{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:41:33Z","timestamp":1785415293138,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,25]]},"reference-count":55,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T00:00:00Z","timestamp":1782345600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["N\/A"],"award-info":[{"award-number":["N\/A"]}],"id":[{"id":"https:\/\/ror.org\/02xhk2825","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001021","name":"Damon Runyon Cancer Research Foundation","doi-asserted-by":"crossref","award":["DRR-65-21"],"award-info":[{"award-number":["DRR-65-21"]}],"id":[{"id":"10.13039\/100001021","id-type":"DOI","asserted-by":"crossref"}]},{"award":["R21NS142779"],"award-info":[{"award-number":["R21NS142779"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["RF1NS125674"],"award-info":[{"award-number":["RF1NS125674"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"award":["Start-up funds"],"award-info":[{"award-number":["Start-up funds"]}],"id":[{"id":"https:\/\/ror.org\/02dxx6824","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,25]]},"abstract":"<jats:p>Mitochondrial function depends on the maintenance of its genome, and disruptions in copy number and distribution are hallmarks of mitochondrial disorders. Mitochondrial DNA (mtDNA) replication is spatially and temporally linked to mitochondrial division (i.e., fission). However, the signal that coordinates these two events, which are physically separated by the barrier of two mitochondrial membranes, remains unknown. To gain insight into this coordination, we employed correlative cryo-electron tomography (cryo-ET) to analyze the microenvironment surrounding replicating nucleoids. Mitochondrial regions containing replicating mtDNA exhibit a unique membrane architecture defined by the presence of clustered, membrane-spanning tethers that traverse the inner membrane space. Using a combination of superresolution microscopy and genetically encoded cryo-ET tagging technology, we identify these tethers as the AAA+ ATPase ATAD3A. We further show that ATAD3A knockdown reduces recruitment of the mitochondrial fission machinery, whereas overexpression promotes its recruitment and subsequent fission. Our work suggests that ATAD3A forms nanoscale linkages that coordinate these two distinct processes, revealing a new structural paradigm for organellar communication across distinct membrane-defined environments.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Highlights<\/jats:title>\n                  <jats:list list-type=\"bullet\">\n                    <jats:list-item>\n                      <jats:p>Replicating mitochondrial DNA (mtDNA) nucleoids are surrounded by a distinct membrane microenvironment.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>ATAD3A forms membrane-spanning tethers enriched at replicating mtDNA sites.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>ATAD3A enrichment is necessary and sufficient to recruit mitochondrial fission machinery and induce fission at mtDNA replication sites.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>ATAD3A structurally couples mtDNA replication state to mitochondrial fission across distinct organellar subcompartments.<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.23.733767","type":"posted-content","created":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T09:45:13Z","timestamp":1782380713000},"source":"Crossref","is-referenced-by-count":0,"title":["ATAD3A structurally links mtDNA replication and mitochondrial fission"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2584-8150","authenticated-orcid":false,"given":"Nitish","family":"Dua","sequence":"first","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7740-7314","authenticated-orcid":false,"given":"Boyuan","family":"Ma","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samantha","family":"Oviedo","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"},{"name":"Department of Molecular and Cellular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0222-6989","authenticated-orcid":false,"given":"Hamidreza","family":"Rahmani","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tumara","family":"Boyd","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2048-6004","authenticated-orcid":false,"given":"Donghyun","family":"Park","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9287-6840","authenticated-orcid":false,"given":"R. Luke","family":"Wiseman","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"},{"name":"Department of Molecular and Cellular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5908-7882","authenticated-orcid":false,"given":"Danielle A.","family":"Grotjahn","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026062711550533000_2026.06.23.733767v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"},{"key":"2026062711550533000_2026.06.23.733767v1.2","doi-asserted-by":"crossref","first-page":"110139","DOI":"10.1016\/j.celrep.2021.110139","article-title":"ATAD3A has a scaffolding role regulating mitochondria inner membrane structure and protein assembly","volume":"37","year":"2021","journal-title":"Cell Reports"},{"key":"2026062711550533000_2026.06.23.733767v1.3","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1301951110"},{"key":"2026062711550533000_2026.06.23.733767v1.4","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.202204093"},{"key":"2026062711550533000_2026.06.23.733767v1.5","doi-asserted-by":"publisher","DOI":"10.1126\/science.adp7114"},{"key":"2026062711550533000_2026.06.23.733767v1.6","unstructured":"Brocher, J. biovoxxel\/BioVoxxel-Toolbox, (2026). URL https:\/\/github.com\/biovoxxel\/BioVoxxel-Toolbox."},{"key":"2026062711550533000_2026.06.23.733767v1.7","doi-asserted-by":"crossref","first-page":"110000","DOI":"10.1016\/j.celrep.2021.110000","article-title":"The TFAM-to-mtDNA ratio defines inner-cellular nucleoid populations with distinct activity levels","volume":"37","year":"2021","journal-title":"Cell Reports"},{"key":"2026062711550533000_2026.06.23.733767v1.8","doi-asserted-by":"publisher","DOI":"10.1152\/ajpendo.00457.2019"},{"key":"2026062711550533000_2026.06.23.733767v1.9","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddx042"},{"key":"2026062711550533000_2026.06.23.733767v1.10","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-genom-091416-035426"},{"key":"2026062711550533000_2026.06.23.733767v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-022-01746-2"},{"key":"2026062711550533000_2026.06.23.733767v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-022-01690-1"},{"key":"2026062711550533000_2026.06.23.733767v1.13","doi-asserted-by":"publisher","DOI":"10.1002\/1873-3468.14021"},{"key":"2026062711550533000_2026.06.23.733767v1.14","doi-asserted-by":"crossref","unstructured":"Friedman, J. R. et al. (2011). Er tubules mark sites of mitochondrial division. Science, 334: 358\u2013362.","DOI":"10.1126\/science.1207385"},{"key":"2026062711550533000_2026.06.23.733767v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2023.09.026"},{"key":"2026062711550533000_2026.06.23.733767v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-023-02053-0"},{"key":"2026062711550533000_2026.06.23.733767v1.17","doi-asserted-by":"crossref","first-page":"1583","DOI":"10.1091\/mbc.e02-07-0399","article-title":"Composition and dynamics of human mitochondrial nucleoids","volume":"14","year":"2003","journal-title":"MBoC"},{"key":"2026062711550533000_2026.06.23.733767v1.18","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.00007-10"},{"key":"2026062711550533000_2026.06.23.733767v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajhg.2016.08.007"},{"key":"2026062711550533000_2026.06.23.733767v1.20","doi-asserted-by":"crossref","first-page":"111774","DOI":"10.1016\/j.celrep.2022.111774","article-title":"Mitochondrial cristae architecture protects against mtDNA release and inflammation","volume":"41","year":"2022","journal-title":"Cell Reports"},{"key":"2026062711550533000_2026.06.23.733767v1.21","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.200609158"},{"key":"2026062711550533000_2026.06.23.733767v1.22","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/bs.mcb.2017.03.014","article-title":"ec-CLEM: A multidimension, multimodel software to correlate intermodal images with a focus on light and electron microscopy","volume":"140","year":"2017","journal-title":"Methods Cell Biol"},{"key":"2026062711550533000_2026.06.23.733767v1.23","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2210730119"},{"key":"2026062711550533000_2026.06.23.733767v1.24","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.abi8886"},{"key":"2026062711550533000_2026.06.23.733767v1.25","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.11553"},{"key":"2026062711550533000_2026.06.23.733767v1.26","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.20945"},{"key":"2026062711550533000_2026.06.23.733767v1.27","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03510-6"},{"key":"2026062711550533000_2026.06.23.733767v1.28","doi-asserted-by":"crossref","unstructured":"Kondadi, A. K. and Reichert, A. S. (2024). Mitochondrial dynamics at different levels: From cristae dynamics to interorganellar cross talk. Annu. Rev. Biophys., 53:annurev\u2013biophys\u2013030822\u2013020736.","DOI":"10.1146\/annurev-biophys-030822-020736"},{"key":"2026062711550533000_2026.06.23.733767v1.29","doi-asserted-by":"publisher","DOI":"10.1101\/2024.01.05.574336"},{"key":"2026062711550533000_2026.06.23.733767v1.30","doi-asserted-by":"crossref","unstructured":"Lewis, S. C. , Uchiyama, L. F. , and Nunnari, J. (2016). Er-mitochondria contacts couple mtdna synthesis with mitochondrial division in human cells. Science, 353.","DOI":"10.1126\/science.aaf5549"},{"key":"2026062711550533000_2026.06.23.733767v1.31","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2215799119"},{"key":"2026062711550533000_2026.06.23.733767v1.32","doi-asserted-by":"crossref","first-page":"106488","DOI":"10.1016\/j.nbd.2024.106488","article-title":"Progressive alterations in polysomal architecture and activation of ribosome stalling relief factors in a mouse model of Huntington\u2019s disease","volume":"195","year":"2024","journal-title":"Neurobiol Dis"},{"key":"2026062711550533000_2026.06.23.733767v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2005.07.007"},{"key":"2026062711550533000_2026.06.23.733767v1.34","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2016.07.011"},{"key":"2026062711550533000_2026.06.23.733767v1.35","doi-asserted-by":"publisher","DOI":"10.2183\/pjab.93.021"},{"key":"2026062711550533000_2026.06.23.733767v1.36","doi-asserted-by":"publisher","DOI":"10.1038\/s41556-023-01343-1"},{"key":"2026062711550533000_2026.06.23.733767v1.37","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.217075"},{"key":"2026062711550533000_2026.06.23.733767v1.38","doi-asserted-by":"publisher","DOI":"10.1002\/PRO.3943"},{"key":"2026062711550533000_2026.06.23.733767v1.39","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-019-0183-6"},{"key":"2026062711550533000_2026.06.23.733767v1.40","doi-asserted-by":"crossref","unstructured":"Purnell, C. , Heebner, J. , Swulius, M. T. , Hylton, R. , Kabonick, S. , Grillo, M. , Grigoryev, S. , Heberle, F. , Waxham, M. N. , and Swulius, M. T. (2023). Rapid synthesis of cryo-ET data for training deep learning models. bioRxiv, page 2023.04.28.538636.","DOI":"10.1101\/2023.04.28.538636"},{"key":"2026062711550533000_2026.06.23.733767v1.41","doi-asserted-by":"crossref","first-page":"6704","DOI":"10.1038\/s41467-022-34205-9","article-title":"Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA","volume":"13","year":"2022","journal-title":"Nat Commun"},{"key":"2026062711550533000_2026.06.23.733767v1.42","doi-asserted-by":"publisher","DOI":"10.1017\/S1551929521001280"},{"key":"2026062711550533000_2026.06.23.733767v1.43","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.12.8.2245"},{"key":"2026062711550533000_2026.06.23.733767v1.44","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-48838-2"},{"key":"2026062711550533000_2026.06.23.733767v1.45","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2024.05.017"},{"key":"2026062711550533000_2026.06.23.733767v1.46","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2006.05.009"},{"key":"2026062711550533000_2026.06.23.733767v1.47","doi-asserted-by":"publisher","DOI":"10.1038\/nrg1606"},{"key":"2026062711550533000_2026.06.23.733767v1.48","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0580-y"},{"key":"2026062711550533000_2026.06.23.733767v1.49","doi-asserted-by":"publisher","DOI":"10.1038\/onc.2015.86"},{"key":"2026062711550533000_2026.06.23.733767v1.50","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.bbabio.2009.09.005","article-title":"Mitochondrial dna mutations and human disease","volume":"1797","year":"2010","journal-title":"Biochimica et Biophysica Acta (BBA) - Bioenergetics"},{"key":"2026062711550533000_2026.06.23.733767v1.51","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-51438-y"},{"key":"2026062711550533000_2026.06.23.733767v1.52","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2671"},{"key":"2026062711550533000_2026.06.23.733767v1.53","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/s41556-025-01829-0","article-title":"MISO regulates mitochondrial dynamics and mtDNA homeostasis by establishing membrane subdomains","volume":"28","year":"2026","journal-title":"Nat Cell Biol"},{"key":"2026062711550533000_2026.06.23.733767v1.54","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09078-0"},{"key":"2026062711550533000_2026.06.23.733767v1.55","doi-asserted-by":"crossref","unstructured":"Zollo, C. , Gomez Suarez, D. , Bararpour, E. P. , Jenner, A. , Verma, P. , Koch, J. , Wilhelm, S. , J\u00fcngst, C. , Faber, L. , White, F. , and Garc\u00eda-S\u00e1ez, A. J. (2026). DRP1 and MID49 co-diffusion scans mitochondria for fission. Nature Cell Biology. Advance online publication.","DOI":"10.1038\/s41556-026-01986-w"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.06.23.733767","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,27]],"date-time":"2026-06-27T18:55:19Z","timestamp":1782586519000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.06.23.733767"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,25]]},"references-count":55,"URL":"https:\/\/doi.org\/10.64898\/2026.06.23.733767","relation":{},"subject":[],"published":{"date-parts":[[2026,6,25]]},"subtype":"preprint"}}