title: Dynamic regulation of a cell adhesion protein complex including CADM1 by combinatorial analysis of FRAP with exponential curve-fitting.
abstract: Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10 min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation.
authors.affiliation: Division of Molecular Pathology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Sakurai-Yageta
authors.affiliation: Division of Molecular Pathology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Maruyama
authors.affiliation: Japan Science and Technology Agency, CREST, 4-5-3, Yonbancho, Chiyoda-ku, Tokyo, 102-8666, Japan; The Division of Mathematical Science, Graduate School of Engineering Science, Osaka University, 1-3, Machikaneyama-cho, Toyonaka, Osaka, 560-8531, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Division of Mathematical Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan; Japan Science and Technology Agency, CREST, 4-5-3, Yonbancho, Chiyoda-ku, Tokyo, 102-8666, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Ichikawa
authors.affiliation: Division of Molecular Pathology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan; Japan Science and Technology Agency, CREST, 4-5-3, Yonbancho, Chiyoda-ku, Tokyo, 102-8666, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Murakami
mesh_terms: D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D007136:Immunoglobulins / Q000378:metabolism*; D007700:Kinetics; D061985:Madin Darby Canine Kidney Cells; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009068:Movement; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D055550:Protein Stability; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: C403514:CADM1 protein, human; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; C116869:DLG3 protein, human; C116887:EPB41L3 protein, human; D007136:Immunoglobulins; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors