title: Live-cell dynamic sensing of Cd(2+) with a FRET-based indicator.
abstract: Cd(2+) causes damages to several human tissues. Although the toxicological and carcinogenetic mechanisms of Cd(2+) have been previously established, some basic questions on this toxicant remain unclear. In this study, we constructed Met-cad 1.57, a new fluorescent resonance energy transfer (FRET)-based Cd(2+) indicator, which contains a portion of a Cd(2+)-binding protein (CadR) obtained from Pseudomonas putida as the Cd(2+) sensing key. We produced a human embryonic kidney cell line HEK-MCD157 which stably expresses the Met-cad 1.57 for further investigations. Both fluorescence spectroscopy and FRET microscopic ratio imaging were used to monitor the Cd(2+) concentration within the living HEK-MCD157 cells. The dissociation constant of Met-cad 1.57 was approximately 271 nM. The function of Ca(2+) channels as a potential Cd(2+) entry gateway was further confirmed in the HEK-MCD157 cells. The organelle-targeted property of the protein-based Cd(2+) indicator directly reveals the nucleus accumulation phenomena. In summary, a human kidney cell line that stably expresses the FRET-based Cd(2+) indicator Met-cad 1.57 was constructed for reliable and convenient investigations to determine the Cd(2+) concentration within living cells, including the identification of the entry pathway of Cd(2+) and sub-cellular sequestration.
authors.affiliation: Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, Republic of China.
authors.forename: Tai-Yu
authors.initials: TY
authors.lastname: Chiu
authors.forename: Po-Hsun
authors.initials: PH
authors.lastname: Chen
authors.forename: Cha-Ling
authors.initials: CL
authors.lastname: Chang
authors.forename: De-Ming
authors.initials: DM
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002104:Cadmium / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C432939:CadR protein, Pseudomonas; D014157:Transcription Factors; D002104:Cadmium