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Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to the lack of native 3D views of cristae morphology. We performin situcryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with well-defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe elongated mitochondria with a notable stacking phenotype, as well as an absence of tubular cristae, when only l-Opa1 is present. In contrast, when mitochondria contain mainly s-Opa1, we observe irregular cristae packing, an increase in globular cristae, and decreased matrix condensation. Notably, we find the absence of l-Opa1 results in mitochondria with wider cristae junctions. BH3 profiling reveals that absence of l-Opa1 reduces cytochrome c release in response to pro-apoptotic stimuli and protects cells from apoptosis induced by anti-cancer agents. We discuss the implications Opa1-dependent cristae morphologies in cell death initiation.HighlightsIn situultrastructural characterization of mitochondrial cristae with different forms of Opa1.Mitochondria with predominantly l-Opa1 show cristae stacking, longer cristae compared to WT, but also a reduction of globular cristae and no tubular cristae.Mitochondria with mostly s-Opa1 showed irregular cristae packing with wider cristae junctions and more narrow cristae than WT.BH3 profiling show Opa1-knock-out cells have reduced apoptotic priming and reduced sensitivity to apoptosis-inducing agents, and the presence l-Opa1 restores a WT protective apoptotic response.