Deleterious effects of mitochondrial ROS generated by KillerRed photodynamic action in human cell lines and C. elegans
Highlights
► mtROS generated by KillerRed could trigger mitochondrial damage. ► mtROS induced caspase-dependent and caspase-independent cell death in human cells. ► mtKillerRed alone showed toxicity in C. elegans under unirradiated conditions. ► Photodynamic action of mtKillerRed induced developmental delay of C. elegans larvae.
Introduction
Photosensitizers are molecules that absorb and re-emit light at a specific wavelength unique to each molecule and produce reactive oxygen species (ROS), such as singlet oxygen 1O2 or other radicals (superoxide, hydroxyl radical) during the photoreaction [1], [2], [3], [4]. Photosensitizers are widely used in photodynamic therapy (PDT), which is a promising treatment for numerous cancers and certain noncancerous diseases. Cancer cells that absorb photosensitizers can be killed by the oxidative damage induced by ROS production following irradiation with light of an appropriate wavelength.
Bulina et al. developed a photosensitizer protein called KillerRed, which is a dimeric red fluorescent protein with excitation and emission maxima at 585 nm and 610 nm, respectively [5], [6], [7]. After irradiation with light at wavelengths of 520–590 nm, KillerRed generates high levels of ROS that can kill Escherichia coli and eukaryotic cells, exceeding the amounts of ROS produced by other green and red fluorescent proteins (e.g. GFP and DsRed) by at least 1000-fold. Because KillerRed is encoded by a gene, unlike other known photosensitizers, it can be expressed in a spatially and temporally regulated manner by choosing an appropriate promoter or by fusing with a protein of interest or a subcellular localization signal. Therefore, genetically encoded KillerRed provides a unique opportunity to not only investigate the downstream effects of ROS generated in subcellular organelles but also the functions of cells in various organs. For example, the effects of peroxisome-generated ROS on the cellular oxidation–reduction balance have been studied in mammalian cells by using peroxisome-targeting KillerRed [8]. More recently, membrane-targeting KillerRed was employed in zebrafish to disrupt neural circuits involving the habenula or to cause cardiac dysfunction following light irradiation [9], [10]. It has also been reported that light irradiation of HeLa or PC12 cells expressing mitochondria-targeting KillerRed induces mitochondrial translocation of mitophagy-related factors, including Parkin and LC3B, suggesting that mitochondria-targeting KillerRed may be a potential tool for studying Parkin-mediated mitophagy [11], [12]. Additionally, Bulina et al. reported that B16 melanoma cells expressing mitochondria-targeting KillerRed were killed by light irradiation, while these cells survived identical irradiation and preserved their normal shape for at least 1.5 h afterward when preincubated with the pan-caspase inhibitor zVAD, suggesting that mtROS might induce apoptosis in B16 melanoma cells [5], [6].
Although previous studies have shown that light irradiation of mammalian cells expressing mitochondria-targeting KillerRed can induce cytotoxicity, it is still unclear exactly how mitochondrial damage is caused by KillerRed-mediated production of ROS in the mitochondria (mtROS) and how mtROS generated by KillerRed affect the fate of cells, that is, whether activation of the cell death mechanisms result in apoptosis and/or necrosis. In the present study, we generated two human cell lines stably expressing mitochondria-targeting KillerRed, and investigated the effects of mtROS generated by KillerRed on the mitochondria and on the fate of these cells. Additionally, several authors have reported that excessive mitochondrial ROS production contributes to the pathogenesis of a wide range of diseases including diabetes, Alzheimer’s disease, and Parkinson’s disease, as well as being involved in the aging process [13]. To investigate role of mtROS in such pathology, we also generated transgenic Caenorhabditis elegans expressing mtKillerRed.
Section snippets
Human cell cultures
HEK293T and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C under 5% CO2.
C. elegans
The C. elegans strains used in this study were maintained at 20 °C on nutrient growth medium (NGM) agar plates seeded with E. coli HB101, according to standard techniques [14]. Worms were grown under dark conditions except when they were irradiated with light. Wild-type Bristol N2, SJ4100 (zcIs13[
mtKillerRed produces ROS after irradiation
We generated human HEK293T and HeLa cell lines that stably expressed mitochondria-targeting signal (MTS)-fused KillerRed protein (mtKillerRed) (293-mtK and HeLa-mtK cells) as described in Section 2 (Fig. S1). The localization of mtKillerRed in the mitochondria was confirmed by colocalization with MitoTracker Green-FM (a mitochondrial membrane-targeting dye). As a negative control, we also generated human cell lines stably expressing MTS-fused DsRed (mtDsRed), which does not produce enough ROS
Mitochondria are morphologically and functionally disrupted by mitochondrial ROS generation in human cells
The present study revealed that the photodynamic action of mtKillerRed, which generated mtROS, led to mitochondrial fragmentation and swelling in human cell lines. During 20 min of incubation after irradiation with light, confocal microscopy revealed that the majority of the mitochondria in HeLa-mtK and 293-mtK cells underwent fragmentation and then gradually became swollen, suggesting that mitochondrial fragmentation probably preceded swelling. In addition, electron microscopy clearly showed an
Abbreviations
ROS reactive oxygen species mtROS mitochondrial ROS mtKillerRed mitochondrial KillerRed PDT photodynamic therapy ΔΨm mitochondrial membrane potential PI propidium iodide NAC N-acetyl-cysteine MPT mitochondrial permeability transition CsA cyclosporine A MTS mitochondrial targeting signal CCCP carbonyl cyanide p-chlorophenylhydrazone NGM nutrient growth medium UPRmt mitochondrial unfolded protein response
Acknowledgements
We thank Drs. Y. Eguchi, K. Shinzawa, Y. Matsuoka, and other members of the Tsujimoto lab for their technical advice and assistance, reagents, and helpful discussion. We acknowledge Drs. Toshihiko Oka and Noriyuki Matsuda for providing the TOM20-GFP plasmid and GFP-Parkin plasmid, respectively. We are grateful to Dr. Hiroki Moribe for providing C. elegans strains, sharing the microinjection system, and to Dr. Takashi Hirose, Ms. Mayumi Shibuya for technical assistance, respectively. This work
References (36)
- et al.
Intracellular signaling mechanisms in photodynamic therapy
Biochim. Biophys. Acta
(2004) - et al.
Molecular effectors of multiple cell death pathways initiated by photodynamic therapy
Biochim. Biophys. Acta
(2007) - et al.
Photodynamic therapy (PDT): a short review on cellular mechanisms and cancer research applications for PDT
J. Photochem. Photobiol. B
(2009) - et al.
The habenula prevents helpless behavior in larval zebrafish
Curr. Biol.
(2010) - et al.
Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy
J. Biol. Chem.
(2011) - et al.
Mitochondrial dysfunction and molecular pathways of disease
Exp. Mol. Pathol.
(2007) - et al.
DAF-7/TGF-beta expression required for the normal larval development in C. elegans is controlled by a presumed guanylyl cyclase DAF-11
Mech. Dev.
(2001) - et al.
Mitochondrial membrane permeability transition and cell death
Biochim. Biophys. Acta
(2006) - et al.
Mitochondrial signaling: the retrograde response
Mol. Cell
(2004) - et al.
Mitochondrial reactive oxygen species control the transcription factor CHOP-10/GADD153 and adipocyte differentiation: a mechanism for hypoxia-dependent effect
J. Biol. Chem.
(2004)
A morphologically conserved nonapoptotic program promotes linker cell death in Caenorhabditis elegans
Dev. Cell
Photodynamic therapy and anti-tumour immunity
Nat. Rev. Cancer
A genetically encoded photosensitizer
Nat. Biotechnol.
Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed
Nat. Protoc.
Fluorescent proteins as light-inducible photochemical partners
Photochem. Photobiol. Sci.
Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk
Mol. Biol. Cell
Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics
BMC Dev. Biol.
Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells
Autophagy
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