title: Desmocollin-2 alone forms functional desmosomal plaques, with the plaque formation requiring the juxtamembrane region and plakophilins.
abstract: The role of the juxtamembrane region of the desmocollin-2 cytoplasmic domain in desmosome formation was investigated by using gene knockout and reconstitution experiments. When a deletion construct of the desmocollin-2 juxtamembrane region was expressed in HaCaT cells, the mutant protein became localized linearly at the cell-cell boundary, suggesting the involvement of this region in desmosomal plaque formation. Then, desmocollin-2 and desmoglein-2 genes of epithelial DLD-1 cells were ablated by using the CRISPR/Cas9 system. The resultant knockout cells did not form desmosomes, but re-expression of desmocollin-2 in the cells formed desmosomal plaques in the absence of desmoglein-2 and the transfectants showed significant cell adhesion activity. Intriguingly, expression of desmocollin-2 lacking its juxtamembrane region did not form the plaques. The results of an immunoprecipitation and GST-fusion protein pull-down assay suggested the binding of plakophilin-2 and -3 to the region. Ablation of plakophilin-2 and -3 genes resulted in disruption of the plaque-like accumulation and linear localization of desmocollin-2 at intercellular contact sites. These results suggest that the juxtamembrane region of desmocollin-2 and plakophilins are involved in the desmosomal plaque formation, possibly through the interaction between this region and plakophilins.
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Sanda-shi, Hyogo-ken 669-1337, Japan;
authors.forename: Miwako
authors.initials: M
authors.lastname: Fujiwara
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Sanda-shi, Hyogo-ken 669-1337, Japan;
authors.forename: Azusa
authors.initials: A
authors.lastname: Nagatomo
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Sanda-shi, Hyogo-ken 669-1337, Japan;
authors.forename: Megumi
authors.initials: M
authors.lastname: Tsuda
authors.affiliation: Department of Liberal Arts, Kitasato University, Sagamihara-shi, Kanagawa-ken, 252-0373, Japan; Department of Histology and Cell Biology, Yokohama City University School of Medicine, Yokohama-shi, Kanagawa-ken, 236-0004, Japan; and.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Obata
authors.affiliation: Department of Mathematics and Life Science, Graduate School of Science, Hiroshima University, Higashihiroshima-shi, Hiroshima-ken, 739-8511, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Mathematics and Life Science, Graduate School of Science, Hiroshima University, Higashihiroshima-shi, Hiroshima-ken, 739-8511, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Sanda-shi, Hyogo-ken 669-1337, Japan; stsuzuki@kwansei.ac.jp.
authors.forename: Shintaro T
authors.initials: ST
authors.lastname: Suzuki
mesh_terms: D015703:Antigens, CD; D064113:CRISPR-Cas Systems; D015820:Cadherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D051187:Desmocollins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051301:Desmoglein 2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003896:Desmosomes / Q000378:metabolism* / Q000648:ultrastructure; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D017353:Gene Deletion; D006801:Humans; D047468:Immunoprecipitation; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051191:Plakophilins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; C081561:DSC2 protein, human; C495314:DSG2 protein, human; D051187:Desmocollins; D051301:Desmoglein 2; D050505:Mutant Proteins; C495337:PKP2 protein, human; C495339:PKP3 protein, human; D010446:Peptide Fragments; D051191:Plakophilins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
keywords: CRISPR; Cas9; DLD-1 cells; desmocollin-2; desmosome; juxtamembrane domain; knockout; plakophilin