Journal: Cell reports

Article Title: Mono- and Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells.

doi: 10.1016/j.celrep.2020.108026

Figure Lengend Snippet: Figure 2. Exocytosis Is Affected at Distinct Stages in Adrenal Chromaffin Cells (A) Mouse chromaffin cells in culture were stimulated with a local application of 100 mM nicotine for 10 s (green bars), and catecholamine secretion was monitored using carbon fiber amperometry. Typical amperometric recordings obtained from chromaffin cells of wild-type (control), Pld1/, or Pld2/ mice are presented. The histogram corresponds to the total number of amperometric spikes recorded per cell in response to 10 s stimulation (101 < n cells < 127; ***p < 0.001 compared with control). (B) Scheme illustrating the parameters of the amperometric spike. (C) Amperometric spike parameters analyzed in chromaffin cells of wild-type (control), Pld1/, and Pld2/ mice. PLD1 knockout affected foot duration and three spike parameters, namely, spike rise time, spike half-width, and spike amplitude (Imax). Data are expressed as means ± SD (n > 100 cells for each condition from four independent cell cultures; *p < 0.05, **p < 0.01, ***p < 0.001 compared with control). (D) PLD1 activity is required for CgA secretion. Bovine chromaffin cells in culture were treated with the PLD inhibitors FIPI, CAY93 (C93), CAY94 (C94), or vehicle (C) for 1 h before stimulation for 10 min with 59 mM K+. Secretion of CgA was estimated by western blot quantification of the CgA present in the cell incubation medium from resting (R) and stimulated (S) cells. Data are given as the mean values ± SD obtained in three experiments performed on cell cultures (n = 3; **p < 0.01 compared with resting condition).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-CgA Home made (Chasserot-Golaz et al., 1996) Anti-PLD1 Cell Signaling Cat# 3832; RRID:AB_2172256 Anti-PLD2 Cell Signaling Cat# 13904; RRID:AB_2798341 HRP-conjugated anti-mouse and anti-rabbit PerBio Cat# 31430; RRID:AB_228307, 31460; RRID:AB_228341 Anti-SNAP-25 Covance Cat# SMI-81; RRID:AB_2315336 Anti-GFP Abcam Cat# ab290; RRID:AB_303395 Gold particle-conjugated anti-rabbit Aurion N/A Chemicals, Peptides, and Recombinant Proteins FIPI Cayman Chemicals Cat# 13563 CAY10593 Cayman Chemicals Cat# 13206 CAY10594 Cayman Chemicals Cat# 13207 Lipofectamine 2000 Thermo Scientific Cat# 11668019 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate PA(36:1) Avanti Polar Lipids Cat# 840861C 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate PA(36:2) Avanti Polar Lipids Cat# 840862C 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphate PA(40:6) Avanti Polar Lipids Cat# 840864C 1,2-didocosahexaenoyl-sn-glycero-3-phosphate PA(44:12) Avanti Polar Lipids Cat# 840887C L-a-phosphatidic acid (Egg, Chicken) PA mix Avanti Polar Lipids Cat# 840101C Hexamethyldisilazane Sigma Aldrich Cat# 440191 Critical Commercial Assays 3-CAT research ELISA kit Eurobio BA E-5600 Basic Primary Neurons Nucleofector Kit Lonza VVPI-1003 Experimental Models: Cell Lines PC12 Laurent Taupenot (Taupenot et al., 1998) Experimental Models: Organisms/Strains Mouse Pld1 / Bernard Nieswandt (Elvers et al., 2010) Mouse Pld2 / Gilbert Di Paolo (Oliveira et al., 2010) Recombinant DNA Spo20p-GFP N/A (Kassas et al., 2017) PDE4A1-GFP N/A (Kassas et al., 2017) Software and Algorithms Adobe Photoshop Adobe https://www.adobe.com SigmaPlot 13 Systat Software https://systatsoftware.com Igor Pro WaveMetrics http://www.wavemetrics.com/ Macro for amperometry analysis Ricardo Borges http://rborges.webs.ull.es/ Icy BioImage Analysis Lab, Institut Pasteur http://icy.bioimageanalysis.org/ Other Carbon-fiber electrode of 5 mm diameter ALA Scientific CFE-2 e1 Cell Reports 32, 108026, August 18, 2020

Techniques: Control, Knock-Out, Activity Assay, Western Blot, Incubation

Journal: Cell reports

Article Title: Mono- and Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells.

doi: 10.1016/j.celrep.2020.108026

Figure Lengend Snippet: Figure 6. Mono-unsaturated PA Rescues the Number of Docked Granules in PLD1-In- hibited Chromaffin Cells (A) Bovine chromaffin cells were incubated with 750 nM FIPI or vehicle (control) for 1 h to inhibit PLD1. Cells were then stimulated for 5 min with 59 mM K+ or maintained under resting conditions. Electron mi- crographs of plasma membrane sheets from resting or stimulated chromaffin cells treated or not with FIPI are shown. Scale bar, 500 nm. (B) Bovine chromaffin cells were incubated for 15 min with 100 mM of the indicated PA in the presence or absence of 750 nM FIPI, before being stimulated for 5 min with 59 mM K+ or maintained under resting conditions. The number of granules morphologically docked on plasma membrane sheets was quantified manually on random fields of 45 mm2. Data are pre- sented as means ± SEM (n > 60 images from three cell cultures for each condition; ***p < 0.001 compared with control, Mann-Whitney test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-CgA Home made (Chasserot-Golaz et al., 1996) Anti-PLD1 Cell Signaling Cat# 3832; RRID:AB_2172256 Anti-PLD2 Cell Signaling Cat# 13904; RRID:AB_2798341 HRP-conjugated anti-mouse and anti-rabbit PerBio Cat# 31430; RRID:AB_228307, 31460; RRID:AB_228341 Anti-SNAP-25 Covance Cat# SMI-81; RRID:AB_2315336 Anti-GFP Abcam Cat# ab290; RRID:AB_303395 Gold particle-conjugated anti-rabbit Aurion N/A Chemicals, Peptides, and Recombinant Proteins FIPI Cayman Chemicals Cat# 13563 CAY10593 Cayman Chemicals Cat# 13206 CAY10594 Cayman Chemicals Cat# 13207 Lipofectamine 2000 Thermo Scientific Cat# 11668019 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate PA(36:1) Avanti Polar Lipids Cat# 840861C 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate PA(36:2) Avanti Polar Lipids Cat# 840862C 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphate PA(40:6) Avanti Polar Lipids Cat# 840864C 1,2-didocosahexaenoyl-sn-glycero-3-phosphate PA(44:12) Avanti Polar Lipids Cat# 840887C L-a-phosphatidic acid (Egg, Chicken) PA mix Avanti Polar Lipids Cat# 840101C Hexamethyldisilazane Sigma Aldrich Cat# 440191 Critical Commercial Assays 3-CAT research ELISA kit Eurobio BA E-5600 Basic Primary Neurons Nucleofector Kit Lonza VVPI-1003 Experimental Models: Cell Lines PC12 Laurent Taupenot (Taupenot et al., 1998) Experimental Models: Organisms/Strains Mouse Pld1 / Bernard Nieswandt (Elvers et al., 2010) Mouse Pld2 / Gilbert Di Paolo (Oliveira et al., 2010) Recombinant DNA Spo20p-GFP N/A (Kassas et al., 2017) PDE4A1-GFP N/A (Kassas et al., 2017) Software and Algorithms Adobe Photoshop Adobe https://www.adobe.com SigmaPlot 13 Systat Software https://systatsoftware.com Igor Pro WaveMetrics http://www.wavemetrics.com/ Macro for amperometry analysis Ricardo Borges http://rborges.webs.ull.es/ Icy BioImage Analysis Lab, Institut Pasteur http://icy.bioimageanalysis.org/ Other Carbon-fiber electrode of 5 mm diameter ALA Scientific CFE-2 e1 Cell Reports 32, 108026, August 18, 2020

Techniques: Incubation, Control, Clinical Proteomics, Membrane, MANN-WHITNEY