Fig 2: Hemifusion and full fusion.(A) Hemifusion (lipid mixing) and full fusion (content release and pore opening) efficiency for homotypic l-Opa1, heterotypic l-Opa1 and s-Opa1 (p<0.001, two-way ANOVA). Bar graphs shown as mean ± SD. Error bars are from five different experiments (50–200 particles were analyzed per bilayer in each experiment). B. Full fusion (pore opening) efficiency at different s-Opa1:l-Opa1 ratios. Data are shown as mean ± SD. Error bars are from 4 to 6 experiments (80–150 particles per bilayer in each experiment). The significance of the data was confirmed using one-way ANOVA (Prism 8.3) where p<0.0001. C. Mean pore opening time in the absence of s-Opa1 and at equimolar s-Opa1. Significance of the difference was confirmed using t-test (Prism 8.3, p<0.0001). D. Representative hemifusion and pore opening fluorescence time series for homotypic l-Opa1 experiment, in the absence of s-Opa1, top and bottom panels, respectively. Scale bar: 1 µm. E: representative traces of TexasRed (liposome signal) and calcein (content signal) intensity for homotypic l-Opa1 experiment. F. Representative hemifusion and pore opening fluorescence traces for a homotypic l-Opa1 experiment in the presence of equimolar s-Opa1. Scale bar: 1 µm. G: Representative trace of TexasRed (liposome signal) and calcein (content signal) intensity for a homotypic l-Opa1 experiment in the presence of equimolar s-Opa1.Figure 6—source data 1.Hemifusion and pore opening.

Fig 3: Immunoblotting analysis of mitochondrial dynamic proteins. Immunoblotting analysis was assessed using lysates prepared from post-mortem brains of seven-month-old hAbKI mice and treated hAbKI mice with Uralithin A and EGCG. (A) Representative immunoblots for hAbKI mice and treated hAbKI mice with Urolithin A and EGCG. (B) Quantitative-densitometry analysis for mitochondrial fission genes Drp1 and Fis1 and fusion proteins, which were significantly decreased in the treated hAbKI mice Urolithin A and EGCG as compared to the hAbKI mice. Mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 were significantly increased in urolithin A-treated hAbKI mice and combined treatment of urolithin A+EGCG in 7-month-old hAbKI mice.

Fig 4: Reconstitution of l-Opa1.(A) Representative size-exclusion chromatograph and SDS-PAGE gel of human l-Opa1 purified from P. pastoris. (B) SDS-PAGE gel of human s-Opa1 purified from P. pastoris. l-Opa1 activity, with velocity (C) and specific activity (D) of GTP hydrolysis in the presence and absence of cardiolipin, while varying protein concentration of Opa1. Data are shown as mean ± SD, with error bars from three independent experiments. Representative single-liposome photobleaching steps (E and F) and histogram of step sizes (distribution for 110 liposomes shown) (G).Figure 2—source data 1.Reconstitution of l-Opa1.

Fig 5: Docking.(A) Homotypic l-Opa1 docks liposomes in a GTP-hydrolysis dependent manner. s-Opa1, alone is insufficient to closely dock liposomes. l-Opa1 in a heterotypic format (on the liposome alone) is competent to closely dock to a bilayer, but this docking is not stimulated by nucleotide. Bar graphs shown as mean ± SD (p<0.0001, one-way ANOVA). Error bars are from 3 to 5 independent experiments (each experiment with >150 particles in a given bilayer). (B) In the presence of cardiolipin on both bilayers, FRET signal reports on close liposome docking mediated by l-Opa1. Left: Green = Cy5 emission signal upon excitation at 543 (TexasRed excitation). Red = Cy5 emission signal in membrane upon excitation at 633 (Cy5 excitation). Right: Green = TexasRed emission upon excitation at 543 nm (TexasRed excitation). Scale bar: 5 µm.Figure 4—source data 1.Docking.