Extended Data Fig. 10 |. S-OPA1 membrane binding and remodelling activities cause lipid bilayer deformations.

a, Representative fluorescent microscopy images show the co-localization of Alexa Fluor 488 labelled WT S-OPA1 onto the Texas Red-DHPE-containing liposomes after approximately 30min. Experiments performed in technical triplicate. Scale bar, 0.5 μm. b, Membrane deformation assays with WT S-OPA1 and liposomes containing 0.25% Nile Red; n = 5 biologically independent experiments and expressed as mean, error bars as ± s.e.m. Statistical analysis was performed using an unpaired two-tailed Student t-test (P = <0.0001, ****), and the Grubbs test removed one outlier from each dataset. c-f, Negative stain TEM analysis of membrane reconstitution assays with c, S-OPA1 alone; d, liposomes with 0.25% Nile Red alone; e, BSA with liposomes containing 0.25% Nile Red; and f, WT S-OPA1 with liposomes containing 0.25% Nile Red. g, Membrane deformation assay with WT S-OPA1 and liposomes containing 1% NBD-PC; n = 5 biologically independent experiments and expressed as mean error bars as ± s.e.m. An unpaired two-tailed Student t-test was performed on the NBD-PC dataset (P = <0.0001, ****). h, liposomes with 1% NBD-PC alone; i, BSA with liposomes containing 1% NBD-PC; and j, WT S-OPA1 with liposomes containing 1% NBD-PC. Inset, close-up view of selected liposomes from negative-stain TEM images. Scale bar, 100 nm. It has been proposed that the outer leaflet perturbations of CL-containing large unilamellar vesicles (LUV) result in vesicle fusion⁸². Atomic force microscopy (AFM) and fluorescence microscopy, moreover, have revealed that the incubation of yeast S-Mgm1 with labelled liposomes results in increased membrane “roughness” on the surface of liposomes³⁷. These observations indicate that the yeast ortholog Mgm1 may utilize a similar mechanism to perturb membrane properties. Together, our structural and functional analyses suggest that OPA1-mediated leaflet perturbations support the membrane remodelling activity of the protein. We note that these conclusions are based on our current structural knowledge and are limited to lipids in synthetic membranes and reconstitution assays in vitro. To what extent these findings apply to mitochondrial membrane remodelling in human cells remains elusive. Our work provides the foundation to further study the complex mechanisms that regulate mitochondrial morphology and function.