Journal: MicrobiologyOpen

Article Title: Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

doi: 10.1002/mbo3.211

Figure Lengend Snippet: Saccharomyces cerevisiae strains used in this study

Article Snippet: pKT1995 [pRS306-GFP-Lact-C2-AAA] , GFP-Lact-C2-AAA URA3 , Takeda et al. ( ) .

Techniques:

Journal: MicrobiologyOpen

Article Title: Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

doi: 10.1002/mbo3.211

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: pKT1995 [pRS306-GFP-Lact-C2-AAA] , GFP-Lact-C2-AAA URA3 , Takeda et al. ( ) .

Techniques:

Journal: MicrobiologyOpen

Article Title: Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

doi: 10.1002/mbo3.211

Figure Lengend Snippet: PS is present on the surface of accumulated SVs and the TGN, but not the ER, in secretory mutant cells. (A) Phenotypes of temperature-sensitive sec and myo2 mutants. (B) Localization of mRFP-Lact-C2 to accumulated SVs and TGN membranes in sec6-4/myo2-12 and sec7-1 cells, respectively. Cells were incubated for 1 h in SD-Leu medium or SD-Leu medium supplemented with 1 mmol/L ethanolamine ( cho1 Δ mutants) or for 2 h in SD-Leu-Ura medium ( myo2-12 mutant) at 30°C (control) or 37°C. The strains used were wild type (WT) (YKT1843), cho1 Δ (YKT1845), sec6-4 (YKT1844), sec6-4 cho1 Δ (YKT1846), myo2-12 (YKT1678), sec7-1 (YKT1857), and sec7-1 cho1 Δ (YKT1858). These strains, all carrying mRFP1-Lact-C2 integrated at the URA3 locus except the myo2-12 mutant, were transformed with pRS315-GFP-SNC1 pm (pKT1491). The myo2-12 mutant was cotransformed with pKT1491 and pRS416-mRFP1-Lact-C2 (pKT1755). Bar, 5 μ m. (C) mRFP-Lact-C2 did not localize to accumulated ER membranes. Cells were incubated for 1 h in SD-Leu-Ura medium at 25°C (control) or 37°C. The strains used were sec12-4 (MBY10-11D), sec21-1 (MBY6-4D), and sec23-1 (MBY8-20C), all cotransformed with pRS315-GFP-SNC1 pm (pKT1491) and pRS416-mRFP1-Lact-C2 (pKT1755). Bar, 5 μ m.

Article Snippet: pKT1995 [pRS306-GFP-Lact-C2-AAA] , GFP-Lact-C2-AAA URA3 , Takeda et al. ( ) .

Techniques: Mutagenesis, Incubation, Control, Transformation Assay

Journal: MicrobiologyOpen

Article Title: Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

doi: 10.1002/mbo3.211

Figure Lengend Snippet: Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in flippase-defective secretory mutant cells. (A) and (B) Localization of GFP-Snc1p-pm and mRFP-Lact-C2 in lem3 Δ crf1 Δ and cdc50 Δ mutants. Cells were incubated in SD-Leu medium at 30°C (control) or 37°C for 1 h. The strains used were lem3 Δ crf1 Δ (YKT1847) , sec6-4 lem3 Δ crf1 Δ (YKT1848) , sec7-1 lem3 Δ crf1 Δ (YKT1860), cdc50 Δ (YKT1849) , sec6-4 cdc50 Δ (YKT1850) , and sec7-1 cdc50 Δ (YKT1859), all carrying URA3::mRFP1-Lact-C2 and pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μ m. (C) Localization of GFP-Snc1p-pm with Sec7p-mRFP or mRFP-Snc1p in the cdc50 Δ mutant. (Left panel) SEC7-mRFP1 (YKT1670) and cdc50 Δ SEC7-mRFP1 (YKT1149) cells, both carrying pRS416-GFP-SNC1 pm (pKT1444), were incubated in SD-Ura medium at 30°C. (Right panel) Wild-type (YKT38) and cdc50 Δ (YKT249) cells, both carrying pRS416-GFP-SNC1 pm (pKT1444) and pRS315-mRFP1-SNC1 (pKT1568), were incubated in SD-Leu-Ura medium at 30°C. Regions labeled with small characters are twofold enlarged to compare GFP and mRFP signal patterns. Bar, 5 μ m.

Article Snippet: pKT1995 [pRS306-GFP-Lact-C2-AAA] , GFP-Lact-C2-AAA URA3 , Takeda et al. ( ) .

Techniques: Mutagenesis, Incubation, Control, Labeling

Journal: MicrobiologyOpen

Article Title: Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

doi: 10.1002/mbo3.211

Figure Lengend Snippet: Quantitative analysis of PS on isolated LDSVs by measurement of mRFP-Lact-C2 fluorescence intensity. Cells were grown at 30°C or shifted to 37°C for 2 h. LDSVs were isolated from the cells by subcellular fractionation followed by Nycodenz gradient fractionation. Relative fluorescence intensity of mRFP-Lact-C2 was measured using a spectrofluorometer, and total phospholipid phosphates were determined. Pma1p and mRFP-Lact-C2 were detected by Western blotting using antibodies against Pma1p and RFP, respectively. The SV-enriched fraction from sec6-4 cells in (A) was loaded as a positive control (PC) for Western blotting in (C) and (D). The strains used were sec6-4 (YKT1844) (A and D), wild type (WT) (YKT1843) (C), and sec6-4 cho1 Δ (YKT1846) (B), all carrying mRFP1-Lact-C2 at the genomic URA3 locus.

Article Snippet: pKT1995 [pRS306-GFP-Lact-C2-AAA] , GFP-Lact-C2-AAA URA3 , Takeda et al. ( ) .

Techniques: Isolation, Fluorescence, Fractionation, Western Blot, Positive Control

Journal: MicrobiologyOpen

Article Title: Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae

doi: 10.1002/mbo3.211

Figure Lengend Snippet: Loss of Lem3p/Crf1p or Cdc50p does not decrease the level of PS in the cytosolic face of LDSVs. (A) Fractionation profile of mRFP-Lact-C2 and total phospholipid phosphates in flippase mutants. Cells were grown in YPDA medium at 30°C and shifted to 37°C for 2 h, whereas P GAL1 -3HA-NEO1 sec6-4 cells were incubated in YPDA medium at 30°C for 8.5 h, followed by a shift to 37°C for 2 h. SVs were isolated and fractionated by Nycodenz density gradient as in Figure . The strains used were sec6-4 (YKT1844), sec6-4 cho1 Δ (YKT1846), sec6-4 lem3 Δ crf1 Δ (YKT1848) , sec6-4 cdc50 Δ (YKT1850), and sec6-4 P GAL1 -3HA-NEO1 (YKT1852), all carrying mRFP1-Lact-C2 at the genomic URA3 locus, and sec6-4 (AAA) (YKT1919) carrying mRFP1-Lact-C2-AAA at the genomic URA3 locus. (B) Lact/Phospholipid in the flippase mutants. Lact/Phospholipid was calculated as the ratio of relative fluorescence intensity of mRFP-Lact-C2 to total phospholipid phosphates in the peak and adjacent four fractions. Data shown are means ± SD of three independent experiments. (C) Localization of GFP-Snc1p-pm and mRFP-Lact-C2-AAA. Cells were incubated in SD-Leu medium at 30°C or 37°C for 1 h. The strains used were mRFP1-Lact-C2-AAA (YKT1918) and sec6-4 mRFP1-Lact-C2-AAA (YKT1919), both carrying pRS315-GFP-SNC1 pm (pKT1491). Bar, 5 μ m.

Article Snippet: pKT1995 [pRS306-GFP-Lact-C2-AAA] , GFP-Lact-C2-AAA URA3 , Takeda et al. ( ) .

Techniques: Fractionation, Incubation, Isolation, Fluorescence

Journal: Cell Reports

Article Title: Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition

doi: 10.1016/j.celrep.2022.111117

Figure Lengend Snippet: Measurements of ANO6 Ca 2+ -activated ion channel and phospholipid scramblase activities (A–C) The inhibitory effect of A6-001 on the ANO6 ion channel activity was analyzed using a whole-cell patch clamp in HEK 293T cells expressing ANO6. The pipette solution contained 10 μM free Ca 2+ . Voltage ramps spanning a range of −100 to +100 mV were delivered from a holding potential of −60 mV every 20 s (A). The current-voltage (I-V) relationships at indicated times (1–5) with increasing concentrations of A6-001 are shown in (B). The numbers in parentheses represent the time points to measure I-V relationships. The inward currents in (A) represent ANO6 tail currents. The half-maximal inhibitory concentration (IC 50 ) of A6-001 on the ANO6 ion channel activity is depicted in (C) (n = 4 at each concentration). (D–F) The effects of ANO6 inhibitors on the surface exposure of phosphatidylserine (PS) were assayed in FRT-ANO6 cells. Cells were pretreated with compounds for 10 min, before ANO6 was activated with 10 μM ionomycin. Exposed PS was stained with Lact-C2-GFP. Representative fluorescence images of A6-001 (10 μM), A6-004 (10 μM), abamectin (10 μM), and ivermectin (10 μM) are shown in (D) (scale bars: 10 μm), and fluorescence intensity values calculated from Lact-C2-GFP images are summarized in (E) (mean ± SEM; n = 6). The dose responses of A6-001 on ANO6 PS scramblase inhibition are analyzed in (F) (mean ± SEM; n = 6). Representative fluorescence images of A6-001 dose response are shown in Figure S2 H. (G and H) The inhibitory effect of A6-001 on PS scrambling was assayed using the four-quadrant dot blot FACS analysis in Jurkat cells, some of which were transfected with hANO6-expressing plasmids using a NEPA21 Super Electroporator (Jurkat-ANO6). Ionomycin evoked an increase in the Lact-C2-positive and propidium iodide (PI)-negative cell population (Q3), which represented the PS-exposed and non-apoptotic cells, respectively. The ionomycin-induced Q3 increase was augmented considerably in cells expressing exogenous ANO6. A6-001 (10 μM) reversed the Q3 increases in Jurkat and Jurkat-ANO6 cells. Representative FACS analyses are shown in (G), and a summary of the multiple experiments is presented in (H) (mean ± SEM; n = 5). ∗ p < 0.05 and ∗∗ p < 0.01: differences from lane 2. Data were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison test. See also Figure S2 .

Article Snippet: After washout, the phosphatidylserine and nuclei were stained with PBS containing 500 nM Lact-C2-GFP, then cells were washed with 200 μL PBS and microscopic images were acquired with a Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA).

Techniques: Activity Assay, Patch Clamp, Expressing, Transferring, Concentration Assay, Staining, Fluorescence, Inhibition, Dot Blot, Transfection

Journal: Cell Reports

Article Title: Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition

doi: 10.1016/j.celrep.2022.111117

Figure Lengend Snippet: ANO6 is responsible for phosphatidylserine externalization evoked by pseudotyped SARS-CoV-2 S virus (SARS2-PsV) HeLa cells expressing ACE2 (HeLa-ACE2) were incubated with a lentivirus-based SARS2-PsV (100 ng p24/mL; 20 MOI) or an authentic SARS-CoV-2 (10 MOI) for 15 min and then with Lact-C2-mCherry for 45 min. (A and B) Chelation of cytosolic Ca 2+ (BAPTA-AM; 10 μM; 1 h) inhibits SARS2-PsV-induced PS externalization. Representative images are shown in (A), and the quantification results of multiple experiments are summarized in (B) (n = 3, each from 3 to 5 fields per experimental condition). (C and D) Control experiments in ACE2-negative HeLa cells. PS externalization was induced by ionomycin (10 μM; 10 min). Representative images are shown in (C), and the quantification results of multiple experiments are summarized in (D) (n = 3). ns, not significant. (E and F) Silencing of ANO6 (siRNAs against ANO6; 100 nM; 24 h) inhibits the phosphatidylserine (PS) externalization evoked by SARS2-PsV. Cell nuclei were stained with DAPI. Representative images are shown in (E), and the quantification results of multiple experiments are summarized in (F) (n = 3, each from 3 to 5 fields per experimental condition). (G and H) ANO6 inhibitors (A6-001 and A6-004, each 10 μM, 1 h) inhibit SARS2-PsV-induced PS externalization. Representative images are shown in (G), and the quantification results of multiple experiments are summarized in (H) (n = 3–4). (I and J) Authentic SARS-CoV-2 virus evokes Ca 2+ - and ANO6-dependent PS externalization. The HeLa-ACE2 cells were pretreated with compounds (10 μM) for 1 h and infected with SARS-CoV-2 for 15 min. Representative images are shown in (I), and the quantification results of multiple experiments are summarized in (J) (n = 3). Bar graph data are shown as the mean ± SEM. ∗ p < 0.05 and ∗∗ p < 0.01. Data were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison test. Scale bars: 50 μm. See also Figure S3 .

Article Snippet: After washout, the phosphatidylserine and nuclei were stained with PBS containing 500 nM Lact-C2-GFP, then cells were washed with 200 μL PBS and microscopic images were acquired with a Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA).

Techniques: Expressing, Incubation, Staining, Infection

Journal: Cell Reports

Article Title: Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition

doi: 10.1016/j.celrep.2022.111117

Figure Lengend Snippet:

Article Snippet: After washout, the phosphatidylserine and nuclei were stained with PBS containing 500 nM Lact-C2-GFP, then cells were washed with 200 μL PBS and microscopic images were acquired with a Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA).

Techniques: Recombinant, Modification, Cell Culture, Protease Inhibitor, Protein Purification, Fluorescence, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Proliferation Assay, CCK-8 Assay, Luciferase, Software, Microscopy, Laser-Scanning Microscopy, Patch Clamp