ROS Generation in Mitochondria and Cytosol during Optogenetic Cytosol Alkalization in Human Cells

Optogenetics allows a precise control of a vast variety of cellular processes in excitable and non-excitable cells, in particular, through their organelles. Microbial rhodopsin-based optogenetic tools could be used to control ion concentrations in various compartments. Recently, induction of apoptosis by rhodopsin-based optogenetics was demonstrated: cytosol alkalization in human cells by outward proton pump Arch3 was shown to induce intrinsic, mitochondria-mediated apoptotic pathway of cell death. It is known that cytosol and mitochondrial matrix alkalization, reactive oxygen species production in mitochondria and Ca²⁺ signaling are tightly interconnected in mammalian cells, and under certain conditions they favor mitochondrial permeability transition pore opening and cell death. However, in the case of optogenetic alkalization reactive oxygen species generation has not been experimentally addressed. In this work we investigated reactive oxygen species generation which occurred under the optogenetic cytosol alkalization. Arch3 was expressed in the plasma membrane of HeLa cells, and optogenetic cytosol alkalization by Arch3 lead to mitochondrial reactive oxygen species generation as well as to elevation of the level of reactive oxygen species in cytosol. We propose that production of reactive oxygen species may be a key step in cell death induced by optogenetic alkalization of cytosol by promoting the opening of mitochondrial permeability transition pores. Our findings may help to better understand the mechanisms of apoptosis induced by reactive oxygen species and shed light on the interplay between the cytosolic pH, mitochondrial dynamics, reactive oxygen species, and some other factors promoting cell death in living cells.