title: Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo.
abstract: Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as 'ectodomain shedding', is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo.
authors.affiliation: Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Aosa
authors.initials: A
authors.lastname: Kamezaki
authors.affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Fuminori
authors.initials: F
authors.lastname: Sato
authors.affiliation: Imaging Platform for Spatio-Temporal Information, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Aoki
authors.affiliation: Division of Molecular and Developmental Biology, National Institute of Genetics, and Department of Genetics, SOKENDAI, Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Asakawa
authors.affiliation: Division of Molecular and Developmental Biology, National Institute of Genetics, and Department of Genetics, SOKENDAI, Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawakami
authors.affiliation: Laboratory of Cell Asymmetry, RIKEN Center of Developmental Biology, Kobe 650-0047, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Matsuzaki
authors.affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Sehara-Fujisawa
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004625:Embryo, Nonmammalian / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D020890:Neuregulin-1 / Q000235:genetics / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D059748:Proteolysis / Q000187:drug effects; D059008:Time-Lapse Imaging / Q000379:methods*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020890:Neuregulin-1; D010703:Phorbol Esters; D000097573:Red Fluorescent Protein; C119222:phorbol-12-myristate