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In situ architecture of Opa1-dependent mitochondrial cristae remodeling

Fry, Michelle Y. and Navarro, Paula P. and Qin, Xingping and Inde, Zintes and Ananda, Virly Y. and Makhlouta Lugo, Camila and Hakim, Pusparanee and Luce, Bridget E. and Ge, Yifan and McDonald, Julie L. and Ali, Ilzat and Ha, Leillani L. and Kleinstiver, Benjamin P. and Chan, David C. and Sarosiek, Kristopher A. and Chao, Luke H. (2023) In situ architecture of Opa1-dependent mitochondrial cristae remodeling. . (Unpublished)

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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 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.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Fry, Michelle Y.0000-0002-3209-5492
Navarro, Paula P.0000-0002-9123-1132
Qin, Xingping0000-0003-3263-0843
Inde, Zintes0000-0001-5564-1698
Ananda, Virly Y.0000-0001-5590-1170
Makhlouta Lugo, Camila0000-0003-4655-8096
Hakim, Pusparanee0000-0002-9018-8179
Luce, Bridget E.0000-0002-0107-5181
Ge, Yifan0000-0001-9135-9569
McDonald, Julie L.0000-0002-3715-9619
Ali, Ilzat0000-0003-2019-5645
Ha, Leillani L.0000-0003-4478-660X
Kleinstiver, Benjamin P.0000-0002-5469-0655
Chan, David C.0000-0002-0191-2154
Sarosiek, Kristopher A.0000-0002-4618-5085
Chao, Luke H.0000-0002-4849-4148
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. We are grateful to Phat Vinh Dip, and Edward Brignole at the MIT.nano cryo-EM facility and Kang Song and Chen Xu at the University of Massachusetts cryo-EM facility for providing access to the cryo-EM microscopes and for all their help, advice, and maintenance of cryo-EM equipment. Electron microscopy was performed in the Microscopy Core of the Center for Systems Biology/Program in Membrane Biology, which is partially supported by an Inflammatory Bowel Disease Grant DK043351 and a Boston Area Diabetes and Endocrinology Research Center (BADERC) Award DK057521. We thank Connor Tou for assistance cloning guide RNAs. P.P.N. was supported by the Swiss National Science Foundation (SNF) Early Postdoc.Mobility P2BSP3_188112 and Postdoc.Mobility P400PB_199252 fellowships. Z.I. was supported by NIA award F32AG077861. This work was supported by a Mass General Hospital ECOR Howard M. Goodman Fellowship (to B.P.K.); Charles H. Hood Foundation Child Health Research Awards to L.H.C. and K.A.S.; and Alex’s Lemonade Stand Foundation for Childhood Cancers Research Award to K.A.S. This work was supported by funding from the National Institutes of Health (R35GM142553 to L.H.C., R01DK125263 and R37CA248565 to K.A.S. Author Contributions. P.P.N. established cryo-FIB/cryo-ET imaging and processing pipeline. P.P.N., M.Y.F., Y.G., and J.L.M. acquired cryo-ET data. P.P.N and M.Y.F processed cryo-ET data and performed data analysis and quantification. P.P.N and I.A. performed STA analyses. P.H., B.L., Y.G., J.L.M., L.L.H., and B.P.K designed and prepared cell lines. D.C.C. generously provided the l-Opa1* and s-Opa1* cells. P.H., Y.G. performed fluorescence imaging. P.P.N. trained and supported V. A. with 3D segmentation and visualization of cryo-electron tomograms. V.A. performed segmentation of tomograms, subcompartment volume analyses, and prepared movies. C.M.L, B.L and J.L.M performed analyses of TEM images. Z.I., X.Q., performed BH3 profiling. M.Y.F., P.P.N., V.A., P.H., B.L., L.H.C. wrote the manuscript. All authors edited the manuscript. Competing Interest Statement. B.P.K is an inventor on patents and/or patent applications filed by Mass General Brigham that describe genome engineering technologies. B.P.K. is a consultant for EcoR1 capital, and is an advisor to Acrigen Biosciences, Life Edit Therapeutics, and Prime Medicine. The remaining authors declare that there are no competing financial interests.
Funding AgencyGrant Number
Swiss National Science Foundation (SNSF)P2BSP3_188112
Swiss National Science Foundation (SNSF)P400PB_199252
NIH Postdoctoral FellowshipF32AG077861
Massachusetts General HospitalUNSPECIFIED
Charles H. Hood FoundationUNSPECIFIED
Alex’s Lemonade Stand Foundation for Childhood CancersUNSPECIFIED
Record Number:CaltechAUTHORS:20230316-182139000.16
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:120134
Deposited By: George Porter
Deposited On:22 Mar 2023 01:09
Last Modified:22 Mar 2023 01:09

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