Author response: Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity

Subcellular Singlet Oxygen and Cell Death: Location Matters

“…The medium was replaced by fresh DMEM medium (without FBS) containing MG2I-ester (Control group: COX-dL5 * * cells, 0 nM, treatment groups: 500 nM). The cells were illuminated for 180 s with 660-nm NIR in a light box using a dispersive, 12-cm distant scattering lens to generate consistent exposure across the plate of 160 mW/cm 2 , similar to our previously published LED illuminators (He et al, 2016;Xie et al, 2020), yielding a total light dose of 28.8 J/cm 2 , followed by addition of medium containing 10% FBS to every well. After incubation for 12 h, the cells were collected and stained with annexin V-FITC (0.4 µg/ml) and propidium iodide (0.5 µg/ml) in binding buffer [0.01 M Hepes (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl 2 solution] for 5 min.…”

“…The genetic encoding of the FAP enables both cellular specificity and subcellular targeting and opens the potential for simultaneously targeting singlet oxygen generation in multiple subcellular locations at the same time, using the same chemical and light exposure. The chemoptogenetic ability to restrict singlet oxygen generation to specific compartments and cells within a complex environment, and to quantitatively activate its generation at a specific time by adding dye and treating with light, enables a range of direct investigations into the role of singlet oxygen in signaling and inducing cell death in a variety of cellular and complex biological contexts (Lan et al, 2014;Fouquerel et al, 2019;Jang et al, 2019;Qian et al, 2019;Binns et al, 2020;Xie et al, 2020).…”

“…Genetically targeted PS Killer Red (Bulina et al, 2006), MiniSOG (Ruiz-González et al, 2013), and SuperNova (Takemoto et al, 2013) have all been used to target intracellular organelles, with varying results on efficacy in directed cell killing, depending on the organelle targeted, expression level, and illumination parameters [reviews: (Wojtovich and Foster, 2014;Trewin et al, 2018)]. Targeted and activated photosensitizers (TAPs) using a genetically encoded fluorogen-activating protein (FAP) enable selective photosensitization directed to targeted cellular sites within genetically or molecularly targeted cells (Wang et al, 2017;Ackerman et al, 2019), as recently illustrated in transgenic zebrafish cardiovascular and nervous systems (He et al, 2016;Xie et al, 2020), and subcellular locations including mitochondria and telomeres, among others (Fouquerel et al, 2019;Jang et al, 2019;Qian et al, 2019). Due to the short range of action of 1 O 2 in living systems (Kuimova et al, 2009), this targeting and activation of the PS protects adjacent normal cells and reduces damage to nearby tissues, because photoactivity requires coincidence of the dye, the activating protein, and the illumination source (Lovell et al, 2010;He et al, 2016).…”

Data_Sheet_1.pdf

“…The medium was replaced by fresh DMEM medium (without FBS) containing MG2I-ester (Control group: COX-dL5 * * cells, 0 nM, treatment groups: 500 nM). The cells were illuminated for 180 s with 660-nm NIR in a light box using a dispersive, 12-cm distant scattering lens to generate consistent exposure across the plate of 160 mW/cm 2 , similar to our previously published LED illuminators (He et al, 2016;Xie et al, 2020), yielding a total light dose of 28.8 J/cm 2 , followed by addition of medium containing 10% FBS to every well. After incubation for 12 h, the cells were collected and stained with annexin V-FITC (0.4 µg/ml) and propidium iodide (0.5 µg/ml) in binding buffer [0.01 M Hepes (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl 2 solution] for 5 min.…”

“…Genetically targeted PS Killer Red (Bulina et al, 2006), MiniSOG (Ruiz-González et al, 2013), and SuperNova (Takemoto et al, 2013) have all been used to target intracellular organelles, with varying results on efficacy in directed cell killing, depending on the organelle targeted, expression level, and illumination parameters [reviews: (Wojtovich and Foster, 2014;Trewin et al, 2018)]. Targeted and activated photosensitizers (TAPs) using a genetically encoded fluorogen-activating protein (FAP) enable selective photosensitization directed to targeted cellular sites within genetically or molecularly targeted cells (Wang et al, 2017;Ackerman et al, 2019), as recently illustrated in transgenic zebrafish cardiovascular and nervous systems (He et al, 2016;Xie et al, 2020), and subcellular locations including mitochondria and telomeres, among others (Fouquerel et al, 2019;Jang et al, 2019;Qian et al, 2019). Due to the short range of action of 1 O 2 in living systems (Kuimova et al, 2009), this targeting and activation of the PS protects adjacent normal cells and reduces damage to nearby tissues, because photoactivity requires coincidence of the dye, the activating protein, and the illumination source (Lovell et al, 2010;He et al, 2016).…”