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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 tpcn2dhkz1a mutant 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.

Original publication

DOI

10.1016/j.ydbio.2017.03.031

Type

Journal article

Journal

Dev Biol

Publication Date

15/05/2017

Volume

425

Pages

109 - 129

Keywords

CRISPR/Cas9, Ca(2+) signaling, Morpholino oligonucleotides, Myofibrillogenesis, STED super-resolution microscopy, Slow skeletal muscle cell differentiation, Two-pore channel 2, Zebrafish, Animals, Base Sequence, Behavior, Animal, Body Patterning, CRISPR-Cas Systems, Caffeine, Calcium, Calcium Channels, Calcium Signaling, Cell Death, Cells, Cultured, Embryo, Nonmammalian, Gene Knockdown Techniques, Gene Knockout Techniques, Inositol 1,4,5-Trisphosphate Receptors, Kinesin, Macrolides, Models, Biological, Morpholinos, Motor Activity, Muscle Cells, Muscle Development, Muscle Fibers, Slow-Twitch, Phenotype, RNA, Messenger, Ryanodine Receptor Calcium Release Channel, Sarcomeres, Zebrafish, Zebrafish Proteins