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• W3103286458 abstract "•Innate immune sensing of cytosolic RNA induces profound mitochondrial fusion•The TBK1-DRP1 axis underlies RNA sensing-triggered mitochondrial reprogramming•TBK1 directly phosphorylates DRP1 to disable its multimeric assembly and function•TBK1-DRP1 signaling enables large MAVS aggregation and healthy antiviral immunity Mitochondrial morphology shifts rapidly to manage cellular metabolism, organelle integrity, and cell fate. It remains unknown whether innate nucleic acid sensing, the central and general mechanisms of monitoring both microbial invasion and cellular damage, can reprogram and govern mitochondrial dynamics and function. Here, we unexpectedly observed that upon activation of RIG-I-like receptor (RLR)-MAVS signaling, TBK1 directly phosphorylated DRP1/DNM1L, which disabled DRP1, preventing its high-order oligomerization and mitochondrial fragmentation function. The TBK1-DRP1 axis was essential for assembly of large MAVS aggregates and healthy antiviral immunity and underlay nutrient-triggered mitochondrial dynamics and cell fate determination. Knockin (KI) strategies mimicking TBK1-DRP1 signaling produced dominant-negative phenotypes reminiscent of human DRP1 inborn mutations, while interrupting the TBK1-DRP1 connection compromised antiviral responses. Thus, our findings establish an unrecognized function of innate immunity governing both morphology and physiology of a major organelle, identify a lacking loop during innate RNA sensing, and report an elegant mechanism of shaping mitochondrial dynamics. Mitochondrial morphology shifts rapidly to manage cellular metabolism, organelle integrity, and cell fate. It remains unknown whether innate nucleic acid sensing, the central and general mechanisms of monitoring both microbial invasion and cellular damage, can reprogram and govern mitochondrial dynamics and function. Here, we unexpectedly observed that upon activation of RIG-I-like receptor (RLR)-MAVS signaling, TBK1 directly phosphorylated DRP1/DNM1L, which disabled DRP1, preventing its high-order oligomerization and mitochondrial fragmentation function. The TBK1-DRP1 axis was essential for assembly of large MAVS aggregates and healthy antiviral immunity and underlay nutrient-triggered mitochondrial dynamics and cell fate determination. Knockin (KI) strategies mimicking TBK1-DRP1 signaling produced dominant-negative phenotypes reminiscent of human DRP1 inborn mutations, while interrupting the TBK1-DRP1 connection compromised antiviral responses. Thus, our findings establish an unrecognized function of innate immunity governing both morphology and physiology of a major organelle, identify a lacking loop during innate RNA sensing, and report an elegant mechanism of shaping mitochondrial dynamics. To defend against pathogen infection and maintain tissue homeostasis, metazoans deploy innate immune-sensing mechanisms via pattern recognition receptors to surveil conserved pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). The RIG-I-like receptor (RLR) family RNA sensors RIG-I (Yoneyama et al., 2004Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (2916) Google Scholar) and MDA5 (Kang et al., 2002Kang D.C. Gopalkrishnan R.V. Wu Q. Jankowsky E. Pyle A.M. Fisher P.B. mda-5: an interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties.Proc. Natl. Acad. Sci. USA. 2002; 99: 637-642Crossref PubMed Scopus (485) Google Scholar) are ubiquitously expressed and constitute essential components of these surveillance systems, in combating viral pathogens that have penetrated physical barriers, such as the RNA viruses influenza A/B, hepatitis C virus (HCV), dengue virus, and West Nile virus (WNV) and some DNA viruses and retroviruses, such as hepatitis B virus (HBV) (Suslov et al., 2018Suslov A. Wieland S. Menne S. Modulators of innate immunity as novel therapeutics for treatment of chronic hepatitis B.Curr. Opin. Virol. 2018; 30: 9-17Crossref PubMed Scopus (25) Google Scholar). RLRs sense viral RNA in the cytosol and trigger a signaling cascade (Sharma et al., 2003Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Triggering the interferon antiviral response through an IKK-related pathway.Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1280) Google Scholar; Fitzgerald et al., 2003Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. 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Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (4860) Google Scholar; Yoneyama et al., 2015Yoneyama M. Onomoto K. Jogi M. Akaboshi T. Fujita T. Viral RNA detection by RIG-I-like receptors.Curr. Opin. Immunol. 2015; 32: 48-53Crossref PubMed Scopus (265) Google Scholar; Chan and Gack, 2015Chan Y.K. Gack M.U. RIG-I-like receptor regulation in virus infection and immunity.Curr. Opin. Virol. 2015; 12: 7-14Crossref PubMed Scopus (109) Google Scholar). Activation of innate RNA sensing can dramatically change the physiological processes and fate of host cells (Franz and Kagan, 2017Franz K.M. Kagan J.C. Innate immune receptors as competitive determinants of cell fate.Mol. Cell. 2017; 66: 750-760Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar; Xu et al., 2014Xu P. Bailey-Bucktrout S. Xi Y. Xu D. Du D. Zhang Q. Xiang W. Liu J. Melton A. 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Treuting P.M. Stetson D.B. Isoforms of RNA-editing enzyme ADAR1 independently control nucleic acid sensor MDA5-driven autoimmunity and multi-organ development.Immunity. 2015; 43: 933-944Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar; Rice et al., 2014Rice G.I. Del Toro Duany Y. Jenkinson E.M. Forte G.M. Anderson B.H. Ariaudo G. Bader-Meunier B. Baildam E.M. Battini R. Beresford M.W. et al.Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling.Nat. Genet. 2014; 46: 503-509Crossref PubMed Scopus (349) Google Scholar). TBK1 and IKKε, the kinases and major signaling mediator downstream of RLR-MAVS signaling, also function as key regulators of apoptosis, autophagy, and inflammatory responses (Baldwin, 2012Baldwin A.S. Regulation of cell death and autophagy by IKK and NF-κB: critical mechanisms in immune function and cancer.Immunol. Rev. 2012; 246: 327-345Crossref PubMed Scopus (201) Google Scholar; Pilli et al., 2012Pilli M. Arko-Mensah J. Ponpuak M. Roberts E. Master S. Mandell M.A. Dupont N. Ornatowski W. Jiang S. Bradfute S.B. et al.TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation.Immunity. 2012; 37: 223-234Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar) and act as important inducers that drive tumorigenesis (Barbie et al., 2009Barbie D.A. Tamayo P. Boehm J.S. Kim S.Y. Moody S.E. Dunn I.F. Schinzel A.C. Sandy P. Meylan E. Scholl C. et al.Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1.Nature. 2009; 462: 108-112Crossref PubMed Scopus (1384) Google Scholar). Nevertheless, the impacts of innate RNA sensing on cell biological functions, such as cellular metabolism, organelle behavior, and cell fate determination, still remain largely undetermined. The mitochondria-associated MAVS (also called VISA, IPS-1, and Cardif) facilitates signaling from the activated sensors RIG-I and MDA5 (Yoneyama et al., 2004Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (2916) Google Scholar; Kawai et al., 2005Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (1898) Google Scholar; Meylan et al., 2005Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1864) Google Scholar; Seth et al., 2005Seth R.B. Sun L. Ea C.K. Chen Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2260) Google Scholar; Xu et al., 2005Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. VISA is an adapter protein required for virus-triggered IFN-beta signaling.Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1426) Google Scholar), which forms prion-like aggregates on the mitochondrial surface (Hou et al., 2011Hou F. Sun L. Zheng H. Skaug B. Jiang Q.X. Chen Z.J. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.Cell. 2011; 146: 448-461Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar; Kowalinski et al., 2011Kowalinski E. Lunardi T. McCarthy A.A. Louber J. Brunel J. Grigorov B. Gerlier D. Cusack S. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA.Cell. 2011; 147: 423-435Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar; Luo et al., 2011Luo D. Ding S.C. Vela A. Kohlway A. Lindenbach B.D. Pyle A.M. Structural insights into RNA recognition by RIG-I.Cell. 2011; 147: 409-422Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), resulting in the activation of TBK1/IKKε and IKKα/β. Localization of MAVS on the mitochondria and K63 polyubiquitination is required for MAVS-instructed activation of TBK1/IKKs (Li et al., 2005Li X.D. Sun L. Seth R.B. Pineda G. Chen Z.J. Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity.Proc. Natl. Acad. Sci. USA. 2005; 102: 17717-17722Crossref PubMed Scopus (617) Google Scholar; Meylan et al., 2005Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1864) Google Scholar; Liu et al., 2013Liu S. Chen J. Cai X. Wu J. Chen X. Wu Y.T. Sun L. Chen Z.J. MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades.eLife. 2013; 2: e00785Crossref PubMed Google Scholar). TBK1/IKKε are activated via intermolecular trans-autophosphorylation (Larabi et al., 2013Larabi A. Devos J.M. Ng S.L. Nanao M.H. Round A. Maniatis T. Panne D. Crystal structure and mechanism of activation of TANK-binding kinase 1.Cell Rep. 2013; 3: 734-746Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar; Li et al., 2011Li S. Wang L. Berman M. Kong Y.Y. Dorf M.E. Mapping a dynamic innate immunity protein interaction network regulating type I interferon production.Immunity. 2011; 35: 426-440Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar; Ma et al., 2012Ma X. Helgason E. Phung Q.T. Quan C.L. Iyer R.S. Lee M.W. Bowman K.K. Starovasnik M.A. Dueber E.C. Molecular basis of Tank-binding kinase 1 activation by transautophosphorylation.Proc. Natl. Acad. Sci. USA. 2012; 109: 9378-9383Crossref PubMed Scopus (131) Google Scholar; Tu et al., 2013Tu D. Zhu Z. Zhou A.Y. Yun C.H. Lee K.E. Toms A.V. Li Y. Dunn G.P. Chan E. Thai T. et al.Structure and ubiquitination-dependent activation of TANK-binding kinase 1.Cell Rep. 2013; 3: 747-758Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and subject to complex regulation (Tojima et al., 2000Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. et al.NAK is an IkappaB kinase-activating kinase.Nature. 2000; 404: 778-782Crossref PubMed Scopus (301) Google Scholar), including regulation by the ion metal phosphatase PPM1A (Xiang et al., 2016Xiang W. Zhang Q. Lin X. Wu S. Zhou Y. Meng F. Fan Y. Shen T. Xiao M. Xia Z. et al.PPM1A silences cytosolic RNA sensing and antiviral defense through direct dephosphorylation of MAVS and TBK1.Sci. Adv. 2016; 2: e1501889Crossref PubMed Scopus (38) Google Scholar), the Hippo signaling effectors YAP and TAZ (Zhang et al., 2017Zhang Q. Meng F. Chen S. Plouffe S.W. Wu S. Liu S. Li X. Zhou R. Wang J. Zhao B. et al.Hippo signalling governs cytosolic nucleic acid sensing through YAP/TAZ-mediated TBK1 blockade.Nat. Cell Biol. 2017; 19: 362-374Crossref PubMed Scopus (94) Google Scholar), and tyrosine phosphorylation (Liu et al., 2017Liu S. Chen S. Li X. Wu S. Zhang Q. Jin Q. Hu L. Zhou R. Yu Z. Meng F. et al.Lck/Hck/Fgr-mediated tyrosine phosphorylation negatively regulates TBK1 to restrain innate antiviral responses.Cell Host Microbe. 2017; 21: 754-768.e5Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Facilitated by TBK1-mediated phosphorylation of MAVS (Liu et al., 2015Liu S. Cai X. Wu J. Cong Q. Chen X. Li T. Du F. Ren J. Wu Y.T. Grishin N.V. Chen Z.J. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.Science. 2015; 347: aaa2630Crossref PubMed Scopus (681) Google Scholar), TBK1 and/or IKKε phosphorylate IRF3 in the MAVS signalosome, which mobilizes IRF3 to dimerize and translocate to the nucleus, where it functions as a transcription factor in coordination with simultaneously activated nuclear factor κB (NF-κB) (Roers et al., 2016Roers A. Hiller B. Hornung V. Recognition of endogenous nucleic acids by the innate immune system.Immunity. 2016; 44: 739-754Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar; Takeuchi and Akira, 2010Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (4860) Google Scholar; Chen et al., 2016Chen Q. Sun L. Chen Z.J. Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing.Nat. Immunol. 2016; 17: 1142-1149Crossref PubMed Scopus (693) Google Scholar). However, the signaling cascade through RNA sensing to IRF3 activation appears extremely slow on the cellular scale, and it remains largely unclear for downstream events upon MAVS activation. Intriguingly, the interference of mitochondrial fusion regulators appears to be deleterious to RNA sensing signaling (Yasukawa et al., 2009Yasukawa K. Oshiumi H. Takeda M. Ishihara N. Yanagi Y. Seya T. Kawabata S. Koshiba T. Mitofusin 2 inhibits mitochondrial antiviral signaling.Sci. Signal. 2009; 2: ra47Crossref PubMed Scopus (173) Google Scholar; Castanier et al., 2010Castanier C. Garcin D. Vazquez A. Arnoult D. Mitochondrial dynamics regulate the RIG-I-like receptor antiviral pathway.EMBO Rep. 2010; 11: 133-138Crossref PubMed Scopus (201) Google Scholar; Onoguchi et al., 2010Onoguchi K. Onomoto K. Takamatsu S. Jogi M. Takemura A. Morimoto S. Julkunen I. Namiki H. Yoneyama M. Fujita T. Virus-infection or 5’ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1.PLoS Pathog. 2010; 6: e1001012Crossref PubMed Scopus (130) Google Scholar). In response to intra- and extracellular cues and the metabolic context, mitochondrial dynamics change rapidly to manage the subtle balance between a hyperfused network that regulates efficient electron transport chain (ETC) supercomplex formation and oxidative phosphorylation (OXPHOS) and cell survival versus mitochondrial fragmentation, which is associated with improved reactive oxygen species (ROS) production, mitophagy, and cell death (Wai and Langer, 2016Wai T. Langer T. Mitochondrial dynamics and metabolic regulation.Trends Endocrinol. Metab. 2016; 27: 105-117Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar; Sprenger and Langer, 2019Sprenger H.G. Langer T. The good and the bad of mitochondrial breakups.Trends Cell Biol. 2019; 29: 888-900Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Mitochondria also function as the key platform in immunity, releasing mitochondrial DNA (mtDNA) as DAMPs, constructing the MAVS signaling complex and the NLRP3 inflammasome, and participating considerably in the maturation, migration, and regulation of various adaptive immune cells (Mills et al., 2017Mills E.L. Kelly B. O’Neill L.A.J. Mitochondria are the powerhouses of immunity.Nat. Immunol. 2017; 18: 488-498Crossref PubMed Scopus (362) Google Scholar; Buck et al., 2017Buck M.D. Sowell R.T. Kaech S.M. Pearce E.L. Metabolic instruction of immunity.Cell. 2017; 169: 570-586Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar; Nagata and Tanaka, 2017Nagata S. Tanaka M. Programmed cell death and the immune system.Nat. Rev. Immunol. 2017; 17: 333-340Crossref PubMed Scopus (170) Google Scholar). Key regulators that mediate the switch between mitochondrial fusion and fission include OPA1, which is responsible for fusion of the inner membrane (Misaka et al., 2002Misaka T. Miyashita T. Kubo Y. Primary structure of a dynamin-related mouse mitochondrial GTPase and its distribution in brain, subcellular localization, and effect on mitochondrial morphology.J. Biol. Chem. 2002; 277: 15834-15842Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar; Olichon et al., 2003Olichon A. Baricault L. Gas N. Guillou E. Valette A. Belenguer P. Lenaers G. Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis.J. Biol. Chem. 2003; 278: 7743-7746Abstract Full Text Full Text PDF PubMed Scopus (825) Google Scholar); MFN1 and MFN2, which are responsible for fusion of the outer membrane (Santel and Fuller, 2001Santel A. Fuller M.T. Control of mitochondrial morphology by a human mitofusin.J. 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Otera H. Nakanishi Y. Nonaka I. Goto Y. et al.Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice.Nat. Cell Biol. 2009; 11: 958-966Crossref PubMed Scopus (675) Google Scholar). Patients with a DRP1 point mutation, mostly in the middle domain (MD) (Waterham et al., 2007Waterham H.R. Koster J. van Roermund C.W. Mooyer P.A. Wanders R.J. Leonard J.V. A lethal defect of mitochondrial and peroxisomal fission.N. Engl. J. Med. 2007; 356: 1736-1741Crossref PubMed Scopus (544) Google Scholar; Vanstone et al., 2016Vanstone J.R. Smith A.M. McBride S. Naas T. Holcik M. Antoun G. Harper M.E. Michaud J. Sell E. Chakraborty P. et al.Care4Rare ConsortiumDNM1L-related mitochondrial fission defect presenting as refractory epilepsy.Eur. J. Hum. Genet. 2016; 24: 1084-1088Crossref PubMed Scopus (80) Google Scholar; Chao et al., 2016Chao Y.H. Robak L.A. Xia F. Koenig M.K. Adesina A. Bacino C.A. Scaglia F. Bellen H.J. Wangler M.F. Missense variants in the middle domain of DNM1L in cases of infantile encephalopathy alter peroxisomes and mitochondria when assayed in Drosophila.Hum. Mol. Genet. 2016; 25: 1846-1856Crossref PubMed Scopus (42) Google Scholar), display dramatic phenotypes with fused and elongated mitochondria and the failure of multiple organs closely related to mitochondrial function (Waterham et al., 2007Waterham H.R. Koster J. van Roermund C.W. Mooyer P.A. Wanders R.J. Leonard J.V. A lethal defect of mitochondrial and peroxisomal fission.N. Engl. J. Med. 2007; 356: 1736-1741Crossref PubMed Scopus (544) Google Scholar; Vanstone et al., 2016Vanstone J.R. Smith A.M. McBride S. Naas T. Holcik M. Antoun G. Harper M.E. Michaud J. Sell E. Chakraborty P. et al.Care4Rare ConsortiumDNM1L-related mitochondrial fission defect presenting as refractory epilepsy.Eur. J. Hum. Genet. 2016; 24: 1084-1088Crossref PubMed Scopus (80) Google Scholar; Chao et al., 2016Chao Y.H. Robak L.A. Xia F. Koenig M.K. Adesina A. Bacino C.A. Scaglia F. Bellen H.J. Wangler M.F. Missense variants in the middle domain of DNM1L in cases of infantile encephalopathy alter peroxisomes and mitochondria when assayed in Drosophila.Hum. Mol. Genet. 2016; 25: 1846-1856Crossref PubMed Scopus (42) Google Scholar). Despite the ultimate importance of mitochondrial dynamics in cellular physiology and adaptive immunity, it remains unknown whether and how this key biological event is intuitively managed by innate immunity. Here, we unexpectedly found that TBK1 directly and massively phosphorylates DRP1 in the MAVS signaling complex upon innate RNA sensing. TBK1-mediated phosphorylation of DRP1 was efficient and exhibited a dominant-negative effect, completely eliminating the mitochondrial incision function of DRP1. This TBK1-DRP1 axis was critical for innate RNA sensing and antiviral immunity, and it linked cellular nutrient sensing to mitochondrial dynamics. Notably, a knockin (KI) strategy to mimic TBK1-mediated DRP1 phosphorylation exerted dominant-negative effects, causing cellular and developmental deficits similar to those seen in DRP1-knockout (DRP1-KO) mice and patients with DRP1 inborn mutations. In summary, our findings describe an indispensable molecular and cellular event governed by innate RNA sensing, and establish a critical link connecting innate immunity to the morphology and physiology of a major cellular organelle. Whether innate RNA sensing intuitively regulates mitochondrial dynamics to facilitate signaling and adapt their diverse functions is unknown. To investigate this possibility, we performed a time-course analysis in HEK293 cells using a super-resolution microscope to surveil mitochondrial morphology upon infection with Sendai virus (SeV), a well-established tool that invokes robust sensing of cytosolic RNA. Notably, mitochondria underwent an intriguing and dramatic alteration in morphology, with striking fusion that began 2 hours post-infection (hpi) and peaked at 6–8 hpi, causing the massive elongation and tubularization of mitochondria, which gradually resolved to their original morphology at 12 hpi (Figure 1A). This SeV-triggered profound induction of mitochondrial fusion was also detected in various epithelial cell types (HaCaT, NMuMG, and HCT116 cells) (Figures 1B and S1A). To exclude that viral factors, such as viral molecules and defective interfering particles, caused this dramatic phenotype, we employed cytosolic exposure to poly(I:C) (polyIC), a widely used stimulus of innate RNA sensing, and 5′ triphosphate double-stranded RNA (5′ppp-dsRNA), which specifically activates RIG-I. Both polyIC and 5′ppp-dsRNA induced similar and robust mitochondrial fusion (Figures 1C and S1B). Furthermore, infection of mammalian orthoreovirus (MRV), a Reoviridae family dsRNA virus that causes subclinical upper respiratory and gastrointestinal symptoms, also resulted in obvious mitochondrial fusion (Figures 1D and S1C). Notably, the expression of activated RIG-I (caRIG-I, RIG-I-N), MAVS, and TBK1, all triggered potent mitochondrial fusion in HEK293T cells (Figures 1E and S1D). Either KO of MAVS alone or double KO (dKO) of MAVS and STING, which abrogated innate RNA sensing stimulated by polyIC and SeV infection (Figures 1F and S1E), attenuated mitochondrial fusion (Figures 1G, S1F, and S1G). Similarly, SeV-induced mitochondrial dynamics was entirely prevented in RIG-I/MDA5 dKO HEK293 cells (Figures 1H and S1H). These consistent observations suggest that innate RNA sensing drives the profound fusion of mitochondria in cells with distinct origins via the RLR-MAVS pathway. We then assessed the particular role of TBK1 and its homolog, IKKε, for mitochondrial fusion in TBK1/IKKε-dKO cells. dKO of TBK1/IKKε obviously decoupled cytosolic RNA sensing and activation of IRF3 (Figure S2A) and abrogated RNA-sensing-triggered mitochondrial fusion upon polyIC and SeV infection (Figures S2B and Figure 2A). Inhibition of TBK1 by a small-molecule inhibitor similarly attenuated RNA-sensing-induced mitochondrial fusion in HCT116 cells (Figures S2C and S2D). By Phos-tag electrophoresis, in which an amplified mobility shift effectively distinguishes proteins with different phosphorylating states, we found that TBK1 robustly modified MFN2, DRP1, and DRP1 receptors (Figures S2E–S2G) when these proteins were coexpressed with TBK1 or MAVS that activated endogenous TBK1. Notably, we also detected a mobility shift for endogenous DRP1 in response to RNA sensing (Figure 2B). As expected, KO of DRP1 in HEK293T cells (Figure S2H) resulted in profoundly elongated and tubular mitochondria at a resting state (Figure 2C). By an antibody recognizing endogenous DRP1, immunofluorescence imaging under super-resolution microscopy also revealed the abundant aggregation of DRP1 in the segmentation interface of mitochondria (Figure S2I). These data suggest that DRP1 may function as a downstream mediator of RNA-sensing- and TBK1-induced alteration of mitochondrial dynamics. Intriguingly, DRP1 interacted with MAVS with high affinity, comparable to those known DRP1 receptors in mitochondria (Figure 2D), but not with endoplasmic reticulum (ER)-associated STING (Figure 2E). An endogenous complex consisting of MAVS and DRP1 in HCT116 cells (Figure 2F), as well as a complex between endogenous DRP1 and stably expressing MAVS in DLD1 cells (Figure S2J), was readily detected via coimmunoprecipitation upon SeV infection assays. DRP1 also interacted with both TBK1 and IKKε (Figure 2G). Immunofluorescence imaging also revealed the intriguing redistribution of endogenous DRP1 upon RNA sensing (Figure 2H). Domain mapping analyses utilizing truncated DRP1 and MAVS indicated that the N-terminal GTPase domain of DRP1, facilitated via its MD, interacted strongly with MAVS through its N-terminal CARD (Figures 2I and S2K). Intriguingly, the kinase activity of TBK1/IKKε appeared to be important for assembly of the endogenous MAVS-DRP1 complex upon SeV infection, and inhibition of TBK1/IKKε activity or genetic ablation of TBK1/IKKε expr" @default.
• W3103286458 created "2020-11-23" @default.
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• W3103286458 creator A5086559201 @default.
• W3103286458 date "2020-12-01" @default.
• W3103286458 modified "2023-10-18" @default.
• W3103286458 title "TBK1-Mediated DRP1 Targeting Confers Nucleic Acid Sensing to Reprogram Mitochondrial Dynamics and Physiology" @default.
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