where the lipid droplets have been stained with BODIPY 493/503 (green). Scale bars are 1 micron. (L) Plot of the distribution of BODIPY 493/503-stained lipid droplets in elongated cdc25-22 lacking Myo51p (green) or Myo52p (purple).
Figure 2
Figure 2
SE lipid droplet formation from the cortical/peripheral ER correlates with the localization of sterols. (A-D) Single focal plane fluorescent micrographs of (A) mYFP-Are1p-cdc25-22, (B) mYFP-Are2p-cdc25-22, (C) mYFP-Are1p, and (D) mYFP-Are2p. All four fusion proteins were expressed from native loci using the P41nmt1 promoter. (E) Single focal plane fluorescent micrograph of elongated cdc25-22 cells at 36°C stained with the sterol marker filipin. (F) Single focal plane fluorescent micrograph of cdc25-22 cells that were elongated at 36°C then allowed to reenter the cell cycle by temperature reduction to 30°C followed by filipin staining. (G) Single focal plane fluorescent micrograph of wild-type fission yeast cells stained with the sterol marker filipin. (H) Single focal plane fluorescent micrographs of cdc25-22 cells expressing mCherry-AHDL that were elongated at 25°C then allowed to reenter the cell cycle by temperature reduction to 25°C followed by BODIPY 493/503 and filipin staining. (I) Confocal microscopy images of cdc25-22 cells expressing mCherry-AHDL (red) where the lipid droplets have been stained with BODIPY 493/503 (green). Top panels are maximum intensities of all z-stacks (green), center panels are the middle z-stack (red), and lower panels are the merger of the top and center panels. (J) Plots showing the distribution of the BODIPY 493/503-stained lipid droplets in the cells in (I). (K) are1Δare2Δ-cdc25-22 cells stained with the sterol marker filipin. (L) are1Δare2Δ fission yeast cells stained with the sterol marker filipin. Scale bars are 1 micron.
Figure 3
Figure 3
TAG lipid droplets form around the nuclear ER in fission yeast cells. (A-N) Sets of single focal plane fluorescent micrographs of fission yeast cells that have been stained with BODIPY 493/503 (left panel) that are expressing the ER luminal marker mCherry-AHDL (center panel) and the merger of these two signals (right panel) with accompanying plots of the positions of lipid droplets and TLC analysis of the neutral lipid content of the whole cell (C,F,I,K,N) and isolated lipid droplets where appropriate (C,F,I). Data plotted in (B,E,H,M) are from ten cells each in either YE5S and YPO. Scale bars are 1 micron.
Figure 4
Figure 4
Localizations of key enzymes in the TAG synthesis and lipolysis pathways. (A-D) Single focal plane fluorescent micrographs of (A) mYFP-Dga1p-cdc25-22, (B) mYFP-Plh1p-cdc25-22, (C) mYFP-Dga1p, and (D) mYFP-Plh1p. All four fusion proteins were expressed from native loci using the P41nmt1 promoter. (E-F) Single focal plane fluorescent micrographs of fission yeast cells expressing Ned1p-eGFP for the native locus grown to late log phase in YE5S (E) and late log phase in YPO (F). (G-H) Phase contrast (left panel) and single plane fluorescent (right panel) micrographs of elongated cdc25-22 (G) and wild-type (H) fission yeast cells expressing Cpt1p-mYFP from the native locus. (I-J) Single focal plane fluorescent micrographs of elongated cdc25-22 (I) and wild-type (J) fission yeast cells expressing mYFP-Dgk1p from the native locus under control of a P41nmt1 promoter. Scale bars are 1 micron.

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