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

Abstract

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.

Photodynamic Physiology—Photonanomanipulations in Cellular Physiology with Protein Photosensitizers

“…KillerRed can also be targeted to the lysosomes (Serebrovskaya et al, 2014), Golgi apparatus (Jarvela and Linstedt, 2014), the plasma membrane, mitochondria, or chromosomes (Shirmanova et al, 2013) in Hela cells. KillerRed has also been targeted to the plasma membrane in C. elegans neurons (Williams et al, 2013), zebrafish neurons and cardiomyocytes (Lee et al, 2010; Teh et al, 2010), or targeted to chromosomes in DU145 cells (Waldeck et al, 2011). The fusion protein TRF1-KillerRed has been used to target the telomeres (Sun et al, 2015).…”

“…Subcellular targeting of protein photosensitizers has been done as mentioned above in different cell types, such as targeted KillerRed expression in Hela cells (Shirmanova et al, 2013; Jarvela and Linstedt, 2014; Serebrovskaya et al, 2014), HEK293 cells (Bulina et al, 2006b), DU145 cells (Waldeck et al, 2011), and miniSOG expression in Hela cells (Ryumina et al, 2013) or in cultured hippocampal neurons (Lin et al, 2013). …”

Chromophore-assisted laser inactivation – towards a spatiotemporal–functional analysis of proteins, and the ablation of chromatin, organelle and cell function

“…Moreover, in transgenic Xenopus embryos that express H2B-tandem KillerRed under control of tissue-specific promoters, tissue development is slowed in tadpoles when illuminated with green-light, suggesting that CALI can be used to elucidate the effect of temporal arrests of cell division during organogenesis (Serebrovskaya et al, 2011). KillerRed fused to either lamin B1, one of the components of inner nuclear membrane, or histone H2A was shown to induce DNA strand breaks dependent on irradiation time and intranuclear chromatin structure (Waldeck et al, 2011). Recently, CALI with KillerRed fused to a Tetrepressor or a transcriptional activator that can bind to integrated DNA cassettes (,90 kb), was shown to cause induction of sitespecific DNA damage (Lan et al, 2013).…”

KillerRed protein basedin vivophotodynamic therapy and corresponding tumor metabolic imaging

“…13 It has the potential to act as a photosensitizer. Some groups reported that this KillerRed protein could produce ROS which has toxicity to the tumor cells 13,15 and its di®erent subcellular component localization also had been found, such as membrane 11,16 or mitochondria 17 which play a major role in photodynamic therapy induced cell death. [18][19][20] During the treatment of photodynamic therapy, the genetically encoded photosensitizer will alter the tumor cell redox state and metabolism, however, the course of tumor redox metabolism during PDT treatment was rarely investigated so far.…”