Journal: Developmental Cell

Article Title: Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization

doi: 10.1016/j.devcel.2019.07.009

Figure Lengend Snippet: Translocation of Lro1 to the INM that Associates with the Nucleolus (A) Schematic of the topology of Lro1. The K/R-rich nucleolar targeting sequences are shown in red. The Ser324 within the GHSXG lipase motif is shown. (B) lro1 Δ cells expressing a nucleolar reporter and the Lro1-GFP mutants shown were imaged at the indicated growth phases. Red stars denote the K/R to A mutations. Arrowheads denote the nucleolus and/or the nucleolar-associated membrane. (C) Quantification of the subcellular localization of the indicated Lro1-GFP mutants in the specified strains. Red stars denote the K/R to A mutations within the extralumenal domain. Three colonies of each strain were analyzed; at least 200 cells were counted for each strain. (D) Lro1-GFP was photobleached, either at the nucleolar-associated membrane or the cortical ER (cER), and fluorescence recovery was measured. Data are means ± SD from three independent experiments (seven cells each); arrow indicates the bleaching event. (E) Nucleolar-associated membrane targeting of 1x-, 2x-, or 3x-MBP-Lro1-GFP fusions during the PDS phase. Right panel: Quantification of the data shown from three experiments, counting only cells with signal in ER or nucleolus; at least 250 cells were quantified for each strain. (F) Localization of the FRB-GFP control (the outlines of cells are shown; vac, vacuole), and the FRB-Lro1-GFP (middle) or FRB-3xMBP-Lro1-GFP (bottom) fusions, before or after the addition of rapamycin. Arrowheads point to the cortical ER membrane. Scale bars in all micrographs, 5 μm. See also Figure S2 .

Article Snippet: Images were recorded using a large chip sCMOS mono camera for sensitive fluorescence imaging (ORCA Flash 4.0v2, Hamamatsu, Hamamatsu, Japan), saved by Zeiss ZEN2.3 software (Blue edition, Carl Zeiss Ltd, Jena, Germany) and exported to Adobe Photoshop (Adobe, San Jose, CA).

Techniques: Translocation Assay, Expressing, Membrane, Fluorescence, Control

Journal: Developmental Cell

Article Title: Compartmentalized Synthesis of Triacylglycerol at the Inner Nuclear Membrane Regulates Nuclear Organization

doi: 10.1016/j.devcel.2019.07.009

Figure Lengend Snippet: Lro1 Is Catalytically Active at the Nucleolar-Associated INM (A) Wild-type or dga1 Δ lro1 Δ are1 Δ are2 Δ (4Δ) cells expressing the indicated plasmids were grown to exponential (EXP) or PDS phases in minimal synthetic medium and spotted on YEPD plates. (B) 4Δ cells expressing Lro1-GFP and Sec63-mCherry were grown from exponential phase to the indicated densities and imaged. (C) 4Δ cells expressing Lro1, or an empty vector, were grown to the indicated densities, labeled with BODIPY 493/503 and their fluorescence was quantified by FACS. Data are representative of two independent experiments. (D) 4Δ cells expressing Lro1-mCherry were grown to the PDS phase and labeled with BODIPY 493/503. Deconvolved through-focus image series were processed to generate 3D image. The full reconstructed field is shown in . (E) Model for the Lro1-mediated regulation of phospholipid homeostasis; see text for details. (F) Co-localization of the indicated GFP fusions with Lro1-mCherry at the PDS phase. (G) Lipidomic quantifications of TG, LPE, LPC, PE, and PC in wild-type (BY4741), ale1Δ, and plb1 Δ plb2 Δ plb3 Δ nte1 Δ lro1 Δ (5Δ) cells expressing the denoted plasmids. Cells were grown in galactose for 5 h. Lipid levels were normalized to the corresponding levels of the wild-type strain expressing the empty vector. Data shown are means of at least 5 experiments ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Scale bars in all micrographs, 5 μm. See also and .

Article Snippet: Images were recorded using a large chip sCMOS mono camera for sensitive fluorescence imaging (ORCA Flash 4.0v2, Hamamatsu, Hamamatsu, Japan), saved by Zeiss ZEN2.3 software (Blue edition, Carl Zeiss Ltd, Jena, Germany) and exported to Adobe Photoshop (Adobe, San Jose, CA).

Techniques: Expressing, Plasmid Preparation, Labeling, Fluorescence