Abstract
We have developed a new genetically encoded tool designed to generate reactive oxygen species (ROS) at target proteins in cultured cells; it is designed using firefly luciferase and photosensitiser protein KillerRed. Targeting this fusion protein, KillerFirefly, to F-actin in live cells and treatment with luciferin induced a characteristic structure, previously reported as a cofilin-actin rod, which is seen in patients with Alzheimer’s disease. This structural change is considered to be elicited by the consistent generation of very low-level ROS by KillerFirefly in the vicinity of F-actin. Moreover, our results suggest the presence of an actin-regulating system, controlled by very low levels of endogenously generated ROS.
Highlights
We have developed a new genetically encoded tool designed to generate reactive oxygen species (ROS) at target proteins in cultured cells; it is designed using firefly luciferase and photosensitiser protein KillerRed
To test whether KillerRed is excited by luciferase via the bioluminescence resonance energy transfer (BRET) effect, the spectrum of emitted light from KillerFirefly was measured and compared with that of luciferase (Fig. 1b)
Because excitation of the KillerRed protein evokes ROS generation[8], we concluded that the KillerFirefly protein generates ROS in response to luciferin treatment in live cells
Summary
We have developed a new genetically encoded tool designed to generate reactive oxygen species (ROS) at target proteins in cultured cells; it is designed using firefly luciferase and photosensitiser protein KillerRed. To F-actin in live cells and treatment with luciferin induced a characteristic structure, previously reported as a cofilin-actin rod, which is seen in patients with Alzheimer’s disease This structural change is considered to be elicited by the consistent generation of very low-level ROS by KillerFirefly in the vicinity of F-actin. KillerRed is a photosensitising fluorescent protein that was developed by mutation of hydrozoan chromoprotein anm2CP8,9 This protein produces O2− in response to light irradiation (maximum excitation at 585 nm) and has been used in the CALI technique. We constructed a fusion protein of KillerRed and firefly luciferase (KillerFirefly, Fig. 1a), which was expected to generate ROS from KillerRed when excited by the bioluminescence resonance energy transfer (BRET) by luciferase in response to the luciferin treatment. We evaluated whether targeted ROS generation by the KillerFirefly protein modifies the function of cellular protein
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