Construction of photoenergetic mitochondria in cultured mammalian cells

Abstract

The proton motive force (PMF) is bio-energetically important for various cellular reactions to occur. We developed PMF-photogenerating mitochondria in cultured mammalian cells. An archaebacterial rhodopsin, delta-rhodopsin, which is a light-driven proton pump derived from Haloterrigena turkmenica, was expressed in the mitochondria of CHO-K1 cells. The constructed stable CHO-K1 cell lines showed suppression of cell death induced by rotenone, a pesticide that inhibits mitochondrial complex I activity involved in PMF generation through the electron transport chain. Delta-rhodopsin was also introduced into the mitochondria of human neuroblastoma SH-SY5Y cells. The constructed stable SH-SY5Y cell lines showed suppression of dopaminergic neuronal cell death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an inducer of Parkinson's disease models, which acts through inhibition of complex I activity. These results suggest that the light-activated proton pump functioned as a PMF generator in the mitochondria of mammalian cells, and suppressed cell death induced by inhibition of respiratory PMF generation.

Principles of designing interpretable optogenetic behavior experiments

“…Glial cells in culture or in visual cortex slices, expressing channelrhodopsin-2, release calcium from intracellular stores upon illumination (Figueiredo et al 2014;Perea et al 2014), as evidenced by the blockade of opsin-mediated calcium signals when such stores are depleted with the drug thapsigargin. Deliberately targeting proton pumps to mitochondria of yeast or mammalian cells has been reported to result in cell survival with less reliance on oxidative phosphorylation (Hoffmann et al 1994;Hara et al 2013). Thus, optogenetic tools may be utilized to study the effects of specific ions on specific physiological processes.…”

Antioxidant Activity of Ge-132, a Synthetic Organic Germanium, on Cultured Mammalian Cells

“…LDH activity was reported as fold induction compared with control cells without Ge-132 treatment as reported previously. 19,20) Absorbance was measured using a plate reader (Powerscan HT, DS Pharma Biomedical, Osaka, Japan).…”

“…21) Total protein extracts were prepared from cultured cells, as described previously. 19) The Western blotting protocol was performed as described previously 22) with minor modification.…”

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

“…We used a standard protocol for immunofluorescence microscopy as described previously [17]. To quantify the ratio of subcellular localization of exogenous CRBN and its deletion mutants, stained cells were classified as reported previously [18e21].…”

“…Total protein extracts were prepared from cultured cells as described previously [17]. We used a standard western blotting protocol as described previously [22].…”