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

Article Title: Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading

doi: 10.1016/j.cell.2019.04.037

Figure Lengend Snippet: (A and B) Schematic of the model transmembrane protein FRTM-Ezrin-AFBD (A) and the mutant FRTM-Ezrin-R579A (FRTM-Ez-AFBD*) (B) that impairs Ezrin-AFBD ability to interact with actin . (C–F) Representative intensity and steady-state anisotropy images and scatter dot plots with mean anisotropy of ROIs obtained from CHO cells stably expressing either FRTM-Ez-AFBD or FRTM-Ez-AFBD* as indicated. The cells were labeled with fluorescent folate, Pteroyl-lysyl-Bodipy(PLB) and plated on FN (blue, green) or glass (red, orange) prior to imaging in the absence (C and D) or after pre-treatment (E and F) with either 20 μM PP2 and 10 μM PF-573228 (red) or 10 μM SMIFH2 (green) or with the vehicle (DMSO; blue). (G–I) Schematic (G) of the supported lipid bilayer functionalized with cRGD that was prepared either on plain (continuous SLB; top) or on 5-nm-tall and 100-nm-wide chromium patterned (nanopatterned SLB, bottom) glass surfaces. (H and I) GFP-GPI-expressing CHO cells plated on glass (red) or on FN (blue) or treated with 10 mM mβCD on FN (green) or plated on either continuous SLBs with mobile ligand (magenta) or SLBs assembled on chromium nano-patterned surfaces. ROIs were drawn either on the pattern (orange) where the ligand is transiently immobile or from regions outside (brown) where the ligand is mobile. Scale bar 10μm in all panels. All error bars represent SD. n.s. p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample size and p values are provided in . See also .

Article Snippet: Lact C2 Ezrin AFBD-YFP , Protein Technology Core (C-CAMP,Bangalore,India) ( ) , N/A.

Techniques: Mutagenesis, Stable Transfection, Expressing, Labeling, Imaging

Journal: Cell

Article Title: Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading

doi: 10.1016/j.cell.2019.04.037

Figure Lengend Snippet: (A–J) Representative intensity and steady-state anisotropy images (A, C, E, G, and I) and scatter dot plot with mean anisotropy values (B, D, F, H, and J) of ROIs obtained from (A and B) vinculin-deficient cells (Vin −/− ) transfected with GFP-GPI (blue, orange) or co-transfected with mCherry-vinculin (+Vin-WT; red, green) and plated on FN or subsequently treated with 10 mM mβCD (orange, green). (C and D) Talin1-deficient cells without (Talin1 −/− ; blue, red) or with co-transfection with Talin2 shRNA (+Talin2 shR; green, orange) and re-plated onto FN after labeling with Alexa-568-FLAER prior to (blue, green) or post-treatment with 10 mM mβCD (red, orange). (E and F) Vin −/− cells alone (blue) or transiently transfected with GFP tagged Vin-WT (green), Vin-A50I (orange), or Vin-A50I-CA (brown) and plated onto FN after labeling with Alexa-568-FLAER. (G and H) Vin −/− cells were transiently transfected with FRTM-Ez-AFBD (FR-EZ; green) or with FR-Ez-AFBD* mutant (FR-EZ*; red), without (open circles) or with Vin-WT (closed circles) and re-plated onto FN after labeling with PLB. (I and J) Vin −/− cells alone (blue) or transfected with Lact C2-Ez-AFBD YFP (red) were labeled with Alexa-568-FLAER and re-plated on FN and directly labeled or treated with 10 mM mβCD (+mβCD; green). Dotted magenta lines in all images outline the transfected cells expressing the indicated constructs. Scale bar, 10 μm in all panels. All error bars represent SD. n.s. p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample size and p values are provided in . See also .

Article Snippet: Lact C2 Ezrin AFBD-YFP , Protein Technology Core (C-CAMP,Bangalore,India) ( ) , N/A.

Techniques: Transfection, Cotransfection, shRNA, Labeling, Mutagenesis, Expressing, Construct

Journal: Cell

Article Title: Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading

doi: 10.1016/j.cell.2019.04.037

Figure Lengend Snippet: Key Resources Table

Article Snippet: Lact C2 Ezrin AFBD-YFP , Protein Technology Core (C-CAMP,Bangalore,India) ( ) , N/A.

Techniques: Purification, Transduction, Recombinant, Clinical Proteomics, Avidin-Biotin Assay, Membrane, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Mutagenesis, Derivative Assay, Control, Construct, shRNA, Software