Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes

Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes View ORCID Profile Xinxin Chen , Siwen Wang , View ORCID Profile Mauricio Torres , Sijie Hao , View ORCID Profile Shengyi Sun , View ORCID Profile Ling Qi doi: https://doi.org/10.1101/2025.06.14.659625 Xinxin Chen 1 Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine , Charlottesville, VA 22903, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xinxin Chen Siwen Wang 2 Department of Molecular and Integrative Physiology, University of Michigan Medical School , Ann Arbor, MI 48109, USA 3 Department of Emergency Medicine, Xiangya Hospital, Central South University , Changsha, Hunan 410008, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mauricio Torres 1 Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine , Charlottesville, VA 22903, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mauricio Torres Sijie Hao 4 Advanced Microscopy Core, University of Virginia School of Medicine , Charlottesville, VA 22903, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shengyi Sun 5 Department of Pharmacology, University of Virginia School of Medicine , Charlottesville, VA 22903, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shengyi Sun For correspondence: bjk5fz{at}virginia.edu xvr2hm{at}virginia.edu Ling Qi 1 Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine , Charlottesville, VA 22903, USA 2 Department of Molecular and Integrative Physiology, University of Michigan Medical School , Ann Arbor, MI 48109, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ling Qi For correspondence: bjk5fz{at}virginia.edu xvr2hm{at}virginia.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Mitochondrial quality control is essential for maintaining cellular energy homeostasis, particularly in brown adipocytes where dynamic mitochondrial remodeling supports thermogenesis. Although the SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD) pathway and autophagy are two major proteostatic systems, how these pathways intersect to regulate mitochondrial integrity in metabolically active tissues remains poorly understood. Here, using adipocyte-specific genetic mouse models combined with high-resolution 2D and 3D ultrastructural imaging technologies, we reveal an unexpected synergy between SEL1L-HRD1 ERAD and autophagy in maintaining mitochondrial structure and function in brown adipocytes. Loss of ERAD alone triggers compensatory autophagy, whereas combined deletion of both pathways (double knockout, DKO) results in severe mitochondrial abnormalities, including the accumulation of hyperfused megamitochondria penetrated by ER tubules, even under basal room temperature conditions. These phenotypes are absent in mice lacking either pathway individually or in SEL1L-IRE1α DKO, highlighting the pathway-specific coordination between ERAD and autophagy. Mechanistically, dual loss of ERAD and autophagy induces ER expansion, excessive ER-mitochondria contact, upregulation of mitochondria-associated membrane (MAM) tethering proteins, impaired calcium transfer, and defective mitochondrial turnover. As a result, DKO adipocytes accumulate dysfunctional mitochondria, exhibit respiratory deficits, and fail to sustain thermogenesis. Collectively, our study uncovers a cooperative and previously unrecognized mechanism of mitochondrial surveillance, emphasizing the critical role of ERAD-autophagy crosstalk in preserving mitochondrial integrity and thermogenic capacity in brown fat. One-sentence summary Our study uncovers a previously unrecognized synergy between SEL1L-HRD1 ERAD and autophagy that is essential for preserving mitochondrial integrity and thermogenic capacity in brown adipocytes, revealing new opportunities for targeting mitochondrial dysfunction in metabolic disease. INTRODUCTION Mitochondria are central hubs of cellular metabolism, playing essential roles in energy production, thermogenesis, and cell survival. Their functional integrity is maintained through continuous remodeling by tightly regulated processes including fusion, fission, biogenesis, and selective degradation, collectively termed ‘mitochondrial quality control’ 1 , 2 . This surveillance is especially critical in brown adipose tissue (BAT), a highly metabolically active organ specialized in non-shivering thermogenesis 3 , 4 . Brown adipocytes are densely populated with mitochondria, whose morphology and activity dynamically respond to environmental cues such as temperature and nutrient status 5 . Disruption of mitochondrial quality control in BAT impairs thermogenic capacity and contributes to metabolic dysfunction, obesity, and related disorders 3 , 6 , 7 ; however, the underlying molecular mechanism remains vague. Cellular proteostasis and organelle quality control are maintained by two highly conserved, principal degradative systems: autophagy and the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. While autophagy has been extensively studied 8 – 11 , ERAD remains relatively underappreciated, despite its critical role in health and disease 12 – 14 . Autophagy is a conserved lysosomal degradation pathway essential for maintaining cellular homeostasis by removing damaged organelles (including the ER and mitochondria), misfolded protein aggregates, and other cytoplasmic components 8 – 11 . Macroautophagy, the most extensively studied form, involves the formation of double-membrane autophagosomes that enclose cytoplasmic cargo and subsequently fuse with lysosomes for degradation 15 . Autophagy plays both constitutive and stress-responsive roles, being upregulated in response to nutrient starvation, hypoxia, and other cellular stresses 16 . Selective forms of autophagy, such as ER-phagy and mitophagy, enable organelle-specific turnover to preserve organellar function and metabolic homeostasis 17 – 20 . Dysregulated autophagy has been implicated in diverse human pathologies, including neurodegenerative diseases, cancer, and metabolic syndromes 21 . In BAT, autophagy plays complex and context-dependent roles. Brown adipocyte-specific knockout (KO) models of core autophagy genes have revealed critical and context-dependent roles for autophagy in regulating mitochondrial homeostasis, thermogenesis, and systemic energy metabolism. Deletion of Atg7 22 , 23 , Atg5 or Atg12 24 using Cre drivers like aP2-Cre, Ucp1-Cre or Ucp1-CreERT2 impairs mitophagy and alters mitochondrial turnover, increased mitochondrial content and enhanced thermogenic potential. In contrast, inducible deletion of Atg3 or Atg16L1 in mature adipocytes using Adipoq-CreERT2 results in mitochondrial dysfunction, oxidative stress, and insulin resistance 25 . These findings suggest that while autophagy restricts excessive mitochondrial accumulation during adipogenesis, it is indispensable for mitochondrial quality and metabolic health in mature BAT. Unlike autophagy, ERAD targets misfolded proteins in the ER for proteasomal degradation in the cytosol 26 – 30 . The SEL1L-HRD1 complex constitutes the most conserved branch of ERAD 31 – 34 . In this complex, SEL1L serves as an essential adaptor for the E3 ubiquitin ligase HRD1, where SEL1L acts as a scaffold, linking substrates to HRD1 and stabilizing HRD1 35 – 39 . Deletion of Sel1L or Hrd1 in mice results in embryonic lethality or premature death 38 , 40 – 42 , and loss of ERAD function in specific cell types leads to functional impairment and disease in mice 12 – 14 , 43 – 49 . In humans, mutations in SEL1L or HRD1 cause ERAD-associated neurodevelopmental disorder with onset in infancy (ENDI) syndrome 50 , 51 . We previously identified SEL1L-HRD1 ERAD as a key regulator of mitochondrial dynamics in brown adipocytes during cold exposure, in part by modulating ER-mitochondria contacts or mitochondria-associated membranes (MAMs) 52 . MAMs are specialized ER-mitochondria contact sites that coordinate mitochondrial dynamics, lipid and calcium exchange, and autophagosome biogenesis 53 – 60 . ERAD deficiency leads to increased levels of the MAM-resident protein Sigma non-opioid intracellular receptor 1 (SIGMAR1), mitochondrial hyperfusion and the accumulation of pleomorphic mitochondria with impaired respiratory function following acute cold stress 52 . While both ERAD and autophagy contribute to ER and mitochondrial homeostasis 14 , how they intersect to maintain mitochondrial integrity in vivo remains unclear. Since damaged mitochondria are typically cleared by mitophagy 61 , and that ERAD deficiency activates autophagy in various cell types including white adipocytes and pancreatic β cells 62 , 63 , we hypothesized that ERAD and autophagy may act cooperatively to preserve mitochondrial quality in BAT. Using adipocyte-specific KO mouse models combined with high-resolution 2D and 3D ultrastructural imaging technologies, we demonstrate a synergistic interplay between SEL1L-HRD1 ERAD and autophagy in regulating mitochondrial morphology, function, and calcium fluxes in brown adipocytes. This coordination occurs through modulation of MAMs and ehanced clearance of damaged mitochondria. Notably, this cooperative regulation is absent in brown adipocytes deficient in both ERAD and IRE1α, a key sensor of the unfolded protein response 12 , 64 . Our findings uncover a previously unappreciated, integrated role for ERAD and autophagy in mitochondrial quality control and thermogenic homeostasis in vivo . RESULTS Activation of autophagy in Sel1L -deficient brown adipocytes Our previous work demonstrated that loss of Sel1L activates autophagy in pancreatic β cells and white adipocytes 62 , 63 . To assess whether a similar response occurs in brown adipocytes, we analyzed brown adipose tissues (BAT) from brown adipocyte-specific Sel1L -deficient ( Sel1L Ucp1Cre ) mice. Compared to WT littermates, Sel1L Ucp1Cre mice exhibited a twofold increase in LC3-II levels, the ATG7-dependent, lipidated form of LC3 associated with autophagosome membrane ( Figure 1A ). Transmission electron microscopy (TEM) revealed a greater number of autophagic-like structures (yellow arrows), often enclosing mitochondria-like organelles (asterisks), in Sel1L Ucp1Cre BAT ( Figure S1A ). Moreover, we observed a marked increase in mitochondrial ubiquitination in purified mitochondria from Sel1L Ucp1Cre BAT ( Figure 1B ), a hallmark of elevated mitophagy. These findings establish that Sel1L deficiency triggers autophagy in brown adipocytes, consistent with previous findings in other metabolically active cell types. Download figure Open in new tab Figure 1. The synergistic role of SEL1L-HRD1 ERAD and autophagy in regulating mitochondrial morphology in brown adipocytes under basal conditions. (A) Immunoblot analysis of LC3 in BAT from 12-week-old WT and Sel1L Ucp1Cre littermates, with quantification of relative LC3-II normalized to HSP90 shown on the right. n = 10 for WT and 5 for Sel1L Ucp1Cre mice, Student’s t -test. (B) Immunoblot analysis of ubiquitin (UB) in purified mitochondria from BAT of 12-week-old WT and Sel1L Ucp1Cre littermates, with quantification of relative UB normalized to citrate synthase (CS) shown on the right. n = 3 per group, Student’s t- test. (C) Representative hematoxylin and eosin (H&E) staining of BAT from 12-week-old male littermates housed at room temperature (RT). n = 3 mice per group. (D-E) Representative TEM images of BAT from 12-week-old male littermates housed at RT. Arrows point to megamitochondria. LD, lipid droplet; M, mitochondrion. n = 3-5 mice per group. (F) Quantification of cumulative probability distribution of mitochondrial size. n = 874 mitochondria for WT, 747 for Sel1L Ucp1Cre , 556 for Atg7 Ucp1Cre , and 1189 for DKO, 3-5 mice per group. (G) Quantification of mitochondrial density (number of mitochondria per μm 2 cytosolic area). n = 5-8 fields, 2-3 mice per group. Data are mean ± SEM. n.s., not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Generation of mice with combined ERAD and autophagy deficiency in brown adipocytes To investigate the physiological significance of autophagy in the context of Sel1L deficiency, we generated brown adipocyte-specific Sel1L and Atg7 double-deficient ( DKO ) mice ( Sel1L f/f ;Atg7 f/f ;Ucp1-Cre ) by crossing Sel1L f/f ;Atg7 f/f mice with Ucp1-Cre transgenic mice. Age-matched WT, Sel1L Ucp1Cre , and Atg7 Ucp1Cre mice were included as controls. While single KO mice grew comparably to WT littermates, DKO mice of both sexes exhibited significant growth retardation ( Figure S1B ). The etiology of this phenotype remains unclear but contrasts with our previous observations in Sel1L;Atg7 AdipoqCre (Sel1L f/f ;Atg7 f/f ;Adipoq-Cre) mice, in which deletion of the same two genes in all adipocytes (under the control of an adipocyte-specific Adiponectin-Cre) did not result in growth abnormalities 63 . These results suggest that the growth defect in DKO mice may be due to leaky expression of Ucp1-Cre in other tissues such as kidneys, adrenal glands, thymus, and brain, as recently reported 65 . At room temperature, BAT weights were significantly increased in both Atg7 Ucp1Cre and DKO mice compared to those of WT and Sel1L Ucp1Cre mice, regardless of normalization to body weight ( Figure S1C-D ). While single deletions of Sel1L or Atg7 did not alter brown adipocyte morphology, combined deficiency led to pronounced lipid droplet hypertrophy, as revealed by hematoxylin and eosin (H&E) staining and Perilipin 1 immunostaining ( Figure 1C and S1E-F ). These changes were consistent across both sexes (data not shown). Western blot analysis confirmed efficient deletion of SEL1L and ATG7 in DKO BAT and demonstrated loss of autophagic activity, as indicated by p62 accumulation and reduced LC3-II formation ( Figure S2A ). Consistent with IRE1α being a known ERAD substrate 66 , IRE1α protein levels were markedly elevated ∼10-fold in Sel1L Ucp1Cre and 44-fold in DKO BAT compared to WT ( Figure S2B ). Splicing of Xbp1 mRNA, a downstream target of IRE1α, was modestly increased, with spliced Xbp1 comprising 21% of total Xbp1 transcripts in DKO BAT vs. ∼ 2-4% in single KO BAT ( Figure S2B ). Similarly, the UPR sensor PERK were elevated by ∼3-fold in Sel1L Ucp1Cre and ∼6-fold in DKO BAT, accompanied by increased phosphorylation of its downstream target eIF2α ( Figure S2C ). Despite this moderate ER stress response, we did not observe evidence of apoptosis, as cleaved caspase-3 remained undetectable in DKO BAT ( Figure S2D ). Megamitochondria formation in DKO brown adipocytes at room temperature Building on our prior work demonstrating a role for SEL1L-HRD1 ERAD in regulating mitochondrial morphology during cold exposure in brown adipocytes 52 , we investigated how concurrent loss of SEL1L-HRD1 ERAD and autophagy affects mitochondrial architecture under basal (room temperature) conditions. Using transmission electron microscopy (TEM), we observed no significant differences in mitochondrial morphology or density between WT and Sel1L Ucp1Cre BAT, consistent with previous findings ( Figure 1D , quantified in Figure 1F-G ). In contrast, Atg7 Ucp1Cre BAT exhibited smaller, more densely packed mitochondria relative to WT controls. Strikingly, brown adipocytes from DKO mice displayed dramatic mitochondrial enlargement and reduced mitochondrial density (arrows, Figure 1D-E , quantified in Figure 1F -G ). Similar megamitochondrial structures were also observed in BAT from Adiponectin-Cre- driven DKO mice (arrows, Figure S1G ), suggesting that this phenotype is a robust consequence of dual ERAD-autophagy loss. 3D FIB-SEM reveals mitochondrial hyperfusion in DKO BAT These ultrastructural findings were corroborated by high-resolution confocal imaging of the mitochondrial outer membrane protein TOMM20 and matrix protein PDH, which further confirmed the presence of enlarged mitochondria in DKO BAT (white arrows, Figure 2A and S3A, quantified in Figure S3B ). To further investigate mitochondrial architecture, we performed focused ion beam-scanning electron microscopy (FIB-SEM). We used convolutional neural network–based image segmentation to reconstruct individual mitochondria at ∼5 nm isotropic resolution in all dimensions (x, y, z), as previously described 52 . Three-dimensional reconstruction revealed that mitochondria in Sel1L Ucp1Cre and Atg7 Ucp1Cre BAT remained largely spherical and morphologically similar to those in WT BAT ( Figure 2B ; Video S1-S3 ). In stark contrast, mitochondria in DKO BAT were significantly enlarged and highly pleomorphic, displaying extensive fusion events ( Figure 2B ; Video S4, quantified in Figure S3C ). Notably, we observed a substantial population of hyperfused megamitochondria in DKO BAT, with the largest reaching a volume of ∼35 μm³— approximately 15-fold greater than the average WT mitochondrial volume of 2.3 μm³. Pseudo-color segmentation of individual mitochondria revealed that this single megamitochondrion alone accounted for ∼25% of the total mitochondrial volume within the imaged DKO field ( Figure 2B ; Video S4 ). These findings demonstrate that combined loss of ERAD and autophagy drives aberrant mitochondrial fusion and the accumulation of pleomorphic megamitochondria in brown adipocytes, even in the absence of external stressors such as cold exposure. Download figure Open in new tab Figure 2. Formation of megamitochondria in Sel1L;Atg7 -deficient brown adipocytes, but not in Sel1L;Ire1a -deficient brown adipocytes. (A) Representative confocal immunofluorescent images of TOMM20 (mitochondrial outer membrane protein) and pyruvate dehydrogenase (PDH, mitochondrial matrix protein) staining in BAT sections from 12-week-old male littermates. Arrows point to megamitochondria. LD, lipid droplet. n = 3 mice per group. (B) Representative FIB-SEM and 3D tomography images of mitochondria in BAT from 12-week-old male littermates. n = 1096 slices for WT, 1425 for Sel1L Ucp1Cre , 1290 for Atg7 Ucp1Cre , and 1039 for DKO, 5 nm/slice. (C) Representative TEM images of BAT from 10-week-old WT and Sel1L;Ire1α Ucp1Cre male littermates. LD, lipid droplet; M, mitochondrion. n = 2 mice per group. (D) Quantification of cumulative probability distribution of mitochondrial size. n = 255 and 187 mitochondria for WT and Sel1L;Ire1α Ucp1Cre mice. n = 2 mice per group Combined Sel1L - Ire1a deficiency does not promote megamitochondria formation To determine whether the mitochondrial phenotype observed in Sel1L-Atg7 deficiency is specific to the loss of these two pathways, we generated brown adipocyte-specific Sel1L and Ire1a DKO ( Sel1L Ucp1Cre ;Ire1a Ucp1Cre ) mice along with their WT littermates ( Figure S3D ). This model was particularly informative given the marked elevation of IRE1α protein levels in Sel1L Ucp1Cre BAT ( Figure S2B and S3D ). As expected, deletion of IRE1α abolished Xbp1 mRNA splicing in both Ire1a Ucp1Cre and Sel1L Ucp1Cre ;Ire1a Ucp1Cre BAT ( Figure S3D ). Importantly, Sel1L Ucp1Cre ;Ire1a Ucp1Cre mice exhibited normal growth compared to WT littermates ( Figure S3E ) and did not display any evidence of megamitochondria in BAT at 10 weeks of age under room temperature conditions ( Figure 2C , quantified in Figure S3G, and Fig. S3F ). These data suggest that mitochondrial remodeling in Sel1L-Atg7 DKO BAT is independent of IRE1α and is unlikely to be a downstream consequence of ER stress. Formation of megamitochondria with perforating ER tubules in DKO brown adipocytes In DKO brown adipocytes, we observed a striking ultrastructural phenotype: in addition to pleomorphic megamitochondria, many of these mitochondria were traversed by perforating tubules (red arrows, Figure 3A and S4A ). These remarkable tubular structures did not puncture the mitochondrial membranes; rather, they exhibited a distinct tri-laminar membrane organization, with two layers derived from the mitochondria (white arrows, Figure 3A ). Notably, mitochondrial cristae radiated outward from the contact sites of these structures ( Figure 3A and S4A ), consistent with a proposed role for the ER in lipid transfer to mitochondria 55 , 67 . Occasionally, we observed a unique configuration in which a donut-shaped megamitochondrion encircled a smaller spherical mitochondrion (asterisk), with intervening perforating tubules (red arrows, Figure 3B ), suggesting a complex interplay between mitochondrial dynamics and ER contacts. Download figure Open in new tab Figure 3. ERAD and autophagy deficiency leads to the formation of megamitochondria with perforating ER tubules and mitochondria under RT. (A) Representative TEM images of BAT from 12-week-old DKO mice showing megamitochondria wrapping around the tubular structures. Red arrows point to mitochondrion-perforating tubules. White arrows point to mitochondrial membranes. n = 5 mice. (B) Representative TEM images of BAT from 12-week-old DKO mice showing megamitochondrion wrapping around the smaller mitochondrion mediated by tubular structures. Red arrows point to mitochondrion-perforating tubules. White arrows point to mitochondrial outer membrane. Asterisk, smaller mitochondrion. n = 5 mice. (C) Representative TEM images of BAT from 12-week-old DKO male mice following BiP-specific immunogold labelling, with pseudo colored blue and red dots shown below. White dotted lines outline megamitochondria. Arrows point to mitochondrion-perforating ER tubules. M, mitochondrion. n = 2 mice per group. To confirm the identity of these tubules, we performed immunogold labeling for the ER chaperone BiP, which revealed dense clusters of BiP-positive signals surrounding and penetrating the mitochondria (white arrows, Figure 3C and S4B ), verifying their ER origin. These findings demonstrate that Sel1L - Atg7 deficiency promotes the formation of pleomorphic megamitochondria penetrated by ER-derived tubules in brown adipocytes under room temperature conditions—a phenotype distinct from that seen in Sel1L Ucp1Cre BAT, where similar structures only emerge under acute cold exposure 52 . Together, these results highlight a synergistic role of SEL1L-HRD1 ERAD and autophagy in regulating mitochondrial architecture, mitochondrial quality control, and ER–mitochondria interactions. ERAD and autophagy synergistically control 3D ER architecture and volume To elucidate the mechanisms underlying the dramatic mitochondrial alterations in DKO brown adipocytes, we next examined ER architecture, as both SEL1L-HRD1 ERAD and autophagy are known to influence ER morphology and homeostasis 19 , 62 , 68 . TEM revealed modest ER dilation in Sel1L Ucp1Cre and increased ER sheet formation in Atg7 Ucp1Cre brown adipocytes ( Figure 4A ) . In contrast, DKO adipocytes displayed extensive expansion of rounded tubular ER structures ( Figure 4A ) , confirmed as the ER via BiP immunogold labeling ( Figure 4B and S5A ). Three-dimensional FIB-SEM further revealed that ER occupied ∼6.3% of the cytoplasmic volume in WT adipocytes (excluding lipid droplets, nuclei, and blood vessels) ( Figure 4C , S5B; Video S1 ). Sel1L or Atg7 deficiency alone led to a moderate increase in ER volume, whereas combined deletion in DKO adipocytes resulted in a pronounced expansion to ∼14% of the cytosolic volume ( Figure 4C ; Video S2-4 ), along with increased ER luminal space (arrows, Figure 4D ). These findings demonstrate a synergistic role for ERAD and autophagy in maintaining ER architecture and controlling ER network volume in brown adipocytes. Download figure Open in new tab Figure 4. ERAD and autophagy synergistically control the ER architecture in brown adipocytes. (A) Representative TEM images of BAT showing the ER (red) from 12-week-old male littermates. n = 3-5 mice per group. (B) Representative TEM images of BAT following BiP-specific immunogold labelling with pseudo-colored blue dots from 12-week-old male littermates. M, mitochondrion. Green dotted lines outline mitochondrial membranes. n = 2 mice per genotype. (C) 3D reconstruction of FIB-SEM images of BAT showing ER from 12-week-old male littermates, with quantification of the ER volume as the percentage of the total cytosolic volume, excluding the volumes of nucleus and lipid droplets in each group. (D) Magnified view of the ER lumen. Yellow lines outline ER membrane. Green arrows indicate the enlarged ER lumen. Enhanced ER-mitochondria contacts in DKO brown adipocytes Given the critical role of MAMs in mitochondrial dynamics and function 54 , 55 , we next explored how SEL1L-HRD1 ERAD and autophagy influence MAM architecture in BAT from mice housed at room temperature. In WT BAT, MAMs appeared as thin ER sheets along mitochondria, with an average distance of 5-25 nm (yellow arrows, Figure 5A ). A modest increase in MAM number was observed in both Sel1L Ucp1Cre and Atg7 Ucp1Cre BAT (yellow arrows, Figure 5A , and quantified in Figure 5B ), whereas DKO BAT exhibited a striking accumulation of MAMs per mitochondrion (yellow arrows, Figure 5A , quantified in Figure 5B ). Download figure Open in new tab Figure 5. ERAD and autophagy deficiency alters ER morphology and increases ER-mitochondria contacts. (A) Representative TEM images of BAT with ER (red) from 12-week-old male littermates. Arrows point to MAMs; green dotted lines outline mitochondrial membranes. n = 3-5 mice per group. (B) Quantification of number of MAMs per mitochondrion across genotypes. n = 291 mitochondria for WT, 294 for Sel1L Ucp1Cre , 231 for Atg7 Ucp1Cre , and 242 for DKO, 3-5 mice per group. (C) 3D reconstruction of FIB-SEM images of BAT showing mitochondria (blue) and ER (purple). (D) 3D visualization of mitochondria volumes marked by areas interacting with the ER at 0-25 nm distance (red). (E-G) Quantification of the number of ER-mitochondrial interacting sites per mitochondrion (E), ER-mitochondrial interacting area per mitochondrion (F), and mitochondrion surface covered by ER (G). n = 273 mitochondria for WT, 160 for Sel1L Ucp1Cre , 375 for Atg7 Ucp1Cre , and 84 for DKO, One-way ANOVA. Data are mean ± SEM. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Three-dimensional FIB-SEM analysis revealed progressive reinforcement of ER–mitochondrial contacts across genotypes, culminating in DKO adipocytes, where mitochondria appeared extensively wrapped by ER membranes ( Figure 5C ; Video S1-S4 ). To visualize these contacts in 3D, ER voxels were computationally expanded by 25 nm, and interacting surfaces were rendered in red ( Figure 5D ; Video S1-S4 ). Quantification confirmed a marked increase in the number, area, and extent of ER–mitochondrial interfaces in DKO BAT compared to the other cohorts ( Figure 5E-G ). To dissect the molecular basis of MAM expansion, we performed subcellular fractionation of BAT to isolate MAMs. The ER–mitochondria tethering proteins vesicle-associated membrane proteins A and B (VAPA/B) were increasingly enriched in the MAM fractions of Sel1L Ucp1Cre and Atg7 Ucp1Cre BAT, with the highest levels in DKO samples ( Figure S6A ). Notably, SIGMAR1, a known substrate of SEL1L-HRD1 ERAD 52 , was similarly elevated in MAM fractions of both single KOs and DKO BAT ( Figure S6A ). In contrast, levels of other MAM-associated proteins, including GRP75, VDAC1 (components of the IP3R1-GRP75-VDAC1 complex 69 ), and MFN2, remained largely unchanged ( Figure S6A ). Total protein levels, but not mRNA, of VAPA/B and SIGMAR1 were significantly increased in DKO BAT ( Figure S6B–C ), suggesting post-translational accumulation. These findings suggest that combined loss of SEL1L-HRD1 ERAD and autophagy drives aberrant expansion of MAMs in brown adipocytes, likely through impaired degradation of ER–mitochondria tethering components. Mitochondrial hyperfusion and impaired quality control in DKO brown adipocytes To investigate the formation of megamitochondria in DKO brown adipocytes, we examined mitochondrial dynamics. In DKO BAT, both total levels and activating phosphorylation of the mitochondrial fission protein DRP1 were markedly reduced compared to WT controls ( Figure 6A ). In contrast, levels of the outer membrane fusion proteins MFN1 and MFN2 were increased in mitochondria isolated from DKO BAT ( Figure 6B ), despite unchanged total cellular levels across genotypes ( Figure S7A ). Download figure Open in new tab Figure 6. Loss of ERAD and autophagy synergistically promotes mitochondrial fusion, p62 aggregation and mitochondrial quality control. (A) Immunoblot analysis of phosphorylated DRP1 at Ser616 (p-DRP1 s616) as a mitochondrial-fission marker in BAT from 12-week-old littermates, with quantification of p-S616 DRP1 normalized to α-TUBULIN (TUB) shown on the right. n = 8-12 per genotype, One-way ANOVA. (B) Immunoblot analysis of mitochondrial fusion markers mitofusin 1 (MFN1) and MFN2 in isolated mitochondria from BAT of 12-week-old littermates, with quantification of MFN1 and MFN2 levels normalized to citrate synthase (CS) shown on the right. n = 3-4 per genotype, One-way ANOVA. (C-D) Representative confocal images of mitochondrial fusion from the PEG-based fusion assay in mature adipocytes. Mitochondrial fusion (yellow signal) is evidenced by co-localization of mito-DsRed (red) and mito-mEmerald (green). Quantitation of mitochondrial fusion assay shown in (D). Classification of cell hybrids as full, partial, or no fusion. Three independent experiments, with more than 50 cell hybrids from each experiment. Statistical analysis using two-way ANOVA, with comparisons made to the WT group. (E) Immunoblot analysis of ubiquitin (UB) levels in purified mitochondria from BAT. The quantification of UB normalized to citrate synthase (CS) shown below. n = 4, One-way ANOVA. (F) Representative confocal images of TOMM20 (green) and p62 (red) in BAT sections from 12-week-old male littermates. White arrows mark the colocalization of p62 and TOMM20. n = 3 mice per genotype. (G) Representative TEM images of p62 inclusions in BAT from 12-week-old Atg7 Ucp1Cre and DKO male mice. Asterisks indicate p62 inclusion. Red arrows point to mitochondrion near p62 inclusion. LD, lipid droplet; M, mitochondrion. n = 3 mice for Atg7 Ucp1Cre and 5 for DKO. (H) Representative TEM images of BAT following p62-specific immunogold labelling from 12-week-old DKO male mice. Asterisks indicate p62-positive inclusion. n = 2 mice per genotype. Data are mean ± SEM. n.s., not significant; * p < 0.1; ** p < 0.01; *** p < 0.001; ****, p < 0.0001. To assess fusion activity functionally, we performed a polyethylene glycol (PEG)-based cell fusion assay 70 using mature brown adipocytes expressing mitochondrially targeted fluorescent proteins. This assay revealed a significant enhancement in mitochondrial fusion in DKO cells relative to all other genotypes, whereas single KO mice displayed only modest changes ( Figure 6C , quantified in Figure 6D ). These findings indicate that simultaneous loss of SEL1L-HRD1 ERAD and autophagy promotes a shift toward excessive mitochondrial fusion. Notably, hyperfused mitochondria in DKO adipocytes exhibited increased ubiquitination ( Figure 6E ), indicative of elevated mitochondrial damage. Given the established role of p62 in mediating mitophagy 71 , 72 , we examined its expression and intracellular distribution. Immunofluorescence staining revealed a marked increase in p62 signal in DKO brown adipocytes, which lacked the typical punctate structures and instead partially aligned along the mitochondrial outer membrane (arrows, Figure 6F and S7B ). Furthermore, TEM showed that cytosolic inclusions – typically formed in the absence of autophagy – were rarely observed in Atg7 Ucp1Cre BAT and completely absent in Sel1L Ucp1Cre BAT (not shown), but were significantly more numerous and enlarged in DKO adipocytes (asterisks, Figure 6G and S7C ). These inclusions were often closely associated with mitochondria exhibiting disrupted cristae, characteristic of damaged mitochondria (red arrows, Figure 6G and S7C ). Immunogold labeling for p62 further confirmed that these inclusions were p62-positive (asterisks, Figure 6H ). Together, these findings indicate that the combined loss of ERAD and autophagy synergistically impairs mitophagic clearance, promotes mitochondrial hyperfusion, and leads to the accumulation of ubiquitinated, dysfunctional mitochondria in brown adipocytes. Mitochondrial hyperfusion disrupts calcium signaling and impairs thermogenesis in DKO brown adipocytes To assess the physiological consequences of accumulated megamitochondria, we evaluated calcium dynamics between the ER and mitochondria, a critical process for mitochondrial metabolism and thermogenic function that is mediated by MAMs 73 , 74 . Using mito-GCaMP6f, a genetically encoded fluorescent calcium indicator targeted to mitochondria 75 , we observed that ER-to-mitochondria calcium transfer was significantly reduced in DKO adipocytes compared to WT cells ( Figure 7A ). In contrast, ER-to-cytosol calcium release measured with Fluo-4 AM, a cytosolic calcium-sensitive dye 76 , remained unchanged in DKO adipocytes ( Figure S7A ), suggesting a selective disruption in MAMs-mediated calcium delivery to mitochondria. Download figure Open in new tab Figure 7. Sel1L;Atg7 deficiency compromises mitochondria function and thermogenesis in brown adipocytes. (A) Representative traces of mitochondrial Ca 2+ responds in WT and DKO differentiated adipocytes challenged with 500 μM ATP using mito-GCaMP6f indicator, with quantification of peak mito-GCaMP6f fluorescence shown on the right. n = 177 cells for WT and 129 for DKO, three repeats, Student’s t -test. (B) Oxygen consumption rate (OCR) of purified mitochondria from BAT of 12-week-old WT and DKO male littermates. Three repeats. (C) qPCR analyses of mitochondrial DNA targeting mitochondrial 1 6s rRNA and Mtco1 genes, normalized to the nuclear-encoded Hk2 gene. n = 4-5 per group, One-way ANOVA. (D) Rectal temperatures of 12-week-old male and female littermates exposed to 4°C for 2 hours. n = 6-21 per group, Two-way ANOVA. (E) Proposed model for a synergic role of SEL1L-HRD1 ERAD and autophagy in mitochondrial quality control in brown adipocytes. See text for details. Data are mean ± SEM. n.s., not significant; * p < 0.1; ** p < 0.01; *** p < 0.001; ****, p < 0.0001. To determine the impact on mitochondrial respiration, we measured oxygen consumption rates in mitochondria isolated from BAT. Purified DKO mitochondria exhibited a markedly reduced maximal respiratory capacity in response to FCCP, comparable to the reduction observed in Sel1L Ucp1Cre mice but contrasting with enhanced respiration seen in Atg7 Ucp1Cre mitochondria ( Figure 7B and S8B-C ). Blue-native PAGE analysis of oxidative phosphorylation (OXPHOS) complexes revealed diminished abundance of complexes III and V in both Sel1L Ucp1Cre and DKO BAT, despite unchanged levels of individual complex subunits by immunoblotting ( Figure S8D and quantitated in Fig. S8E ). Consistent with compromised mitochondrial function, DKO BAT displayed elevated mitochondrial DNA (mtDNA) content ( Figure 7C ), suggestive of compensatory mitochondrial biogenesis. Lastly, we assessed thermogenic capacity under cold stress. At room temperature, body temperatures were comparable across genotypes. However, during acute cold exposure, DKO mice rapidly developed hypothermia, exhibiting a significant drop in core body temperature within 2 hours ( Figure 7D ). In contrast, WT and single-KO mice maintained euthermia over this interval. Notably, Sel1L Ucp1Cre mice only became hypothermic after prolonged cold exposure (4–6 hours), as previously reported 52 . Together, these findings highlight the cooperative role of SEL1L-HRD1 ERAD and autophagy in preserving mitochondrial calcium homeostasis, respiratory competence, and thermogenic function in brown adipocytes. DISCUSSION Our study uncovers a previously unrecognized interplay between SEL1L-HRD1 ER-associated degradation (ERAD) and autophagy in the maintenance of mitochondrial quality control and function in brown adipocytes. In the absence of Sel1L , compensatory autophagy is activated, supporting both ER homeostasis and mitochondrial integrity. However, the combined deficiency of ERAD and autophagy leads to the formation of pleomorphic “megamitochondria,” impaired mitochondrial respiration, and severe thermogenic dysfunction ( Figure 7E ). These megamitochondria are characterized by increased ER-mitochondria contacts and elevated levels of MAM proteins, such as VAPA/B and SIGMAR1. Given the established role of these contacts in mitochondrial fission 54 , our findings suggest that these alterations disrupt mitochondrial dynamics by inhibiting fission and promoting fusion. This is evident even at room temperature, where megamitochondria are perforated by ER tubules. Notably, these structural and functional abnormalities are absent in brown adipocytes of single KO 52 or Sel1L - Ire1a double-deficient mice housed at room temperature, highlighting the distinct and synergistic roles of ERAD and autophagy in regulating mitochondrial dynamics. Collectively, our findings demonstrate the integrated roles of ERAD and autophagy in preserving ER and mitochondrial integrity, their interactions, and mitochondrial function. The ERAD-autophagy interaction in brown adipocytes is distinct from its role in white adipocytes and pancreatic β cells, where these pathways primarily collaborate to clear protein aggregates, such as lipoprotein lipase and proinsulin, ensuring ER homeostasis without noticeable impact on mitochondrial morphology 62 , 63 . In brown adipocytes, however, the effect extends to mitochondria, likely due to the high mitochondrial content and dynamic nature of mitochondria. Previously, we demonstrated that ERAD regulates ER-mitochondrial contacts via MAM proteins, with megamitochondria only observed in Sel1L -deficient mice after 4-6 hours of cold exposure 52 . In contrast, here we show that megamitochondria formation in ERAD-autophagy DKO cells occurs even at room temperature, independent of cold exposure. We speculate that this synergistic effect depends not only on ER expansion and the accumulation of damaged mitochondria, but also from increased ER-mitochondria contacts and elevated MAM protein levels, which likely stall mitochondrial fission. Our finding of increased VAPA/B protein abundance at MAMs in cells deficient in both ERAD and autophagy adds a new dimension to the understanding of ER-mitochondrial dynamics. VAPA/B is a key ER receptor involved in interactions with other organelles, such as the Golgi, mitochondria, and endosomes/autophagosomes 77 – 79 , facilitating lipid exchange and maintaining membrane integrity. The elevated levels of VAPA/B may reflect a cellular attempt to enhance lipid transfer between organelles under stress, given their critical role in organelle function. However, this adaptive response likely has both beneficial and detrimental effects. While increased ER-mitochondrial contacts may temporarily support mitochondrial lipid requirements, the resulting mitochondrial hyperfusion—exacerbated by the accumulation of damaged mitochondria due to autophagy deficiency—may impair respiratory function 80 . This highlights the critical balance between mitochondrial fusion and fission, which is critical for mitochondrial health. Dysregulation of this balance, in the context of combined ERAD and autophagy deficiencies, likely contributes significantly to mitochondrial dysfunction. The impaired mitochondrial respiration and thermogenic responses observed in our study highlight the broader metabolic consequences of combined SEL1L-HRD1 ERAD and autophagy deficiencies. As brown adipose tissue (BAT) plays a central role in energy expenditure and thermogenesis in humans 81 , 82 , and considering that mitochondrial function and dynamics are critical to the pathogenesis of various diseases 83 , 84 , our findings have significant implications for metabolic disorders such as obesity and type 2 diabetes, as well as for neurodegeneration and aging. These results suggest that the interplay between ERAD and autophagy in mitochondrial quality control could serve as a promising therapeutic target, especially in light of our recent identification of patients with hypomorphic ERAD mutations 50 , 51 . Enhancing autophagic activity in ERAD-deficient cells may restore mitochondrial function and improve metabolic outcomes. Further investigation into the molecular mechanisms driving ERAD-autophagy synergy is crucial for developing therapeutic strategies to maintain mitochondrial integrity and address metabolic dysfunction. Future studies should focus on translating these findings into actionable treatments to improve mitochondrial function and metabolic health. Limitations of the Study Our study identifies a synergistic role for SEL1L-HRD1 ERAD and autophagy in maintaining mitochondrial quality control in brown adipocytes. However, two limitations should be acknowledged. First, although we observed enhanced ER-mitochondria contacts and increased expression of MAM-associated tethering proteins in DKO BAT, we did not directly establish a causal relationship between altered MAM dynamics and mitochondrial hyperfusion. Further mechanistic studies, such as manipulating specific MAM components, will be necessary to delineate their direct contribution to mitochondrial remodeling. Second, our use of the Ucp1-Cre driver to generate adipocyte-specific knockouts may introduce unintended effects, as Ucp1-Cre is known to exhibit off-target recombination in certain non-adipose tissues, including the central nervous system and skeletal muscle. This could confound the interpretation of brown adipocyte-specific phenotypes, especially for systemic or whole-tissue analyses. Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ling Qi ( xvr2hm{at}virginia.edu ). Materials Availability All unique/stable reagents generated in this study are available from the lead contact without restriction. Data and Code Availability This study did not generate large-scale datasets or original code. All data supporting the findings of this study are included in the main text or Supplemental Materials and are available from the Lead Contact upon reasonable request. AUTHOR CONTRIBUTIONS X.C. and S.W. collaboratively designed and performed most experiments; M.T. assisted with EM experiments. L.Q. and S.S. supervised the study and wrote the manuscript with help from X.C. X.C. wrote the methods and figure legends; S.H. provided technical support for data analysis. All authors commented on and approved the final manuscript. Declaration of interests The authors have declared that no conflict of interest exists. ACKNOWLEDGEMENTS We acknowledge Drs. Ming-Feng Tsai and Yanzhuang Wang for providing reagents; Allen Hunter for assistance with FIB-SEM analysis; Dave Castle, members of the Qi–Sun and Arvan laboratories for technical assistance and insightful discussions. We acknowledge the use of Electron Microscopy Core, Advanced Microscopy Core, Research Histology Core at the University of Virginia, and the University of Michigan Comprehensive Cancer Center (UMCCC) Tissue Core, Imaging laboratory, the Microscopy Core of Michigan Biomedical Research Core Facilities, and Michigan Center for Materials Characterization. This work was supported by NIH grants 1R01DK11174, R01DK117639, R35GM130292 and 1R01DK120047 (L.Q.). X.C. is supported by American Diabetes Association (ADA) Postdoctoral Fellowship (11-23-PDF-62). Funder Information Declared National Institute of Diabetes and Digestive and Kidney Diseases, https://ror.org/00adh9b73 , 1R01DK11174 , R01DK117639 , 1R01DK120047 National Institute of General Medical Sciences, https://ror.org/04q48ey07 , R35GM130292 American Diabetes Association, https://ror.org/04f6cgz95 , 11-23-PDF-62 REFERENCES 1. ↵ van der Bliek , A.M. , Shen , Q. , and Kawajiri , S. ( 2013 ). 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Share Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes Xinxin Chen , Siwen Wang , Mauricio Torres , Sijie Hao , Shengyi Sun , Ling Qi bioRxiv 2025.06.14.659625; doi: https://doi.org/10.1101/2025.06.14.659625 Share This Article: Copy Citation Tools Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes Xinxin Chen , Siwen Wang , Mauricio Torres , Sijie Hao , Shengyi Sun , Ling Qi bioRxiv 2025.06.14.659625; doi: https://doi.org/10.1101/2025.06.14.659625 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17690) Bioengineering (13892) Bioinformatics (41935) Biophysics (21451) Cancer Biology (18587) Cell Biology (25499) Clinical Trials (138) Developmental Biology (13377) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24318) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88601) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15152) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)