Journal: bioRxiv

Article Title: Identification and characterization of a synaptic active zone assembly protein

doi: 10.1101/2024.04.08.588536

Figure Lengend Snippet: A, B) Shown are confocal images depicting muscle 4 neuromuscular junctions (NMJs) from third instar larvae of w 1118 labeled with the indicated antibodies. Further details on genotypes can be found in the main text. C) gSTED images of muscle 4 neuromuscular junctions of Blobby-GFP (magenta) animals stained for BRP NC82 (green) and Blobby C-term (blue). Magnified images of individual AZs imaged in planar (I) or vertical (II) orientation. Blobby GFP - BRP NC82 20 ± 2 nm, n=150 AZs, 4 animals; Blobby GFP – Blobby C-Term 14 ± 2 nm, n=47 AZs, 4 animals; Blobby C-Term – BRP NC82 17 ± 2 nm, n=47 AZs, 4 animals. D) Representative confocal images of muscle 4 NMJs from w 111 8 and brp Null mutants stained with the Blobby ex8b antibody (magenta) and RIM-BP C-term (green), individual boxed boutons shown in higher magnifications. E, F) Quantification of overlap between BRP and Blobby ex8b signals in controls and brp Null . E) Plot of Pearsońs coefficients: w 111 8 0.59 ± 0.03, n=31, N=4; brp Null 0.14 ± 0.05, n=31, N=4); F) Plot of Manders coefficients: w 1118 0.5 ± 0.04, n=31, N=4; brp Null 0.35 ± 0.04, n=31, N=4). Graphs show mean ± SEM. ****p<0.0001; **p<0.01 (Kolmogorov-Smirnov test was applied). N: number of animals, n: number of boutons.

Article Snippet: The following primary antibodies were used: mouse anti-BruchpilotNc82/ BRP Cterm (1:100, DSHB, catalog #nc82; RRID:AB_2314866), rabbit-anti BlobbyC-term (1:300, this manuscript), anti-rabbit Blobbyex8b (1:500, this manuscript); guinea pig-anti Unc13A (1:300, this manuscript); rabbit-anti GluRIID (1:500, Qin et al., 2005).

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Identification and characterization of a synaptic active zone assembly protein

doi: 10.1101/2024.04.08.588536

Figure Lengend Snippet: A-J : Confocal imaging analysis comparing larval NMJs of blobby Null animals to controls (A-G) and blobby- STOP - ALFA to ok6>blobby- ALFA (F-J). A, F) Representative confocal images of muscle 4 NMJ images of third instar larvae stained with BRP NC82 (green) and GluRIID (magenta) of indicated genotypes; B-E) and G-J) : Quantification of NMJ morphology and AZ numbers of indicated genotypes. B, G) Normalized NMJ area (in µm 2 ): w 1118 100 ± 8.1, n=16, N=4; blobby Null 74.58 ± 7.26, n=16, N=4; ok6>blobby- ALFA 100 ± 7.9, n=16, N=4; blobby- STOP - ALFA 72.78 ± 7.66, n=15, N=4; unpaired student t-test); C, H) Number of AZs identified as discrete BRP positive clusters per NMJ, values normalized to wild type: w 1118 100 % ± 9,6 % n=16, N=4; blobby Null : 70.7 % ± 9.4 %, n=16, N=4; ok6>blobby- ALFA: 100 % ± 9.49 %, n=15, N=4; blobby- STOP - ALFA 74.29 % ± 6.78 %, n=14, N=4, unpaired student t-test. D, I) Number of AZs normalized to NMJ area from projected images, values normalized to respective controls: w 1118 100 ± 5.71, n=16, N=4; blobby Null 92.35 ± 6.89, n=17, N=4; ok6>blobby- ALFA 100 ± 4.35, n=15, N=4; blobby- STOP - ALFA 113.7 ± 7.7, n=16, N=4, unpaired student t-test) E, J) Quantification of ectopically distributed BRP signal as BRP signal not apposed to GluRIID signals in projected confocal images; blobby Null in comparison to control: w1 118 0.7% ± 0.07%, n=32, N=5; blobby Null 11.2% ± 1.7%, n=38, N=5); ok6>blobby- ALFA 0.59% ± 0.1%, n=15, N=4; blobby- STOP - ALFA 3.5% ± 1.2%, n=15, N=4, Kolmogorov-Smirnov test). Graphs show mean ± SEM. ****p<0.0001; *p<0.05. N: number of animals, n: number of NMJs analyzed. K-L) Representative time-gated (g)STED images highlighting BRP NC82 -positive aggregates (“blobs”) of blobby Null mutants in synaptic boutons stained with (K) BRP NC82 (green) showing apposed signal of postsynaptic GluRIID (magenta) and (L) BRP NC82 (green) and RIM-BP C-term (magenta) indicating co-labeling of both epitopes in the blobs.

Article Snippet: The following primary antibodies were used: mouse anti-BruchpilotNc82/ BRP Cterm (1:100, DSHB, catalog #nc82; RRID:AB_2314866), rabbit-anti BlobbyC-term (1:300, this manuscript), anti-rabbit Blobbyex8b (1:500, this manuscript); guinea pig-anti Unc13A (1:300, this manuscript); rabbit-anti GluRIID (1:500, Qin et al., 2005).

Techniques: Imaging, Staining, Comparison, Control, Labeling

Journal: Neural Development

Article Title: Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation

doi: 10.1186/1749-8104-7-11

Figure Lengend Snippet: Panx1 is expressed in nestin-positive periventricular NSC/NPCs throughout postnatal development in vivo. ( A ) Mouse brain schematic with the neurogenic ventricular zone (VZ) and subgranular zone (SGZ) regions highlighted ( i ). Cell types throughout neurogenesis and the lineage markers expressed at each stage. Relatively quiescent NSCs become highly proliferative NPCs, and neuronally committed migratory neuroblasts before exiting the cell cycle as immature neurons ( ii ). Schematic of cell types in the periventricular region. The ependymal cell layer is interdigitated by thin processes or whole luminal surfaces of the radial glia-like NSCs ( iii ). This figure is a partial reproduction of another figure created by the same authors that appears in and is reproduced with permission. ( B ) Western blot of lysates from HEK293T cells overexpressing EGFP, Panx1EGFP, or Panx1, showing specificity of the Panx1 Cterm antibody. ( C ) Confocal images of murine brain slices at the periventricular region. Nestin-positive NSCs exhibit high levels of Panx1 co-expression in postnatal day 15 (P15) ( i ), P30 ( ii ) and P60 ( iii ) brains. Arrows indicate areas of co-expression. ( D ) Image from a confocal z-stack with orthogonal side-views of a P30 periventricular region. Panx1 expression is noted in ependymal/sub-ependymal layers. ( E ) Confocal image from a P15 periventricular region. Panx1 is absent from DCX-positive neuroblasts. Hoechst 33342 was used as a nuclear counterstain in C-E. All scalebars 10 μm. DCX, doublecortin; NPC, neural progenitor cells; NSC, neural stem cells; Panx1, pannexin 1; V, ventricle.

Article Snippet: Primary antibodies were: rabbit polyclonal anti-Panx1 Cterm (1:40 Invitrogen), rat monoclonal anti-GFAP (1:40, Invitrogen/Life Technologies, Burlington, Ontario, Canada), mouse monoclonal anti-nestin (1:100, Chemicon/Millipore, Temecula, California, USA), guinea pig polyclonal anti-doublecortin (DCX; 1:800, Chemicon/Millipore), mouse monoclonal anti-Tuj1 class III β-tubulin (1:250, RDI).

Techniques: In Vivo, Western Blot, Expressing

Journal: Neural Development

Article Title: Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation

doi: 10.1186/1749-8104-7-11

Figure Lengend Snippet: Panx1 blockage decreases ATP release from N2a cells and blocking P2 receptors reduces cell proliferation. ( A ) Panx1 is endogenously expressed in N2a cells, as demonstrated by RT-PCR ( i ) and western blot analysis ( ii ). To the left of the western blot, the asterisk (*) above the arrow denotes high-molecular weight species likely representing various Panx1 oligomeric forms, while the asterisk below the arrow denotes low molecular weight Panx1 cleavage products consistent in size with those reported in the literature . ( B ) ATP release from N2a cells was stimulated upon treatment with 20 mM KCl buffer compared to control (5.33 mM) and 0 mM KCl conditions ((**) P < 0.01 for one-way ANOVA with post-hoc Tukey’s multiple comparison test, N = 3). ( C ) Blockage of Panx1 channels with 0.5 mM probenecid significantly decreases stimulated ATP release from N2a cells compared to controls (60.50 ± 7.913% and 99.17 ± 6.831%, respectively, N = 5, P = 0.0159 for non-parametric t test). ( D ) N2a cell numbers were quantified at 0, 24 and 48 hours for cells treated with 30 μM PPADS, 1 mM probenecid, or vehicle control. PPADS and probenecid treatments significantly reduced N2a numbers at 24 and 48 hours ((*) P < 0.05 and (***) P <0.001 for one-way ANOVA with post-hoc Tukey’s multiple comparison test, N = 3). ANOVA, analysis of variance; Panx, pannexin1, PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid.

Article Snippet: Primary antibodies were: rabbit polyclonal anti-Panx1 Cterm (1:40 Invitrogen), rat monoclonal anti-GFAP (1:40, Invitrogen/Life Technologies, Burlington, Ontario, Canada), mouse monoclonal anti-nestin (1:100, Chemicon/Millipore, Temecula, California, USA), guinea pig polyclonal anti-doublecortin (DCX; 1:800, Chemicon/Millipore), mouse monoclonal anti-Tuj1 class III β-tubulin (1:250, RDI).

Techniques: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Molecular Weight

Journal: Neural Development

Article Title: Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation

doi: 10.1186/1749-8104-7-11

Figure Lengend Snippet: Panx1 regulates N2a cell proliferation. ( A ) Image from a confocal z-stack with orthogonal side-views of N2a cells overexpressing Panx1EGFP. Panx1 is highly localized to the plasma membrane, stained with wheat germ agglutinin (WGA), as well as to intracellular membranes. ( B ) N2a cells overexpressing Panx1 exhibited a reduced doubling time (14.7 hours) compared to control cells overexpressing EGFP (26.2 hours) ((*) P < 0.05 and (**) P < 0.01 for one-way ANOVA with post-hoc Tukey’s multiple comparison test, N = 3). ( C ) Treatment of transfected N2a cells with the Panx1 channel blocker, probenecid (1 mM), significantly reduced cell proliferation at 48 hours in both Panx1 and EGFP overexpressing cells ((**) P < 0.01 and (***) P < 0.001 for one-way ANOVA with post-hoc Tukey’s multiple comparison test). ANOVA, analysis of variance; Panx1, pannexin1.

Article Snippet: Primary antibodies were: rabbit polyclonal anti-Panx1 Cterm (1:40 Invitrogen), rat monoclonal anti-GFAP (1:40, Invitrogen/Life Technologies, Burlington, Ontario, Canada), mouse monoclonal anti-nestin (1:100, Chemicon/Millipore, Temecula, California, USA), guinea pig polyclonal anti-doublecortin (DCX; 1:800, Chemicon/Millipore), mouse monoclonal anti-Tuj1 class III β-tubulin (1:250, RDI).

Techniques: Staining, Transfection

Journal: Neural Development

Article Title: Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation

doi: 10.1186/1749-8104-7-11

Figure Lengend Snippet: Panx1 expression in NSC/NPCs is re-capitulated in vitro in neurosphere cultures. ( A ) Outline of neurosphere culture generation from P0 to P3 hippocampus or microdissected VZ. Spheres are cultured for seven days in vitro (DIV) with addition of growth factors (hEGF and bFGF) every two DIV, before harvesting for subsequent analyses, or differentiation by removal of growth factors and plating on a poly-D-lysine coated surface. ( B ) Panx1 mRNA ( i ) and protein ( ii ) are expressed in VZ and SGZ neurospheres. To the left of the western blot, the asterisk (*) above the arrow denotes high-molecular weight species likely representing various Panx1 oligomeric forms, while the asterisk below the arrow denotes low molecular weight Panx1 cleavage products consistent in size with those reported in the literature . ( C ) Confocal images showing Panx1 expression in a cryosectioned undifferentiated VZ neurosphere ( i ) and a digitally zoomed z-stack with orthogonal side-views ( ii ) in nestin-positive/GFAP-positive and nestin-positive/GFAP-negative cells. ( D ) Image from a confocal z-stack with orthogonal side-views of Panx1 expression in DCX-negative cells in a differentiated VZ neurosphere. ( E ) Image from a confocal z-stack with orthogonal side-views showing high levels of Panx1 co-expression with Tuj-1 in immature neurons from a differentiated VZ neurosphere. Hoechst 33342 was used as a nuclear counterstain in C-E. Scalebars 10 μm. DCX, doublecortin; GFAP, glial fibrillary acidic protein; NPC, neural progenitor cells; NSC, neural stem cells; Panx1, pannexin1; SGZ, subgranular zone; VZ, ventricular zone.

Article Snippet: Primary antibodies were: rabbit polyclonal anti-Panx1 Cterm (1:40 Invitrogen), rat monoclonal anti-GFAP (1:40, Invitrogen/Life Technologies, Burlington, Ontario, Canada), mouse monoclonal anti-nestin (1:100, Chemicon/Millipore, Temecula, California, USA), guinea pig polyclonal anti-doublecortin (DCX; 1:800, Chemicon/Millipore), mouse monoclonal anti-Tuj1 class III β-tubulin (1:250, RDI).

Techniques: Expressing, In Vitro, Cell Culture, Western Blot, Molecular Weight

Journal: Neural Development

Article Title: Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation

doi: 10.1186/1749-8104-7-11

Figure Lengend Snippet: Panx1 regulates primary NSC/NPC proliferation. ( A ) Outline of neurosphere treatments in vitro . Growth factors (hEGF and bFGF) were added every two DIV beginning at DIV0, while the Panx1 blocker probenecid was added at DIV1 to a concentration of 1 mM. Sphere diameter was measured at DIV7. ( B ) Brightfield images of vehicle and probenecid treated spheres ( i ). A significant reduction in neurosphere diameter was observed following probenecid treatment compared to vehicle treatment (41.85 ± 1.649 μm and 93.97 ± 5.089 μm respectively, P < 0.0001 for non-parametric t test, N = 12) ( ii ). DIV, days in vitro ; NPC, neural progenitor cells; NSC, neural stem cells; Panx1, pannexin 1.

Article Snippet: Primary antibodies were: rabbit polyclonal anti-Panx1 Cterm (1:40 Invitrogen), rat monoclonal anti-GFAP (1:40, Invitrogen/Life Technologies, Burlington, Ontario, Canada), mouse monoclonal anti-nestin (1:100, Chemicon/Millipore, Temecula, California, USA), guinea pig polyclonal anti-doublecortin (DCX; 1:800, Chemicon/Millipore), mouse monoclonal anti-Tuj1 class III β-tubulin (1:250, RDI).

Techniques: In Vitro, Concentration Assay