Photosensitizer KillerRed

- Genetically-encoded photosensitizer, direct expression in cells
- No exogenous chemical compounds or cofactors required
- No cell toxicity before light activation
- Induction by green light irradiation
- Successful performance in fusions
- Recommended for selective in vivo cell killing and CALI applications

Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They can be used for precise inactivation of selected proteins in chromophore-assisted light inactivation (CALI) technique and for the light-induced cell killing, for example in photodynamic therapy. Besides KillerRed protein, all known to date photosensitizers are chemical compounds that must be introduced into living systems exogenously. Red fluorescent protein KillerRed is the first genetically-encoded photosensitizer [Bulina et al., 2006a]. Unlike chemical analogs, KillerRed can be directly expressed by target cells, both individually and in fusion with a target protein. It shows no cell toxic effects before light activation. Upon green-light irradiation, KillerRed generates ROS that damage the neighboring molecules.

Main properties

Normalized excitation (thin line) and emission (thick line) spectra.

CHARACTERISTIC *Brightness is a product of extinction coefficient and quantum yield, divided by 1000. Molecular weight, kDa 27 Polypeptide length, aa 239 Structure dimer Aggregation no Maturation rate at 37°C slow Activating light green (e.g. 540-580 nm) Fluorescence color red Excitation maximum, nm 585 Emission maximum, nm 610 Quantum yield 0.25 Extinction coefficient, M-1cm-1 45 000 Brightness* 11.3

Recommended antibodies, filter sets and activating lasers

KillerRed can be recognized using Anti-KillerRed antibody (Cat.# AB961-AB962) available from Evrogen.

Before light activation, KillerRed can be detected using TRITC filter set or similar. Recommended Omega Optical filter sets are QMAX-Red and XF174.

KillerRed phototoxicity is induced by green-light irradiation at 540-580 nm and depends on light intensity irradiation time and KillerRed concentration. Importantly, we recommend that you use arc-lamp irradiation. Laser-light irradiation in confocal mode is much less efficient.

In CALI, mild illumination of KillerRed-tagged protein for a limited time results in precise inactivation of this protein only. Upon more prolonged and intensive irradiation, KillerRed can be effectively used for damaging the organelles and killing the target cells. Intensity of green light and irradiation time must be individually determined for particular biological system and instrumentation.

KillerRed-mediated ROS production is accompanied by profound KillerRed photobleaching. The resulting cell events (cell fate after irradiation, effect on protein localization) can be monitored using another fluorescent reporter, for example a green fluorescent protein. We recommend that you use TurboGFP for cell and organelle, or TagGFP for protein labeling.

Performance and use

KillerRed can be used for in vivo killing of selected cells and CALI applications. It can be expressed and induced in various experimental systems, including bacteria, Xenopus, zebrafish, and mammalian cells.

KillerRed is 2000-fold more phototoxic than EGFP; however, it is not as effective as chemical probes. KillerRed-mediated ROS production is accompanied by profound KillerRed photobleaching.

Despite it dimerization capacity, KillerRed demonstrates successful performance in many fusions including that with mitochondrial targeted signal, cytoplasmic β-actin, fibrillarin, dopamine transporter, Tau34, etc.

KillerRed suitability to generate stably transfected cells has been proven by Marinpharm company. Variuos cell lines expressing KillerRed are commercially available.

KillerRed expression in mammalian cells. (A) Stably transfected ARPE-19 cells expressing membrane-targeted KillerRed. Image was kindly provided by Dr. Christian Petzelt (Marinpharm); (B) Transiently transfected HeLa cells expressing KillerRed fusion with Tau34.

KillerRed suitability for light-induced killing of prokaryotic cells has been demonstrated using E. coli XL1-Blue strain. A single E. coli colony was picked out, diluted into 1 ml of PBS buffer and divided into two equal portions. One of them was irradiated with white light (1W/cm², light source Fiber-Light from Dolan-Jenner Industries, Inc) for different periods of time, whereas the other was kept in darkness. Both sample aliquot were then plated onto Petri dishes at different dilutions. The number of growing colonies corresponded to the number of bacteria cells surviving after irradiation (i.e. colony forming units, CFU). CFU number for the irradiated E. coli portion was compared with non-irradiated one, thus allowing estimation of the relative phototoxic effect for KillerRed. In control experiments, E. coli cells expressing different fluorescent and non-fluorescent proteins were used.

KillerRed killed 96% of bacterial cells after 10 min and almost 100% of cells after 20 min of irradiation with white light.

		KillerRed-mediated killing of bacteria cells. Left graph: Blue and green rectangles show relative phototoxic effect from irradiation with blue (460-490 nm) and green (540-580 nm) light of 35 mW/cm2. Numbers above the rectangles represent decrease in viable E. coli cells after 30-min irradiation (folds). Right graph: Time-course of light-induced killing of E. coli expressing KillerRed.

KillerRed-mediated killing of eukaryotic cells: Following two ways have been found to be effective for killing the eukaryotic cells using KillerRed: (1) via apoptotic pathway using KillerRed targeted to mitochondria, and (2) via membrane lipid oxidation using membrane-localized KillerRed. Irradiation of KillerRed, localized in cell cytosol, has a weak effect on cell survival.

Effects of KillerRed localized in mitochondria: Mitochondrial localization increases the phototoxic effect of photosensitizers (primarily by provoking the apoptosis). Use of KillerRed targeted to mitochondria allows effective cell killing through apoptotic pathway as has been demonstrated in the following experiments:

HeLa cells expressing cytoplasmic TurboGFP and mitochondria-localized KillerRed-dMito were generated. 10-min irradiation of selected cells with green light resulted in profound KillerRed photobleaching. 60 min after irradiation, cells had an abnormal shape and "bubbles" typical of apoptotic pathway. These cells disrupted within next 30-60 min.

Confocal image of HeLa cells expressing KillerRed in mitochondria (red) and TurboGFP in cytosol (green). Lower left cells were pre-irradiated with green light (515-560 nm, 7W/cm2) light for ten minutes. This led to profound KillerRed photobleaching. Cells are shown in 60 minutes after irradiation. It is clearly visible that irradiated cells have abnormal shape and "bubbles", characteristic for apoptotic cell-death pathway.

In another experiment, nearly 100% of B16 melanoma cells expressing KillerRed targeted to mitochondria died within 45 min after 15-min of irradiation (40x objective, 535-575 nm excitation filter, 3.3 W/cm²). It is noticeable that when preincubated with the pancaspase inhibitor zVAD-fmk (10 μM), the cells were resistant to the same green-light exposure and held their shape for at least 1.5 hours after illumination.

Apart from the immediate phototoxic effect, photosensitizers can mediate postponed cellular responses such as cell growth arrest or cell death via long-term apoptotic mechanism: B16 melanoma cells expressing mitochondria-targeted KillerRed or EGFP were mixed together and irradiated by green light of low intensity (3.7x objective, 535-575 nm excitation filter, 115 mW/cm²) for 45 min. No red fluorescent cells were observed in 16 hrs after irradiation, whereas green fluorescent cells remained viable. It confirms that mitochondria-localized KillerRed can mediate cell death through long-term mechanisms in response to low-intensity illumination. This effect can be used in different applications.

Effects of KillerRed localized in membrane: Comparing with the mitochondria-targeted KillerRed, irradiation of membrane-localized KillerRed causes a more effective and fast cell death within 10-30 min, presumably because of lipid oxidation [Bulina et al., 2006b].

	Light-induced killing of HeLa cell using membrane-targeted KillerRed (movie). First image: intact cell features red fluorescent cellular membrane and green fluorescent cytosol due to membrane-targeted KillerRed and TurboGFP expression, respectively. Further images: 40 minute time-course of cell fragmentation induced by 10-min green light irradiation. Movie from [Bulina et al., 2006a].

In addition, membrane-targeted KillerRed was shown to be an effective tool for the light-induced cell killing within a developing zebrafish. Zebrafish embryo was microinjected with a mixture of vectors driving expression of membrane-targeted KillerRed and a green fluorescent protein at the single- cell stage. A muscle cell expressing both proteins was irradiated with green light (40x objective, TRITC filter set, 10 min) at 48 hrs after fertilization. By the end of 10-min irradiation, the cell already started to change its shape. Within 20 min after irradiation was stopped, the cell was disrupted completely. Mitochondria targeted KillerRed was shown to be of low efficiency in similar experiments.

Light-induced killing of a muscle cell within a developing zebrafish embryo. Left image: A region expressing membrane-targeted KillerRed and green fluorescent marker; right images: time course of light-induced killing of a muscle cell within a developing zebrafish. Fluorescence was collected using standard FITC and TRITC filter sets. Figure was first published in [Bulina et al., 2006b].

KillerRed use for CALI applications: KillerRed use for CALI application has been demonstrated on the model of β-galactosidase inactivation in bacterial cells and inactivation of pleckstrin homology (PH) domain of phospholipase C delta-1 (PLC delta-1) in mammalian cells. In the first experiment, KillerRed was fused to β-galactosidase (β-gal) enzyme and expressed in E. coli. Upon green-light irradiation (540-580 nm, 30 min, 360mW/cm²) β-gal activity was effectively suppressed in living E. coli streaks. On the contrary, no effect of green light on the enzyme activity was detected in control cells containing unmodified β-gal gene. In vitro test showed that in E. coli cell extract β-gal fused to KillerRed lost 99,4% of enzymatic activity within 25 min of white light exposure (1W/cm²), with half inactivation time of about 5 min. Irradiation of E. coli extracts containing unfused β-gal protein alone or β-gal mixed with KillerRed had no effect on enzyme activity. To verify specificity of KillerRed phototoxic effect, horse-radish peroxidase (HRP) was added to the sample. Upon 15 min of illumination (white light, 1W/cm²) only 2% of HRP activity was lost, showing high specificity of the phototoxic effect.

	Time-course of CALI of β-galactosidase.

In the second experiment, a triple EGFP-PH-KillerRed fusion protein that allows both protein visualization and CALI was transiently expressed in mammalian cell line. Intracellular localization of EGFP signal was evaluated before and after CALI of the PH domain using confocal and fluorescence microscopy. In intact cells, the fusion is located predominantly at the plasma membrane because of specific affinity of PH domain to phosphatidylinositol 4,5-bisphosphate.

Irradiation with intense green light led to KillerRed-mediated ROS production, PH domain damage, and fusion protein dissociation from the membrane. After 10 sec of green-light irradiation (63x objective, mercury lamp, 515-560 nm filter, 7W/cm²), translocation of the PH domain into cytosol was clearly visible. When irradiated for a longer period of time, considerable amount of PH domain translocated into cytosol, increasing the cytoplasm-to-membrane green fluorescent signal ratio to 0.5-0.9.

In the negative control experiments, the cellular location of a DsRedExpress (Clontech) containing construct, GFP-PH-DsRedExpress, showed no dependence on green-light irradiation. Similarly, no detectable CALI of the PH domain was achieved when KillerRed was expressed in the cell separately from PH domain, in either the membrane or cytosol.

KillerRed-mediated light-induced inactivation of PLC delta-1 PH domain. (A) Schematic outline of the experimental system; (B, C) confocal images of a cell expressing EGFP-PH-KillerRed triple fusion (EGFP green fluorescent signal) before (B) and after (C) 10-s irradiation with green light. Note considerable increase in cytoplasmic signal.

Available variants and fusions

KillerRed id developed on the basis of the chromoprotein from Anthomedusae sp.[Shagin et al., 2004]. Its codon usage is optimized for high expression in mammalian cells [Haas et al., 1996], but it can be successfully expressed in other heterological systems.

KillerRed-dMito fusion: Mitochondrial targeting sequence (MTS) was derived from subunit VIII precursor of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. Two MTS were fused to KillerRed N-terminus. When expressed in mammalian cells, this variant is localized in mitochondria. Mitochondrial localization of KillerRed increases its light-induced cell toxity and makes it effective for cell killing presumably through apoptotic pathway.

KillerRed-mem fusion comprises KillerRed linked with membrane localization signal (MLS) of neuromodulin. The neuromodulin MLS (N-terminal 20 amino acid residues) contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets KillerRed to cellular membranes [Skene and Virag, 1989]. Irradiation of membrane-localized KillerRed leads to high effective and fast cell death, presumably due to lipid oxidation. Comparing to the mitochondrially targeted KillerRed, irradiation of membrane-localized KillerRed leads to even more effective and fast cell death (within 10-30 min). Membrane-targeted KillerRed was shown to be suitable for the light induced cell killing within a developing zebrafish.