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. Author manuscript; available in PMC: 2025 Sep 5.
Published in final edited form as: Nature. 2023 Aug 23;620(7976):1109–1116. doi: 10.1038/s41586-023-06462-1

Fig. 4: Examining the functional impact of OPA1 interface and membrane binding mutants.

Fig. 4:

(a-d) Images of HeLa-M cells transfected with WT or mutant OPA1-GFP. Left, mitochondria immunostained against Tom20. Middle, OPA1-GFP localization to the mitochondrial network. Right, colocalization of OPA1-GFP and mitochondria immunostained against Tom20. The scale bar measures 10 μm and 3 μm in the zoomed insets. A, WT Opa1-GFP display filamentous mitochondria. b-d, OPA1-GFP interface-1(b) mutant (E626A:T630A:K668A:E671A) (b), OPA1-GFP interface-7 mutant (K819A, N820A) (c), and the membrane-inserting loop, Pα2, OPA1-GFP mutant (W771A:K772A:K773A:R774A:W775A:L776A:Y777A:W778A:K779A:N780A: R781A) (d) all cause fragmentation of the mitochondrial network. E-g, Models of the OPA1 mutants transfected in (b-d) with s-OPA1 colored by domain (see Fig. 1), mutated residues labeled and colored as red sticks and other H-bonding residues in the interface shown as sticks colored by domain. Residues in red associated with DOA mutations are bolded. H, Table summarizing mitochondrial network fragmentation for each mutant. Mitochondrial networks were measured and categorized as filamentous (blue), intermediate (orange), or fragmented (gray) and plotted as a percentage of total cells. The number (N) of cells counted appear above each bar. The alanine mutations introduced into the OPA1-GFP sequence were: R627A:K663A:R683A (I1(a), Interface-1), E626A:T630A:K668A:E671A (I1(b), Interface-1), Q659A (I1(c), Interface-1), E679A (I1(d), Interface-1), H631A (I1(e), Interface-1), K614A:H615A:D835A (I2, Interface-2), F860A:Y861A:Y862A:Y863A:Q864A:R865A:H866A:F867A (I4(a)/Pα6, Interface-4 and Pα6 membrane binding), I735A (I4(b), Interface-4), E444A (I5, Interface-5), D716A (I6, Interface-6), K819A:N820A (I7, Interface-7), R235A (I8, Interface-8), E213A:Q217A:R557A:D565A (I9, Interface-9), W771A:K772A:K773A:R774A:W775A:L776A:Y777A:W778A:K779A:N780A: R781A (Pα2, Pα2 membrane binding), L243A (C1, non-interfacial control 1), L248A (C2, non-interfacial control 2), K369A (C3, non-interfacial control 3), T651A (C4, non-interfacial control 4). Detailed images and statistics for each interface mutant can be found in Extended Data Fig. 9 and 10. Experiments were repeated 3 times, each experiment was repeated independently with similar results.