Journal: The Journal of Clinical Investigation

Article Title: OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca 2+ release, exocytosis, and pancreatic tissue damage

doi: 10.1172/JCI169428

Figure Lengend Snippet: ( A ) Model indicating localization of OCaR1 to secretory granules and lysosomes in acinar cells. NAADP-mediated Ca 2+ release via TPC2 channels (blue) is monitored by the activity of adjacent Ca 2+ -activated chloride channels (brown). ( B ) CCK-8–induced (2 pM) Ca 2+ -activated Cl – currents in C57BL/6N and OCaR1 –/– acinar cells in the absence of extracellular Ca 2+ . ( C ) AUC of inward currents over 20 minutes and average amplitude ( n = 16 cells, 5 mice per genotype). ( D ) Model of C-terminal fusion construct of TPC2 and GCaMP6m. ( E ) Representative (17 images, 3 mice) confocal microscopy images of TPC2-GCaMP6m–expressing acinar cells costained with anti-GFP and anti-Rab27B and merged image. Scale bars: 5 μm. ( F ) CCK-8 (2 pM)–induced oscillations in GCaMP6m fluorescence in acinar cells from mice expressing TPC2-GCaMP6m. Acute application of Ned-19 (50 μM) abolishes CCK-8–induced responses ( n = 99). ( G ) Representative traces ( n = 20, from 100 cells per mouse, 5 mice) from CCK-8–evoked global Ca 2+ oscillations in WT acinar cells. ( H ) Representative traces ( n = 20) of CCK-8 (2 pM)–evoked oscillations in GCaMP6m fluorescence in TPC2-GCaMP6m–expressing acinar cells. ( I ) Frequency and duration of Ca 2+ oscillations detected by TPC2-GCaMP6m during stimulation with 2 pM CCK-8 of acinar cells from C57BL/6N ( n = 77 cells, 3 mice) and OCaR1 –/– mice ( n = 135 cells, 4 mice). ( J ) Current densities elicited by coapplication of PI(3,5)P 2 and TPC2-A1-N in vacuolin-enlarged endo-lysosomal vesicles isolated from HEK293 cells overexpressing mTPC2-RFP with or without OCaR1-YFP . Activated currents were blocked with 1 mM ATP (overlapping the basal current) ( n = 4–6). Right: Average current densities at –100 mV. Comparison was done by 2-sample t test ( C and I ) or 1-way ANOVA and Tukey’s post hoc test ( J ) (* P < 0.05, *** P < 0.001). IM, 10 μM ionomycin.

Article Snippet: TPC2-A1-N and TPC2-A1-P were purchased from MedChemExpress.

Techniques: Activity Assay, CCK-8 Assay, Construct, Confocal Microscopy, Expressing, Fluorescence, Isolation, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Two-pore channels regulate endomembrane tension to enable remodeling and resolution of phagolysosomes.

doi: 10.1073/pnas.2309465121

Figure Lengend Snippet: Fig. 2. TPC activity is required for phagosome resolution. (A and B) RAW 264.7 cells expressing TPC1-tdTomato or TPC2-mCherry challenged with Alexa Fluor 488 anti-IgG labeled sRBC for indicated times. xy plane, Left. Extended focus, Left. (Scale bar, 5 µm.) Quantification in (B) for individual phagosomes. n = 3. (C and D) wt and TPC1/2−/− BMDM challenged with sRBC. Resolution assessed up to 4 h. (Scale bar, 10 µm.) Phagosome volume quantified in (D). Dots represent individual fields containing >3 cells, >9 phagosomes. n = 3. (E) As in (C), using 25× magnification. (Scale bar, 25 µm.) (F) wt and TPC1/2−/− BMDM and TPC1/2−/− BMDM expressing wtTPC2-mCherry or TPC2(N257A)-mCherry, quantified as in (D). Dots represent individual phagosomes. >30 cells, >100 phagosomes. n = 2.

Article Snippet: Mammalian expression vectors were described in the following sources: wtTPC1- tdTomato (13), wtTPC2- mCherry (Addgene; plasmid no. 135183), TPC2(N257A)- mCherry (Addgene; plasmid no. 135186), LAMP1- GFP (35), and EEA1- eGFP (77).

Techniques: Activity Assay, Expressing, Labeling

Journal: The International journal of developmental biology

Article Title: Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca 2+ signaling during myogenesis in intact zebrafish

doi: 10.1387/ijdb.150206am

Figure Lengend Snippet: Embryos were fixed between 16 hpf and 24 hpf and then dual immunolabelled with a (Ai-Ii) myosin heavy chain and (Aii–Iii) TPC2 antibody. (F-I) Some embryos were injected with (F) standard control-MO, (G) p53-MO, (H) TPCN2-MO + p53-MO or (I) TPCN2-MO + p53-MO + TPCN2-mRNA between the 1-4-cell stage and then fixed at 24 hpf prior to immunolabeling. (Aiii–Iiii) Line-scan analyses were performed along individual myofibers and (Aiv-Iiv) graphs were plotted to show the development of TPC2 expression over time. The regions bounded by the blue rectangles in panels Ei and Eii are shown at higher magnification in panels Ji and Jii. (Jiii) The myosin heavy chain and TPC2 images when merged. (Jiv) Line-scan analysis showing the localization of TPC2 in relation to that of the myosin heavy chain in more detail. (K) Schematic representation of two sarcomeres. (Li) End view of several myofibers showing the localization of TPC2 in relation to the myosin heavy chain. (Lii) Schematic transverse section through an embryo at 24 hpf showing the location of TPC2 in relation to the SMCs and other cell types (modified from Fig. 1B in Du et al., 1997). Scale bars, 10 μm (A-I); 5 μm (J, L).

Article Snippet: Cells were then incubated sequentially with the 2137A rabbit anti-TPC2 antibody (used at 1:10; custom-made by CovalAb UK Ltd., Cambridge, UK) and either the F59 mouse anti-myosin heavy chain antibody (used at 1:10; Developmental Studies Hybridoma Bank, Iowa, USA) or the 34C mouse anti-ryanodine receptor (RyR) antibody (used at 1:500; Sigma-Aldrich Corp.).

Techniques: Injection, Control, Immunolabeling, Expressing, Modification

Journal: The International journal of developmental biology

Article Title: Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca 2+ signaling during myogenesis in intact zebrafish

doi: 10.1387/ijdb.150206am

Figure Lengend Snippet: (A) Schematic representations of two sarcomeres showing the localization of (Aa) myosin in green and (Ab) TPC2 (in red), RyR (in green) and IP3R (diagonal shading). (B-E, panels a-f) Series of optical sections projected as single images to show the localization of TPC2 with regards to other proteins in the myofibrils or SR. Cells were dual-immunolabeled with: (B) myosin heavy chain and TPC2 antibodies; (C) myosin heavy chain and LAMP1 antibodies; (D) RyR and TPC2 antibodies; or (E) RyR and IP3R type III antibodies. The pattern of localization is shown in cells cultured for either (B-E, panels a-c) ~24 h or (B-E, panels d-f) ~48 h, and for each series of antibodies used, the pattern of localization of each protein is shown both alone (panels a,d and b,e) and when superimposed on the image of the other protein labeled (panels c,f). Scale bars, 1 μm. (B-E, panel g) Line-scan analyses. See key to symbols in Fig. 3 for explanation of yellow dashed lines and the blue and pink arrowheads; white arrowheads indicate TPC2-exclusion zones around the sarcomeric z-line and yellow arrowheads indicate regions of TPC2 and myosin heavy chain overlap. (F,G) Side (panel a) and end (panel b) views of SMCs to show the localization of (F) TPC2 and (G) LAMP1 in relation to the myosin heavy chain. (F,G, panel c) Schematics to illustrate the different views observed.

Article Snippet: Cells were then incubated sequentially with the 2137A rabbit anti-TPC2 antibody (used at 1:10; custom-made by CovalAb UK Ltd., Cambridge, UK) and either the F59 mouse anti-myosin heavy chain antibody (used at 1:10; Developmental Studies Hybridoma Bank, Iowa, USA) or the 34C mouse anti-ryanodine receptor (RyR) antibody (used at 1:500; Sigma-Aldrich Corp.).

Techniques: Immunolabeling, Cell Culture, Labeling

Journal: The International journal of developmental biology

Article Title: Two-Pore Channel 2 activity is required for slow muscle cell-generated Ca 2+ signaling during myogenesis in intact zebrafish

doi: 10.1387/ijdb.150206am

Figure Lengend Snippet: Single optical sections (taken at the widest part of the nucleus) to show the localization of TPC2 and other proteins in the nuclear region of SMCs. Cells were dual-immunolabeled with: (A,B) myosin heavy chain and TPC2 antibodies; (C,D) myosin heavy chain and LAMP1 antibodies; (E,F) RyR and IP3R type III antibodies, (G) myosin heavy chain and IP3R type I antibodies or (H) myosin heavy chain and IP3R type II antibodies, and then counterstained with DAPI to label the nucleus. In panels A-D, G and H the myosin heavy chain antibody was simply used to identify SMCs in the mixed cell culture (and is therefore not shown). In the case of TPC2, LAMP1, IP3R type I and IP3R type II, the localization pattern of these proteins was shown alone (A-D,G,H, panel a) and when superimposed on the image of the nucleus (A-D,G,H, panel c). In the case of RyR and IP3R, the localization pattern of each protein was shown alone (E,F panels a and b, respectively), and when superimposed together and with the image of the nucleus (E,F panel d). Scale bars, 5 μm. (Ad-Dd,Ee,Fe,Gd,Hd) Line-scan analyses. The 0 and 18 in panel Ac indicate the start and end point of the line-scan, which makes up the x-axis of panel Ad.

Article Snippet: Cells were then incubated sequentially with the 2137A rabbit anti-TPC2 antibody (used at 1:10; custom-made by CovalAb UK Ltd., Cambridge, UK) and either the F59 mouse anti-myosin heavy chain antibody (used at 1:10; Developmental Studies Hybridoma Bank, Iowa, USA) or the 34C mouse anti-ryanodine receptor (RyR) antibody (used at 1:500; Sigma-Aldrich Corp.).

Techniques: Immunolabeling, Cell Culture