Journal: The Journal of biological chemistry

Article Title: OCA7 is a melanosome membrane protein that defines pigmentation by regulating early stages of melanosome biogenesis.

doi: 10.1016/j.jbc.2022.102669

Figure Lengend Snippet: Figure 1. OCA7 localizes to melanosomes. A, spinning disc confocal fluorescence microscopy analysis of live primary human melanocytes coexpressing OCA7-EGFP with mCherry-Rab32 or RFP-SKL. The scale bars indicate 10 μm for unmagnified images and 1 μm for magnified insets. B, Pearson correlation coefficient (PCC) for images from (A). Error bars represent the 95% confidence interval (mCherry-Rab32, n = 55 cells; RFP-SKL, n = 34 cells). C, colocalization quantification as determined by PCC for images of MNT1 cells expressing OCA7-EGFP with organelle markers from Fig. S1, showing median with error bars representing the 95% confidence interval. n = 50, n = 30, n = 16, n = 45, n = 24, n = 13, n = 29, n = 33, n = 31, n = 33, n = 25, and n = 32 cells for TPC2-iRFP, mCherry-Rab32, mCherry-Rab38, mCherry-CD63, tyrosinase-mCherry, tyrosinase-P1-mCherry, mCherry-Rab27a, mCherry-Rab7a, mCherry-Rab11a, mCherry- Rab5a, BFP2-KDEL, and RFP-SKL, respectively. D, live cell Airyscan super-resolution and brightfield images of MNT1 melanocytes expressing OCA7-EGFP showing a cohort of OCA7 localizes to membranes surrounding melanin pigment. The scale bars indicate 10 μm for unmagnified image and 1 μm for magnified inset. EGFP, enhanced GFP; iRFP, infrared fluorescent protein; OCA7, oculocutaneous albinism type 7; RFP, red fluorescent protein.

Article Snippet: To generate TPC2BioID2-HA, TPC2 was amplified from pEGFP-N3-TPC2 (Addgene #80153) and inserted into MCS-Linker-BioID2-HA (gifted from Kyle Roux, Addgene #74224) linearized by inverse PCR.

Techniques: Microscopy, Expressing

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

Journal: Journal of Virology

Article Title: Neuromasts and Olfactory Organs of Zebrafish Larvae Represent Possible Sites of SARS-CoV-2 Pseudovirus Host Cell Entry

doi: 10.1128/jvi.01418-22

Figure Lengend Snippet: Effect of pharmacologically mediated inhibition of TPC2 activity or CRISPR/Cas9-mediated tpcn2 knockout on PEG-pseudovirus entry in zebrafish larvae. (A) (a and b) Immunolabeling analysis of TPC2 localization in the head at 3 dpf and 5 dpf (viewed from a dorsal orientation) showing distinct expression in the olfactory organ (black arrowheads), neuromasts (white arrowheads), and epiphysis (brown arrowheads). (c) Schematic showing a dorsal view of the head of a zebrafish larva at 5 dpf indicating the position of the olfactory organs (OO), nasal (N) and supraorbital (SO1 to SO3) neuromasts, and epiphysis. (d and e) Surface plots showing the fluorescence intensity in neuromast SO2 and the olfactory organ, respectively. Bar, 100 μm. (B) RT-PCR analysis of cDNA obtained at 2 dpf from intact wild-type embryos that were pretreated with naringenin or DMSO (control) prior to exposure to live PEG-pseudovirus for 48 h. (a) Representative electrophoresis gel showing luc expression and (b) quantification of the relative expression of luc in the naringenin group compared to the DMSO group. (C) RT-PCR analysis of cDNA obtained from intact wild-type and tpcn2 dhkz1a embryos, which were exposed to live PEG-pseudovirus at 2 dpf for 48 h. (a) Representative electrophoresis gel showing luc expression and (b) quantification of the relative expression of luc in the tpcn2 mutant compared to the wild-type control. (D) Representative electrophoresis gel of luc expression from RT-PCR analysis of cDNA extracted from the excised heads alone of wild-type and tpcn2 dhkz1a embryos that had been exposed to live PEG-pseudovirus at 2 dpf for 48 h.

Article Snippet: They were then incubated overnight at 4°C with an anti-ACE2 antibody (PA5-86636; Thermo Fisher Scientific) or an anti-TPC2 antibody (A17271; ABclonal Inc., Woburn, MA, USA), both of which were diluted 1:50 with blocking buffer.

Techniques: Inhibition, Activity Assay, CRISPR, Knock-Out, Immunolabeling, Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Mutagenesis

Journal: Journal of Virology

Article Title: Neuromasts and Olfactory Organs of Zebrafish Larvae Represent Possible Sites of SARS-CoV-2 Pseudovirus Host Cell Entry

doi: 10.1128/jvi.01418-22

Figure Lengend Snippet: Schematic illustrations to show the suggested mechanism of PEG-pseudovirus entry into the anterior neuromasts of zebrafish larvae. (A) Structure of a neuromast. The regions bounded by the black and red rectangles are shown at higher magnification in panels B and C, respectively. (B) Proposed endocytic pathway illustrating the localization of TPCs (both TPC1 and TPC2) on different endolysosomal compartments and the location of bafilomycin A1 inhibition. (C) Proposed endocytic mechanism in the anterior neuromasts of (a) DMSO-treated (control) and (b) bafilomycin A1-treated larvae, after incubation with Alexa Fluor 488-dextran (green). Panel A was modified from reference with permission of Elsevier, and panel B was modified from reference with permission of the Federation of European Biochemical Societies.

Article Snippet: They were then incubated overnight at 4°C with an anti-ACE2 antibody (PA5-86636; Thermo Fisher Scientific) or an anti-TPC2 antibody (A17271; ABclonal Inc., Woburn, MA, USA), both of which were diluted 1:50 with blocking buffer.

Techniques: Inhibition, Incubation, Modification