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.