Oxidative bursts of single mitochondria mediate retrograde signaling toward the ER

Abstract

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP₃Rs), we show that Ca²⁺ channels directly sense oxidative bursts and respond with Ca²⁺ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca²⁺ transport via elimination of IP₃Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca²⁺ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.

Keywords: Ca2+ microdomain; Inositol-1,4,5-trisphosphate receptor; Mitochondrial retrograde signaling; Organelle contacts; Redox nanodomain.

Figure 1.. Spontaneous and stimulated mPTP events underpin the intra- and extra-mitochondrial features of mitochondrial flickers

(A) Airyscan imaging of TMRE and calcein-labeled HepG2 cells. Plots of whole-cell ROI of TMRE (inset; red) and calcein (inset; teal). (B) Calcein decline following transient depolarization events in single mitochondria. (C) Decline in calcein proportional to duration of depolarization event. (D) Oligo increased mitochondrial membrane potential (ΔΨ_m). (E) Increased flicker frequency induced by Oligo. (F) ST promoted global depolarization. (G) Increased flicker frequency induced by ST (Pearson’s correlation: Ctrl, 0.4075; ST, 0.927). (H) ΔΨ_m flickers accompanied by stepwise declines in calcein fluorescence. (I) Mitochondrial calcein decreases sensitivity to CsA. (J) ΔΨ_m flickers and calcein decrease sensitivity to CsA. (K) ST-induced ΔΨ_m flickers accompanied by transient elevation in matrix pH measured by SypHer. (L) OMM-targeted SypHer reports acidification at the cytosolic surface of the OMM during flickers. (D)–(G) and (J) present mean ± SEM. Scale bars, 10 µm.

Figure 2.. Mitochondrial flickers generate dynamic redox signals at the ER-mitochondrial interface

(A) Schematic of hypothesis, flicker-induced oxidative redox shift (green) of interface. (B) Redox poise (GSSG/GSH ratio) in unstimulated cells measured by Grx1-roGFP2 targeted to the cytosol (Cyto), mitochondrial matrix (Matrix), and ER-mitochondrial interface (Interface); *p < 0.05, two-way ANOVA. (C) Redox poise following addition of ST. (D) Kymograph of ΔΨ_m (TMRE, upper) and Interface GSSG/GSH ratio (Grx1roGFP2, lower) from a 1 × 4 µm ROI crossing three adjacent mitochondria. Line plots (lower) of the region split into three ROIs (ROI1–ROI3). (E) Spontaneous transient depolarization (−ve ΔΨ_m: −ve ΔF TMRE, red) of single mitochondrion (upper) during flicker. Interface GSSG/GSH ratio adjacent to the parent organelle (left, lower row: Grx1roGFP2 overlay; 415 nm, red; 485 nm, green). ΔΨ_m (right) (TMRE 577 nm F/F₀, deep red) and GSSG/GSH (Grx1roGFP2, green). (F) ΔΨ_m and interface GSSG/GSH in unstimulated cells, single example event (left) and flickers synchronized to half ΔTMRE (TMRE F/F₀ mean ± SEM, deep red; Grx1roGFP Ratio/Ratio0, green, right). (G) Flicker (−ve ΔΨ_m: −ve ΔF TMRE, red) of single mitochondrion (upper row) during ST treatment. Interface GSSG/GSH. (H) ΔΨ_m and interface GSSG/GSH during flicker. Example event (left) and synchronized events (right) to half ΔTMRE (TMRE F/F₀, deep red; Grx1roGFP Ratio/Ratio0, green). (I) Matrix (left) and Interface (right) of GSSG/GSH responses to transient (filled) and sustained (empty) flickers. (J) Time to peak of Grx1roGFP2 response normalized to half ΔTMRE. Matrix (left) and Interface divided into responses from transient ΔΨ_m (<20 s) and sustained (>20 s) flickers. (K) Mean and logistic fits of Matrix and Interface GSSG/GSH normalized to half ΔTMRE recovery (F), (H) right, (I), and (K) present mean ± SEM. (J) Mean, median, 25th/75th percentile and outliers. Scale bars, 10 µm.

Figure 3.. Mitochondrial flickers promote local Ca²⁺ signals via oxidation of the IP₃R

(A) Functional calibration of HyPer-IP₃R1 versus cytosolic HyPer (Cyto) oxidized with stepwise H₂O₂ and fully reduced with DTT, mean ± SEM, with dose response (logistic) curve (fit). Inset: sensor dynamic range, ratio (R_min: DTT) to (R_max: H₂O₂). (B) Baseline oxidation of HyPer cytosol (Cyto) or IP₃R (IP₃R1) in HepG2. **Significance, Mann-Whitney U = 9, p = 0.003. (C) IP₃R and cytosolic H₂O₂ levels following ST-induced flickers. (D) ST-induced flicker oxidation of HyPer-IP₃R and SypHer-IP₃R control in the presence of CsA versus FK506 control. (E) Flicker and local Ca²⁺ transient. Difference image analysis of matrix pH (SypHer_480nm +ve and −ve change: first row +DF, green; −ΔF, blue). Difference imaging of interface Ca²⁺ (second row [Ca²⁺]_Int RCaMP_577nm +ve +DF, blue). pH (SypHer R/R₀; black) and [Ca²⁺]_Int (RCaMP F/F₀; blue) (ROI 1, right upper; ROI 2, right lower). Lower: 3D renderings of matrix pH (SypHer 480/414 nm) and [Ca²⁺]_Int (RCaMP, F₅₇₇ nm) at 16 s time point. (F) Independent subcellular release sites, maximum [Ca²⁺]_Int in 300 s period increase (RCaMP_577nm, ΔF_max). Traces of three independent release sites (ROI1–ROI3; and whole cell; lowest). (G) Individual [Ca²⁺]_Int transients, amplitude versus time. (H) [Ca²⁺]_Int amplitude (peak height, F/F₀), area of [Ca²⁺]_Int transient (peak area, µm²), and duration at half maximum amplitude. Repeated [Ca²⁺]_Int with first (upper) and second iteration (lower) assessed separately. (A) inset, (B), and (D), mean, median, 25th/75th percentile and outliers. Scale bars, 10 µm.

Figure 4.. Mitochondrial flicker activity interacts with ER-mitochondrial Ca²⁺ transport

(A) Flicker frequency in MCU-KO and cells rescued with MCU-FLAG (MCU-Rescue; filled) following stimulation with ST. (B) Matrix Ca²⁺ rise assessed with mtCEPIA3 ([Ca²⁺]_Mt: CEPIA 485 nm F/F₀) in MCU KO (empty) and Rescue (filled). (C) Kaplan-Meier plots of cell survival indicated by permanent global depolarization of ΔΨ_m in MCU-KO (empty) and MCU-Rescue cells (filled). (D) Flicker frequency in IP₃R-TKO without (empty) and with YFP-IP₃R1 rescue (IP₃R Rescue; filled) following stimulation with ST (addition: 5 min). (E) Changes in cytoplasmic Ca²⁺ measured with Fura2 ([Ca²⁺]_Cyt Fura2 340/380 nm). (F) Kaplan-Meier plots global ΔΨ_m loss in IP₃R TKO and IP₃R1 Rescue. (G) ΔΨ_m (top) flicker (arrow) and terminal depolarization (right) of the mitochondrial network and oligomerization of Bax CFP-Bax and YFP-Bax FRET pair (lower). (H) ΔΨ_m flickers (TMRM F/F₀ red) with Bax FRET (F_FRET/F_CFP, black). (I) Continuation of (H), Bax FRET increase (black) coincident with global ΔΨ_m loss (red). (J) Bax oligomerization in IP₃R TKO and IP₃R Rescue cells treated with ST. (K) Schematic of flicker-induced oxidation of the interface (green) engaging targets of the Ca²⁺ signaling network (IP₃R&MCU). Scale bars, 10 µm. (A), (B), (D), and (E) present mean ± SEM.