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: To create uas : cas9mkate-u6 : piezo1gRNA , we recombined a p5E-UAS (BseRI cleavage site removed), pME-cas9mkate (Addgene 109547), and p3E-pA constructs into a pDestTol2CG2 containing a U6 promoter sequence flanked with BseRI cleavage sites [ , ].

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: To create uas : cas9mkate-u6 : piezo1gRNA , we recombined a p5E-UAS (BseRI cleavage site removed), pME-cas9mkate (Addgene 109547), and p3E-pA constructs into a pDestTol2CG2 containing a U6 promoter sequence flanked with BseRI cleavage sites [ , ].

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

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: Effects of LRRC37B overexpression on functional and synaptic properties of mouse CPNs, related to Figure 2 (A) Immunodetection of LRRC37B and ankyrin-G in the mouse cerebral (barrel) cortex after transfection of EGFP only or bicistronic expression of EGFP and human(h)LRRC37B-HA or chimpanzee(c)LRRC37B-HA cDNAs (arrows at the AIS). (B) Human (n = 62 from 9 animals from 3 litters) and chimpanzee (n = 18 neurons from 6 animals from 1 litter) LRRC37B colocalizes with ankyrin-G in mouse neurons transfected for LRRC37B and EGFP; note that in next panels, LRRC37B is for human LRRC37B (mean + SEM). (C) Corresponding quantification of the AIS length and position of mouse neurons transfected for LRRC37B (n = 62 from 9 animals from 3 litters) compared with control neurons (n = 59 from 9 animals from 3 litters) (B, mean + SEM; C, lines at median; Mann-Whitney tests). (D) Immunodetection of LRRC37A2-HIS in the mouse cerebral cortex after transfection of LRRC37A2-HIS and EGFP cDNAs. (E) Intrinsic properties of LRRC37B neurons (36 neurons from 12 animals from 4 litters) compared with control neurons (18 neurons from 7 animals from 4 litters) complementary to Figures 2 B–2F (lines at median; Mann-Whitney tests). (F) Phase plot analysis of single evoked AP from control versus LRRC37B transfected neurons complementary to Figure 2 F (see in ). (G) Phase plot analysis of single evoked AP of LRRC37B neurons (n = 10 neurons from 2 animals from 1 litter) and control neurons (n = 9 neurons from 2 animals from 1 litter) (see in ) (lines at median; Mann-Whitney tests). (H) Phase plot analysis of single evoked AP of LRRC37B neurons (n = 48 neurons from 16 animals from 8 litters) and control neurons (n = 25 neurons from 10 animals from 8 litters) (see in ) (lines at median; Mann-Whitney tests). (I) IV-curves (left, ionic currents) and maximum currents (right) of LRRC37B transfected neurons (n = 43 neurons) compared with of control neurons (n = 26 neurons) (left, mean + SEM; right, lines at median; Mann-Whitney tests). (J and K) Quantification of VGAT puncta, gephyrin-tdTomato puncta, and VGAT/gephyrin-tdTomato puncta in mouse neurons in utero electroporated for plasmids leading to a bicistronic expression of LRRC37B and EGFP (40 neurons for VGAT, 33 neurons for gephyrin quantifications, from 11 animals from 4 litters) or EGFP only (30 neurons for VGAT, 26 for gephyrin quantifications, from 8 animals from 3 litters) as well as gephyrin-tdTomato expression (lines at median; Mann-Whitney tests). (L) Excitatory and inhibitory postsynaptic potentials (E/I PSP) frequency and amplitude in LRRC37B transfected mouse neurons (10 neurons from 4 animals from 2 litters) versus control neurons (8 neurons from 4 animals from 2 litters) (lines at median; Mann-Whitney tests). ns, non-significant; ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: All constructs were verified by DNA sequencing. p-SCN1B-FLAG plasmid originates from Origene (RC209565); pSCN8A plasmid originates from Addgene (#162280) and previously described in DeKeyser et al.. pCAG-cre is a gift from Franck Polleux laboratory (United States) previously described in Hand et al.. pCAG-GEPH.FingR-tdTomato-IL2RGTC is a gift from Juan Burrone (United Kingdom), derived from pCAG_GPHN.FingR-mKate2-IL2RGTC (Addgene #46297) previously described Gross et al.. pdisplay-GPR158_ECTO, pdiplsay-SLIRTK2_ECTO and pdisplay-LRRTM1_ECTO were previously described., pCD4_ECTO is a gift from Luís Ribeiro (Joris de Wit’s laboratory), with the cDNA of the predicted extracellular domain of CD4, originating from pdisplay-CD4 (Addgene #51604) previously described, inserted into the pDisplay™ Mammalian Expression Vector (ThermoFisher V66020).

Techniques: Over Expression, Functional Assay, Immunodetection, Transfection, Expressing, MANN-WHITNEY, In Utero

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet:

Article Snippet: All constructs were verified by DNA sequencing. p-SCN1B-FLAG plasmid originates from Origene (RC209565); pSCN8A plasmid originates from Addgene (#162280) and previously described in DeKeyser et al.. pCAG-cre is a gift from Franck Polleux laboratory (United States) previously described in Hand et al.. pCAG-GEPH.FingR-tdTomato-IL2RGTC is a gift from Juan Burrone (United Kingdom), derived from pCAG_GPHN.FingR-mKate2-IL2RGTC (Addgene #46297) previously described Gross et al.. pdisplay-GPR158_ECTO, pdiplsay-SLIRTK2_ECTO and pdisplay-LRRTM1_ECTO were previously described., pCD4_ECTO is a gift from Luís Ribeiro (Joris de Wit’s laboratory), with the cDNA of the predicted extracellular domain of CD4, originating from pdisplay-CD4 (Addgene #51604) previously described, inserted into the pDisplay™ Mammalian Expression Vector (ThermoFisher V66020).

Techniques: Recombinant, Plasmid Preparation, Magnetic Beads, Expressing, Comparison, Software

Journal: Nature Communications

Article Title: ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

doi: 10.1038/s41467-018-07586-z

Figure Lengend Snippet: ATP- and curvature-sensitive membrane binding of EHD1. a Schematic of generation of membrane templates used in the study. b Distribution of tube radii from five independent preparations of templates. c Representative fluorescence micrographs showing distribution of EGFP-LactC2 on SLBs and tubes. Asteriks mark the bare glass surface. Scale bars = 5 μm. d Protein:membrane fluorescence ratios of EHD1-EGFP with various nucleotides (blue) and GFP-LactC2 (red) for the indicated numbers of tubes and SLBs sampled. Black lines denote means. Statistical significance was assessed using Mann–Whitney test and **** P < 0.0001. e Protein:membrane fluorescence ratios of AMP-PNP-bound EHD1-EGFP on tubes of varying starting sizes for the indicated numbers of tubes. Red lines denote means. Statistical significance was assessed using Mann–Whitney’s two-tailed test and * P = 0.010, ** P = 0.005, **** P < 0.0001

Article Snippet: LactC2 was a gift from Sergio Grinstein (Addgene plasmid # 22852).

Techniques: Membrane, Binding Assay, Fluorescence, MANN-WHITNEY, Two Tailed Test