Live-Cell Dynamic Sensing of Cd2+ with a FRET-Based Indicator

Abstract

Cd2+ causes damages to several human tissues. Although the toxicological and carcinogenetic mechanisms of Cd2+ 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 Cd2+ indicator, which contains a portion of a Cd2+-binding protein (CadR) obtained from Pseudomonas putida as the Cd2+ 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 Cd2+ concentration within the living HEK-MCD157 cells. The dissociation constant of Met-cad 1.57 was approximately 271 nM. The function of Ca2+ channels as a potential Cd2+ entry gateway was further confirmed in the HEK-MCD157 cells. The organelle-targeted property of the protein-based Cd2+ indicator directly reveals the nucleus accumulation phenomena. In summary, a human kidney cell line that stably expresses the FRET-based Cd2+ indicator Met-cad 1.57 was constructed for reliable and convenient investigations to determine the Cd2+ concentration within living cells, including the identification of the entry pathway of Cd2+ and sub-cellular sequestration. © 2013 Chiu et al.