Autofluorescent Proteins as Photosensitizer in Eukaryontes

Waldeck, Waldemar; Mueller, Gabriele; Wießler, Manfred; Brom, Manuela; Tóth, Katalin; Braun, Klaus

doi:10.7150/ijms.6.365

Cited by 18 publications

(20 citation statements)

References 25 publications

(26 reference statements)

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“…This microscopic studies show the evidence that SuperNova may not interfere with normal cell division rate. Multimerisation may account for this observation as cell cycle could be suppressed and cell death promoted by the abnormal oligomerisation of histones via the KillerRed dimer tendency as cell division is sensitive to the monomerisation state of histones15.…”

Section: Resultsmentioning

confidence: 99%

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

Takemoto

Matsuda

Sakai

et al. 2013

Sci Rep

134

189

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Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

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Section: Resultsmentioning

confidence: 99%

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

Takemoto

Matsuda

Sakai

et al. 2013

Sci Rep

134

189

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“…Use of diphtheria toxin A for cell killing in C. elegans can also be problematic due to its high toxicity. The phototoxic protein KillerRed, developed from a red chromoprotein homolog of GFP, has been used to kill cultured cells when targeted to mitochondria, membranes, or histones and illuminated with green light (12,38). KillerRed works through generation of radicals or hydrogen peroxide rather than through singlet oxygen (15,39).…”

Section: Discussionmentioning

confidence: 99%

Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG

Garren

Shu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

193

203

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We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mitominiSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.light inducible | locomotion | optogenetics | reactive oxygen species | pre-motor interneurons

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“…The permanent cloning of a pH2A-KillerRed transfected DU145 cell line was not successful. The cells could not survive as clones; they changed their phenotype and their lost morphological structures, as already documented by Waldeck 17. For this reason the fluorescence images combined with the images, as depicted in the Figure 3, could solely be generated in nuclei of DU 145 cells pH2A-KillerRed transiently transfected.…”

Section: Resultsmentioning

confidence: 72%

Positioning Effects of KillerRed inside of Cells correlate with DNA Strand Breaks after Activation with Visible Light

Waldeck¹,

Mueller²,

Wießler³

et al. 2011

Int. J. Med. Sci.

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Fluorescent proteins (FPs) are established tools for new applications, not-restricted to the cell biological research. They could also be ideal in surgery enhancing the precision to differentiate between the target tissue and the surrounding healthy tissue. FPs like the KillerRed (KRED), used here, can be activated by excitation with visible day-light for emitting active electrons which produce reactive oxygen species (ROS) resulting in photokilling processes. It is a given that the extent of the KRED's cell toxicity depends on its subcellular localization. Evidences are documented that the nuclear lamina as well as especially the chromatin are critical targets for KRED-mediated ROS-based DNA damaging. Here we investigated the damaging effects of the KRED protein fused to the nuclear lamina and to the histone H2A DNA-binding protein. We detected a frequency of DNA strand breaks, dependent first on the illumination time, and second on the spatial distance between the localization at the chromatin and the site of ROS production. As a consequence we could identify defined DNA bands with 200, 400 and (600) bps as most prominent degradation products, presumably representing an internucleosomal DNA cleavage induced by KRED. These findings are not restricted to the detection of programmed cell death processes in the therapeutic field like PDT, but they can also contribute to a better understanding of the structure-function relations in the epigenomic world.

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Autofluorescent Proteins as Photosensitizer in Eukaryontes

Cited by 18 publications

References 25 publications

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation

Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG

Positioning Effects of KillerRed inside of Cells correlate with DNA Strand Breaks after Activation with Visible Light

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