Optogenetic system induces mito-contacts to increase MMP

(a) Schematic representation of the blue light induced mitochondrial contacts. The light-sensitive proteins CRY2PHR were anchored to mitochondria via the specific organelle-targeting transmembrane domain Miro1TM. Under blue light illumination, the CRY2PHR interacted with each other to form mito-contact. Meanwhile, the fluorescent protein mCherry served as a marker of the plasmid’s expression. (b) Representative SIM images of living HeLa cells expressing CRY2PHR-mCherry-Miro1TM and stained with MitoTrackerTM Green (MTG). The left one was incubated in the dark condition and the right one was under blue light exposure with the power density of 300 μW/cm2 for 20 min. (c) The percentage contact ratio of mitochondria from SIM images of HeLa cells corresponding to (b). Data are given as M ± SEM (n = 10). (d) Representative TEM images of HeLa cells expressing CRY2PHR-mCherry-Miro1TM. The left one was incubated in the dark condition and the right one was under blue light exposure with the power density of 300 μW/cm2 for 20 min. (e) The percentage contact ratio of mitochondria from the TEM images of HeLa corresponding to (d). Data are given as M ± SEM (n = 10). (f) Representative SIM images of a living HeLa cell expressing CRY2PHR-mCherry-Miro1TM and staining MitoTrackerTM Deep Red FM (MTDR) with blue light exposure at 300 μW/cm2 for 20 min. (g) The zoom in images of different mito-contact area from (f). The red dots were light induced CRY2PHR aggregate. (h) Representative SIM images of transfected + 20 min light exposed HeLa cells staining with Rhodamine 123 to reveal real-time mitochondial membrane potatioal (MMP) in a live cell and the zoom in images are mito-contact area. (i) Relative MMP between whole cells and mito-contact area (CRY2PHR aggerate dots enriched area) corresponding to (h). Data are given as M ± SEM (n = 10). Statistical differences between the experimental groups were analyzed using a double-tailed Student’s t test. All P values less than 0.05 were considered to indicate statistical significance.

Mito-contacts increase local MMP to support ATP production upon blue-light damage

(a) Schematic representation of four groups of treatment for HeLa cells. (b) Representative SIM images of living HeLa cells exposed under blue light of 300 μW/cm2 for 3 h. The left one was from the control group and the right one was from the transfected group. (c) The percentage contact ratio of mitochondria from SIM images of HeLa cells corresponding (b). Data are given as M ± SEM (n = 10). (d) Representative SIM images of transfected + 3 h light exposed HeLa cells staining with Rhodamine 123 to reveal real-time MMP in a live cell and the zoom in images are mito-contact area. (e) Relative MMP between whole cells and mito-contact area (CRY2PHR aggerate dots enriched area) corresponding to (d). Data are given as M ± SEM (n = 10). (f) The ATP production of HeLa cells with different treatments. Data are given as M ± SEM (n = 3). (g) Quantitative analysis of mitochondrial morphology in (b). Data are given as M ± SEM (n = 10). (h) The differential gene expression between dark and 3h light treated cells from RNA-sequence data of all four groups HeLa cells. The left one was Control groups (light vs dark), mapping the 139 upregulated genes (red) and 401 downregulated genes (blue); the right one was Transfected groups (light vs dark), mapping the 30 upregulated genes (red) and 133 downregulated genes (blue). (i) The blue light-specific regulated DEGs and related pathways. (j) Schematic representation of mito-contact maintaining functions. Statistical differences between the experimental groups were analyzed using a double-tailed Student’s t test. All P values less than 0.05 were considered to indicate statistical significance.

Mito-contacts preserve MMP for mitochondrial functions upon long-time blue-light eye damage

(a) Representative SIM images of living human retinal cells (ARPE-19) before and after exposed to blue light of 300 μW/cm2 for 24 h with or within the transfection of CRY2PHR-mCherry-Miro1TM. Mitochondria stained with MTDR. (b) The percentage of mitochondria contact ratio corresponding to (a). Data are given as M ± SEM (n = 5). (c) Representative SIM images of transfected + 24 h light exposed ARPE-19 cells staining with Rhodamine 123 to reveal real-time MMP in a live cell and the zoom in images are mito-contact area. (d) Relative MMP between whole cells and mito-contact area (CRY2PHR aggerate dots enriched area) corresponding to (c). Data are given as M ± SEM (n = 10). (e) The real time OCR curves of ARPE-19 cells expressing CRY2PHR-mCherry-Miro1TM in mitochondria stress test. The OCR measured before the addition of oligomycin A represents the basal respiration of the cells; the OCR measured after the injection of FCCP reflects the maximal mitochondrial respiration capacity; and the OCR measured after the injection of rotenone and antimycin A represents non-mitochondrial respiration. Data are given as M ± SEM (n = 3). (f) The OCR of maximum respiration (i.e., cells’ maximum achievable respiration rate) of ARPE-19 cells after different treatments. (g) The OCR of the reserve capacity (i.e., the capability of the cell to respond to an energetic demand) of ARPE-19 cells after different treatments. (h) The ATP production of ARPE-19 cells after 24 h light exposure. Data are given as M ± SEM (n = 3). Statistical differences between the experimental groups were analyzed using a double-tailed Student’s t test. All P values less than 0.05 were considered to indicate statistical significance.

Mito-contact system extends lifespan of C. elegans under constant blue-light exposure

(a) The fluorescent image of C. elegans with the transgenic array containing CRY2PHR-mCherry-Miro1TM. (b) The fluorescent image of C. elegans stained with MTG and exposed to blue light. (c) The fluorescent images of C. elegans with the transgenic array containing CRY2PHR-mCherry-Miro1TM, stained MTG and exposed to blue light. (d) Survival curves of control animals (n = 60) with or without exposure to blue light. (e) Survival curves of animals (n = 60) with or without the CRY2PHR-mCherry-Miro1TM transgenic array. (f) Survival curves of animals (n = 60) exposed to blue light with or without the CRY2PHR-mCherry-Miro1TM transgenic array.