TY - JOUR
T1 - A Luminescent Cyclometalated Iridium(III) Complex Accumulates in Mitochondria and Induces Mitochondrial Shortening by Conjugation to Specific Protein Targets
AU - Wang, Baojiang
AU - Liang, Yimin
AU - Dong, Hongjuan
AU - Tan, Tianfeng
AU - Zhan, Bao
AU - Cheng, Jinping
AU - Lo, Kenneth Kam Wing
AU - Lam, Yun Wah
AU - Cheng, Shuk Han
PY - 2012/12/21
Y1 - 2012/12/21
N2 - We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)2(phen-ITC)](PF6) (Ir-ITC; Hpq=2-phenylquinoline, phen-ITC=5-isothiocyanate-1,10-phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir-ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir-ITC with live-cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two-dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage-dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir-ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.
AB - We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)2(phen-ITC)](PF6) (Ir-ITC; Hpq=2-phenylquinoline, phen-ITC=5-isothiocyanate-1,10-phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir-ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir-ITC with live-cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two-dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage-dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir-ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.
KW - 2DGE
KW - Fluorescent probes
KW - Iridium
KW - Luminescence
KW - Mitochondrial fission
UR - http://www.scopus.com/inward/record.url?scp=84871247176&partnerID=8YFLogxK
U2 - 10.1002/cbic.201200517
DO - 10.1002/cbic.201200517
M3 - Article
C2 - 23148033
AN - SCOPUS:84871247176
VL - 13
SP - 2729
EP - 2737
JO - ChemBioChem
JF - ChemBioChem
SN - 1439-4227
IS - 18
ER -