Journal: Molecular Biology of the Cell

Article Title: Calcium influx promotes PLEKHG4B localization to cell–cell junctions and regulates the integrity of junctional actin filaments

doi: 10.1091/mbc.E23-05-0154

Figure Lengend Snippet: MSCs are involved in PLEKHG4B localization to basal membranes and cell-cell junctions. (A) Treatment with Yoda1 or Jedi2 promotes PLEKHG4B localization to cell-cell junctions. MDCK cells were transfected with YFP-PLEKHG4B, cultured for 24 h, and then treated with 10 µM Yoda1, 50 µM Jedi2, or vehicle for 15 min. Cells were fixed and stained with rhodamine-phalloidin and anti-β-catenin antibodies. (B) The x-z images of the cells along a dashed yellow line in (A). (C) Quantification of the percentage of cells with diffuse, central, or cell-cell junctional localization of YFP-PLEKHG4B at the basal plane in Yoda1- or Jedi2-treated cells. (D) Effects of GsMTx4 on PLEKHG4B localization to the basal membrane. MDCK cells were transfected with YFP-PLEKHG4B, cultured for 48 h, and then treated with 10 µM GsMTx4 or vehicle for 15 min. Cells were fixed and stained with rhodamine-phalloidin and anti-β-catenin antibody. (E) Quantification of the percentage of cells with diffuse, central, or cell–cell junctional localization of YFP-PLEKHG4B at the basal plane in GsMTx4-treated and -untreated cells. In (A) and (D), single confocal sections at the basal plane of the cells are shown. Yellow arrowheads indicate the positions of PLEKHG4B localization at cell–cell junctions. Scale bars: 20 µm. In (C) and (E), P values were calculated by ordinary one-way ANOVA followed by Dunnett’s test (C) or two-tailed paired t test (E), with over 25 cells from three independent experiments. *, P < 0.05; ***, P < 0.001.

Article Snippet: Jedi2 (HY-131018) was obtained from MedChem Express.

Techniques: Transfection, Cell Culture, Staining, Membrane, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: Calcium influx promotes PLEKHG4B localization to cell–cell junctions and regulates the integrity of junctional actin filaments

doi: 10.1091/mbc.E23-05-0154

Figure Lengend Snippet: Proposed model for the role of Ca 2+ influx, PI(4,5)P 2 , ANXA2, and MSCs in PLEKHG4B localization. PLEKHG4B localizes to the basal membrane in normal Ca 2+ medium. Ionomycin treatment causes PLEKHG4B accumulation at cell–cell junctions. Ionomycin-induced PLEKHG4B localization at cell–cell junctions is suppressed by treatment with BAPTA-AM, inhibition of its binding to ANXA2 or PI(4,5)P 2 , indicating that Ca 2+ influx and association with ANXA2 and PI(4,5)P 2 are required for junctional localization of PLEKHG4B. Treatment with low Ca 2+ or BAPTA-AM or inhibition of PI(4,5)P 2 binding suppresses PLEKHG4B localization to the basal membrane in normal Ca 2+ medium, indicating that the suitable level of Ca 2+ and PI(4,5)P 2 -binding are required for its basal membrane localization. Yoda1 and Jedi2 promote PLEKHG4B localization to cell–cell junctions and GsMTx4 suppresses its localization to cell–cell junctions and the basal membrane, indicating that MSC-mediated Ca 2+ influx is involved in PLEKHG4B localization to these regions. See text for details.

Article Snippet: Jedi2 (HY-131018) was obtained from MedChem Express.

Techniques: Membrane, Inhibition, Binding Assay

Journal: PLOS Biology

Article Title: Piezo1-mediated spontaneous calcium transients in satellite glia impact dorsal root ganglia development

doi: 10.1371/journal.pbio.3002319

Figure Lengend Snippet: (A) LEFT Depiction of mechanical compression assay where animal is mounted dorsally on inverted spinning disk confocal with a dextran loaded microneedle mounted above the animal. RIGHT image of mechanical compression assay apparatus. (B) Depiction of mechanical compression assay with needle placing force on DRG. (C) Quantification of the percent of DRG responding to mechanical force in animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) (labeled sox10) or expressing Tg(neurod : gal4+myl7); Tg(uas : GCaMP6s) (labeled neurod) and treated with either DMSO or GsMTx4 at both 2 and 3 dpf (sox10 2 dpf DMSO: n = 7 animals, 7 DRG, sox10 2 dpf GsMTx4: n = 5 animals, 5 DRG neurod 2 dpf DMSO: n = 5 animals 5 DRG neurod 2 dpf GsMTx4: n = 4 animals, 4 DRG sox10 3 dpf DMSO: n = 11 animals, 11 DRG sox10 3 dpf GsMTx4: n = 4 animals, 4 DRG neurod 3 dpf DMSO: n = 9 animals, 9 DRG neurod 3 dpf GsMTx4: n = 4 animals, 4 DRG). (D) Confocal image taken of the mechanical compression assay in 2 dpf animal expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) . Images show an inactive time point, a time point with tissue compression, and an active time point in response to tissue compression. Inactive and active DRG marked with an arrow. (E) Quantification of the change in integrated density of fluorescence during each phase of mechanical compression assay of a DRG in a 3 dpf animal treated with either DMSO or GsMTx4. Change in integrated density of fluorescence is scored as time point subtracting the initial time point divided by time point ( Δf / f ). (F) Quantification of the average change in fluorescence during the phases of mechanical compression following treatment with either 2% DMSO or 1 μM GsMTx4 for 30 min (Resting, Compression, and Decompression DMSO: n = 9 DRG, 9 animals, Resting, Compression, and Decompression GsMTx4: n = 4 DRG, 4 animals). (G) Confocal z-projection of peripheral DRG axon in an animal expressing Tg(ngn1 : GFP) at 2 and 3 dpf. Arrow notes the end processes of the peripheral axon. Arrowhead denotes peripheral axons from Rohon beard neurons. (H) Average distance (μM) of DRG displacement needed to elicit a response ( n = 16 animals, 16 DRG). (I) Confocal images of RNAscope- piezo1 and Immunohistochemistry-GFP in Tg(sox10 : meGFP) animals. GFP is shown in magenta and piezo1 is shown in cyan. Arrowheads indicate piezo1 puncta. Arrows indicate autofluorescence. (J) Quantification of DRG at 3 dpf with piezo1 puncta and without piezo1 puncta ( n = 8 animals, 24 DRG). (K) Confocal images of 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) . Red colors indicate a higher intensity of fluorescence and blue colors indicate a lower intensity of fluorescence. Images depicted are of animals either treated with 2% DMSO, 40 μM Jedi2, or 1 μM GsMTx4. Arrows note active cells. (L) Line graphs of z score of integrated density of fluorescence for a 1-h time period in 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) that were treated with either 2% DMSO, 40 μM Jedi2, or 1 μM GsMTx4. A z score greater than 2.58 indicates an active Ca 2+ event. Red scale bar shows a z score of 2.58. (M) Heatmaps of the z score of individual sox10 + cells from animals in G and H during a 1-h period of Ca 2+ imaging. Yellow notes a high z score (2.58 or greater) (DMSO: n = 8 animals, 17 DRG, 52 cells, Jedi2: n = 7 animals, 18 DRG, 79 cells, GsMTx4: n = 6 animals, 16 DRG, 44 cells). (N) Quantification of the average number of Ca 2+ events per sox10 + cell in animals treated with either 2% DMSO, 1 μM GsMTx4, 100 μM Yoda1, or 40 μM Jedi2 (DMSO: n = 8 animals, 17 DRG, 52 cells, Jedi2: n = 7 animals, 18 DRG, 79 cells, GsMTx4: n = 6 animals, 16 DRG, 44 cells, Yoda1: n = 4 animals, 9 DRG, 35 cells). (O) Quantification of the average number of Ca 2+ events per neurod + cell in 3 dpf animals expressing Tg(neurod : gal4+myl7); Tg(uas : GCaMP6s) that were treated with either 2% DMSO, 1 μM GsMTx4, 40 μM Jedi2, or 100 μM Yoda1 (DMSO: n = 10 animals, 24 DRG, 33 cells, Jedi2: n = 5 animals, 18 DRG, 35 cells, GsMTx4: n = 4 animals, 8 DRG, 8 cells, Yoda1: 4 animals, 7 DRG, 8 cells). (P) Quantification of the average number of Ca 2+ events per sox10 + cell following genetic manipulation via injection of uas : cas9mkate-u6 : piezo1gRNA or uas : cas9mkate-u6 : emptygRNA into animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) at 3 dpf. Additionally, a group of Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) injected with uas : cas9mkate-u6 : piezo1gRNA and treated with 40 μM Jedi2 treatment was also included in the experiment (u6:emptygRNA: n = 6 animals, 16 DRG, 40 cells, u6:piezo1gRNA: n = 4 animals, 13 DRG, 57 cells, u6:piezo1gRNA+Jedi2: n = 4 animals, 4 DRG, 7 cells). Scale bar is 10 μM (D, G, I, K). Statistical tests: unpaired t test (H, N, O), Fisher’s exact (C), multiple unpaired t tests (F). The data underlying this figure can be found in . dpf, days post fertilization; DRG, dorsal root ganglia.

Article Snippet: For this study, we used chemical treatments of HMR1556 20 μM (Sigma-Aldrich), Thapsigargin 10 μM (Sigma-Aldrich), Apyrase 10U (Sigma-Aldirch), Carbenoxolone 100 μM (Tocris), GsMTx4 1 μM (Tocris), Yoda1 100 μM (Tocris), and Jedi2 40 μM (Tocris).

Techniques: Expressing, Labeling, Fluorescence, RNAscope, Immunohistochemistry, Imaging, Injection

Journal: PLOS Biology

Article Title: Piezo1-mediated spontaneous calcium transients in satellite glia impact dorsal root ganglia development

doi: 10.1371/journal.pbio.3002319

Figure Lengend Snippet: (A) Confocal z-projection of DRG in 3 dpf animal expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) and treated with either 2% DMSO or 40 μM Jedi2. Individual cells are traced for ROIs and labeled with a number. (B) Line graphs of the z score of the integrated density of fluorescence over a 1 h period. Each numbered line graph corresponds to a numbered ROI. Red scale bar represents a z score of 2.58. Blue arrowheads note simultaneously active time points. Red arrowheads note isolated active time points. (C) Quantification of the average number of isolated Ca 2+ transient events per sox10 + cell in 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) that were treated with either 2% DMSO, 1 μM GsMTx4, 100 μM Yoda1, or 40 μM Jedi2 (DMSO: 4 animals, 5 DRG, 24 cells, GsMTx4: n = 3 animals, 5 DRG, 23 cells, Jedi2: n = 7 animals, 18 DRG, 59 cells, Yoda1: n = 3 animals, 6 DRG, 28 cells). (D) Quantification of the average number of simultaneous Ca 2+ transient events per sox10 + cell in 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) that were treated with either 2% DMSO, 1 μM GsMTx4, 100 μM Yoda1, or 40 μM Jedi2 (DMSO: n = 4 animals, 5 DRG, 24 cells, GsMTx4: n = 3 animals, 5 DRG, 23 cells, Jedi2: n = 7 animals, 18 DRG, 59 cells, Yoda1: n = 3 animals, 6 DRG, 28 cells). (E) Quantification of the average number of isolated Ca 2+ transient events following genetic manipulation via CRISPR/Cas9 targeting piezo1 or empty gRNA cassette in 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) uas : cas9mkate-u6 : piezo1gRNA or uas : cas9mkate-u6 : emptygRNA (u6:emptygRNA: n = 6 animals, 14 DRG, 38 cells, u6:piezo1gRNA: n = 4 animals, 13 DRG, 57 cells). (F) Quantification of the average number of simultaneous Ca 2+ transient events following genetic manipulation via CRISPR/Cas9 targeting piezo1 or empty gRNA cassette in 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) uas : cas9mkate-u6 : piezo1gRNA or uas : cas9mkate-u6 : emptrygRNA (u6:emptygRNA: n = 6 animals, 14 DRG, 38 cells, u6:piezo1gRNA: n = 4 animals, 13 DRG, 57 cells). (G) Quantification of the average number of microdomains per DRG in 3 dpf animals expressing Tg(sox10 : gal4+myl7); uas : GCaMP6s-caax that were treated with either 2% DMSO, 1 μM GsMTx4, or 40 μM Jedi2 for 30 min prior to imaging (DMSO: n = 7 animals, 7 DRG, GsMTx4: n = 7 animals, 7 DRG, Jedi2: n = 6 animals, 8 DRG). (H) Quantification of the average duration of microdomains in 3 dpf animals expressing Tg(sox10 : gal4+myl7); uas : GCaMP6s-caax that were treated with either 2% DMSO or 40 μM Jedi2 for 30 min prior to imaging (DMSO: n = 8 animals, 8 DRG, Jedi2: n = 5 animals, 7 DRG). Scale bar is 10 μM (A). Statistical tests: unpaired t test (C, D, E, F, H), one-way ANOVA followed and represented by post hoc Dunnett test (G). The data underlying this figure can be found in . dpf, days post fertilization; DRG, dorsal root ganglia; ROI, regions of interest.

Article Snippet: For this study, we used chemical treatments of HMR1556 20 μM (Sigma-Aldrich), Thapsigargin 10 μM (Sigma-Aldrich), Apyrase 10U (Sigma-Aldirch), Carbenoxolone 100 μM (Tocris), GsMTx4 1 μM (Tocris), Yoda1 100 μM (Tocris), and Jedi2 40 μM (Tocris).

Techniques: Expressing, Labeling, Fluorescence, Isolation, CRISPR, Imaging

Journal: PLOS Biology

Article Title: Piezo1-mediated spontaneous calcium transients in satellite glia impact dorsal root ganglia development

doi: 10.1371/journal.pbio.3002319

Figure Lengend Snippet: (A) Timeline of experimental process where animals were treated with either 2% DMSO, 1 μM GsMTx4, or 40 μM Jedi2 for 30 min each day at 2 and 3 dpf. (B) Confocal z-projection of DRG in animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) following consecutive days of treatment with either DMSO or Jedi2. ROIs are traced individual cells labeled with a number. Arrows denote active cells. (C) Line graphs of the z score of the integrated density of fluorescence over a 1-h period. Numbers correlate with ROIs in (B). Red line signifies a z score of 2.58. Blue arrowheads note simultaneous active time points. Red arrowheads note isolated active time points. (D–F) Quantification of the average number of isolated (D), simultaneous Ca 2+ activity events per sox10 + cells (E), or average percent of high correlation coefficient (F) per sox10 + cells in 4 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) that were treated at 2 and 3 dpf with DMSO, GsMTx4, or Jedi2 (DMSO: n = 4 animals, 6 DRG, 32 cells, GsMTx4: n = 3 animals, 4 DRG, 22 cells, Jedi2: n = 4 animals, 5 DRG, 25 cells). (G) Timeline of experimental process where animals were treated with either 2% DMSO, 1 μM GsMTx4, 100 μM Yoda1, or 40 μM Jedi2 for 30 min each day at 2–4 dpf. Animals were then fixed and processed for imaging at 5 dpf. (H) Confocal z-projections of 5 dpf DRG in animals expressing Tg(neurod : tagRFP) and stained for Sox10. Magenta displays Tg(neurod : tagRFP) , and cyan displays Sox10. (I, J) Quantification of the average number of neurod + (I) and Sox10 (J) cells in 5 dpf animals treated with either 2% DMSO, 1 μM GsMTx4, 40 μM Jedi2, or 100 μM Yoda1 for 30 min each day from 2–4 dpf (DMSO: n = 15 animals, 53 DRG, GsMTx4: n = 10 animals, 34 DRG, Jedi2: n = 10 animals, 39 DRG, Yoda1: n = 7 animals, 18 DRG). (K) Timeline of experimental process where animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCamp6s); uas : Chr2-tdTomato were exposed to 488 nm light for 30 min each day during targeted developmental period. Animals were fixed, stained, and processed for imaging at 5 dpf. (L) Confocal z-projections of 5 dpf DRG in animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCamp6s); uas : Chr2-tdTomato and stained for HuCD expression. Magenta displays HuCD + neurons. Cyan displays sox10 + satellite glia. (M, N) Quantification of the average number of HuCD + neurons (M) and sox10 + satellite glia (N) in 5 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s); uas : Chr2-tdTomato and either exposed or not exposed to 488 nm light for 30 min each day during development (exposed: n = 4 animals, 9 DRG, not exposed: n = 4 animals, 4 DRG). (O–Q) Quantification of the percent of DRG with cell divisions (O) and/or cell deaths (P) in 24-h time lapses of Tg(sox10 : meGFP); Tg(neurod : tagRFP) animals treated either with 2% DMSO or 40 μM Jedi2 from 2–3 dpf or 2–4 dpf (2 dpf DMSO: 6 animals, 17 DRG, 2 dpf Jedi2: n = 8 animals, 31 DRG, 3 dpf DMSO: n = 8 animals, 24 DRG, 3 dpf Jedi2: n = 8 animals, 24 DRG). (Q) Quantification of the average duration (seconds) of shivering in 5 dpf animals that were treated with 2% DMSO, 1 μM GsMTx4, or 40 μM Jedi2 for 30 min each day from 2–4 dpf (DMSO: n = 12 animals, GsMTx4: n = 11 animals, Jedi2: n = 13 animals). Scale bar is 10 μM (B, H, L). Statistical tests: one-way ANOVA followed and represented by post hoc Dunnett test (D, E, F, I, J), Fisher’s exact: (O, P), unpaired t test: (M, N, Q). The data underlying this figure can be found in . dpf, days post fertilization; DRG, dorsal root ganglia; ROI, regions of interest.

Article Snippet: For this study, we used chemical treatments of HMR1556 20 μM (Sigma-Aldrich), Thapsigargin 10 μM (Sigma-Aldrich), Apyrase 10U (Sigma-Aldirch), Carbenoxolone 100 μM (Tocris), GsMTx4 1 μM (Tocris), Yoda1 100 μM (Tocris), and Jedi2 40 μM (Tocris).

Techniques: Expressing, Labeling, Fluorescence, Isolation, Activity Assay, Imaging, Staining

Journal: Nature Communications

Article Title: A mechanism for the activation of the mechanosensitive Piezo1 channel by the small molecule Yoda1

doi: 10.1038/s41467-019-12501-1

Figure Lengend Snippet: Coupling between chemical activation pathways in Piezo1. ΔPZ1 cells were co-transfected with GC6 and one of the indicated A1718 and A2094 mutant construct or with GC6 only (control). The relative amplitude of calcium signals (Δ F/F 0 ) obtained by application of 100 µM Yoda1 ( a ) or 1 mM Jedi2 ( b ) is shown as a dotted box plot. For statistical analysis, cells from at least three independent experiments were pooled and considered as independent points (n values between 12 and 877). The box upper and lower limits represent standard error of mean values (shown as horizontal inner lines). Source data are provided as a Source Data file. Comparison of the mean values between WT/mutants and control was done using two-tails t -tests. Asterisks indicate standard p -value range. *, 0.01 < p < 0.05; **, 0.00 1 < p < 0.01; ***, p < 0.001 and n.s. (non-significant): p > 0.05. Statistical results are only shown for mutants exhibiting Δ F/F 0 larger than control conditions. For clarity, the number of analyzed cells is from (a) and (b) are plotted as function of the range of Δ F/F 0 values obtained with 100 µM Yoda1 ( c ) or 1 mM Jedi2 ( d ) for each tested mutant (gray rectangle: A1718W, red circles: A1718G, blue triangles: A1718I, green triangles: A1718L, purple diamonds: A1718V, gold triangles: A2094D, cyan triangles: A2094F, brown hexagons: A2094V, olive stars: A2094W, orange pentagons: R2135A, blue spheres: control and green crosses: WT)

Article Snippet: A similar protocol was used for Jedi2 (purchased from MolPort) where the final agonist concentration was 1 mM and the stock concentration was 100 mM (in DMSO).

Techniques: Activation Assay, Transfection, Mutagenesis, Construct, Control, Comparison