In situ architecture of Opa1-dependent mitochondrial cristae 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 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 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. HighlightsO_LIIn situ ultrastructural characterization of mitochondrial cristae with different forms of Opa1. C_LIO_LIMitochondria with predominantly l-Opa1 show crista stacking, longer cristae, reduced globular cristae and an absence of tubular cristae. C_LIO_LIMitochondria with mostly s-Opa1 showed irregular cristae packing with wider cristae junctions and narrower cristae. C_LIO_LIl-Opa1 expressing cells with WT-like cristae junction properties, show wild-type apoptotic response and calcium handling. C_LIO_LIImbalance in Opa1 processing show compromised respiratory function and an increase in amorphous cristae. C_LI