title: Ca2+release via two-pore channel type 2 (TPC2) is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos.
abstract: We recently demonstrated a critical role for two-pore channel type 2 (TPC2)-mediated Ca2+release during the differentiation of slow (skeletal) muscle cells (SMC) in intact zebrafish embryos, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO). Here, we extend our study and demonstrate that knockdown of TPC2 with a non-overlapping splice-blocking MO, knockout of TPC2 (via the generation of a tpcn2dhkz1amutant line of zebrafish using CRISPR/Cas9 gene-editing), or the pharmacological inhibition of TPC2 action with bafilomycin A1 or trans-ned-19, also lead to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. When the morphants were injected with tpcn2-mRNA or were treated with IP3/BM or caffeine (agonists of the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), respectively), many aspects of myofibrillogenesis and myotomal patterning (and in the case of the pharmacological treatments, the Ca2+signals generated in the SMCs), were rescued. STED super-resolution microscopy revealed a close physical relationship between clusters of RyR in the terminal cisternae of the sarcoplasmic reticulum (SR), and TPC2 in lysosomes, with a mean estimated separation of ~52-87nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca2+release via TPC2 might trigger the generation of more global Ca2+release from the SR via Ca2+-induced Ca2+release.
authors.affiliation: Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, PR China.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Kelu
authors.affiliation: Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, PR China.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Webb
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
authors.forename: John
authors.initials: J
authors.lastname: Parrington
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
authors.forename: Antony
authors.initials: A
authors.lastname: Galione
authors.affiliation: Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, PR China; Marine Biological Laboratory, Woods Hole, MA, USA. Electronic address: almiller@ust.hk.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Miller
mesh_terms: D000818:Animals; D001483:Base Sequence; D001522:Behavior, Animal / Q000187:drug effects; D019521:Body Patterning / Q000187:drug effects; D064113:CRISPR-Cas Systems / Q000235:genetics; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D055786:Gene Knockout Techniques; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D008954:Models, Biological; D060172:Morpholinos / Q000494:pharmacology; D009043:Motor Activity / Q000187:drug effects; D032342:Muscle Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D024510:Muscle Development / Q000187:drug effects; D018657:Muscle Fibers, Slow-Twitch / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012518:Sarcomeres / Q000187:drug effects / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; C499749:Kif7 protein, zebrafish; D018942:Macrolides; D060172:Morpholinos; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel; D029961:Zebrafish Proteins; C000622478:tpcn2 protein, zebrafish; D002110:Caffeine; C040929:bafilomycin A1; D016547:Kinesins; D002118:Calcium
keywords: CRISPR/Cas9; Ca(2+) signaling; Morpholino oligonucleotides; Myofibrillogenesis; STED super-resolution microscopy; Slow skeletal muscle cell differentiation; Two-pore channel 2; Zebrafish