A Luminescent Cyclometalated Iridium(III) Complex Accumulates in Mitochondria and Induces Mitochondrial Shortening by Conjugation to Specific Protein Targets

Abstract

We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)(2)(phen-ITC)](PF(6)) (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.

Mitochondria-targeting cyclometalated iridium(III)–PEG complexes with tunable photodynamic activity

“…Thus, the complex is electrostatically attracted to the mitochondrial interior membrane as a result of the greater transmembrane potential. In line with these results, we have also recently observed similar mitochondrial-staining properties of a related iridium(III) complex as revealed by proteomic analyses [52].…”

Dinuclear iridium(iii) complexes as phosphorescent trackers to monitor mitochondrial dynamics

“…4-6 Several evidence suggest that dysfunctions of mitochondrial dynamics are engaged in several diseases, such as Parkinson's disease, Alzheimer's disease, neurodegeneration, multiple sclerosis and cancer. [7][8][9][10] Although the concept that bolstering mitochondrial health and/or replacing dysfunctional 30 and potentially toxic mitochondria with functional mitochondria to confer diseases is attracted, the current state of knowledge related to mitochondria as a dynamic organelle is nascent. In addition, tools are currently too limited to satisfactorily track the dynamics.…”

Synthesis and characterization of green-to-yellow emissive Ir(III) complexes of pyridylbenzothiadiazine ligand

“…Cationic Ir(III) complexes, which have found wide use as emitters in electroluminescent devices [3], as photocatalysts [4] and in biolabeling [5], have been derivatized mostly by incorporation of electron donating or withdrawing groups onto either the cyclometallating or ancillary ligands [6,7]. Though several pyridylazole ancillary ligands have been explored as ancillary ligands, the overall structural diversity of heterocycles employed remains relatively poor.…”