The spatiotemporal dynamics of MAMs: mechanisms, pathologies, and therapeutic rewiring.

The spatiotemporal dynamics of MAMs: mechanisms, pathologies, and therapeutic rewiring - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Cell Mol Biol Lett . 2026 Mar 7;31:54. doi: 10.1186/s11658-026-00887-y Search in PMC Search in PubMed View in NLM Catalog Add to search The spatiotemporal dynamics of MAMs: mechanisms, pathologies, and therapeutic rewiring Dongxue Xu Dongxue Xu 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China Find articles by Dongxue Xu 1, # , Yinye Huang Yinye Huang 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China Find articles by Yinye Huang 1, # , Xiaoyu Zhang Xiaoyu Zhang 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China Find articles by Xiaoyu Zhang 1 , Benzheng Liu Benzheng Liu 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China Find articles by Benzheng Liu 1 , Mingying Wang Mingying Wang 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China Find articles by Mingying Wang 1 , Yiming Li Yiming Li 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China Find articles by Yiming Li 1, ✉ , Zhiyong Peng Zhiyong Peng 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China 2 Clinical Research Center of Hubei Critical Care Medicine, Wuhan, China 3 Department of Critical Care Medicine, The Second Affiliated Hospital of Hainan Medical University, Haikou, China Find articles by Zhiyong Peng 1, 2, 3, ✉ Author information Article notes Copyright and License information 1 Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China 2 Clinical Research Center of Hubei Critical Care Medicine, Wuhan, China 3 Department of Critical Care Medicine, The Second Affiliated Hospital of Hainan Medical University, Haikou, China ✉ Corresponding author. # Contributed equally. Received 2025 Nov 27; Accepted 2026 Feb 11; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13081536  PMID: 41794668 Abstract Mitochondria-associated endoplasmic reticulum membranes (MAMs) constitute highly dynamic signaling hubs that coordinate a spatiotemporal network regulating calcium flux, lipid trafficking, and innate immune activation. Beyond functioning as physical organelle tethers, the plasticity of MAMs is essential for cellular resilience. Notably, maladaptive remodeling of these contacts, which presents a spatiotemporal paradox in that both pathological tightening and excessive dissociation can precipitate dysfunction, underlies the pathogenesis of diverse complex diseases, including neurodegeneration, cardiovascular failure, and kidney injury. In this review, we provide an integrated synthesis of the molecular architecture of MAMs and highlight the indispensable role of endoplasmic reticulum (ER)–mitochondria coupling in sustaining physiological homeostasis. We further dissect how MAM dysregulation operates as a central convergence point for metabolic stress and inflammatory signaling. Additionally, we summarize technological advances such as super-resolution imaging and multi-omics frameworks that increasingly resolve the structural and functional heterogeneity of MAMs. Importantly, emerging evidence indicates a therapeutic paradigm shift: several widely used clinical agents, including sodium-glucose cotransporter 2 (SGLT2) inhibitors and metformin, appear to exert their renoprotective and metabolic benefits by restoring or stabilizing MAM integrity. Together, these insights reposition MAMs not as passive structural bystanders but as actionable, high-value therapeutic targets for next-generation precision medicine and drug repurposing strategies. Graphical Abstract Keywords: Mitochondria-associated membranes, ER stress, Neurodegenerative disease, Cancer, Diabetes, Kidney disease, Therapeutics Introduction The dynamic interplay between ER and mitochondria Within the highly compartmentalized environment of eukaryotic cells, organelles are not isolated entities but rather engage in dynamic material exchange and signal communication through membrane contact sites (MCSs), thereby coordinating the cell’s overall function [ 1 ]. Among these, the physical connections between the endoplasmic reticulum (ER) and mitochondria are particularly noteworthy. These specialized subcellular domains, known as mitochondria-associated membranes (MAMs), form a core regulatory platform for cellular life activities [ 2 , 3 ]. At MAMs, a multitude of critical cellular processes converge, including lipid synthesis [ 4 , 5 ] and trafficking, calcium (Ca 2+ ) signaling [ 6 , 7 ], mitochondrial dynamics [ 8 ], and the initiation of autophagy [ 9 ], and ferroptosis [ 10 , 11 ]. The integrity of this platform is maintained by specific tethering protein complexes, such as the inositol 1,4,5-trisphosphate receptor (IP3R)–glucose-regulated protein 75 (GRP75)–VDAC1 and VAPB–protein tyrosine phosphatase interacting protein 51 (PTPIP51) complexes, which physically bridge the two organelles and facilitate their functional crosstalk. While recent reviews have provided excellent summaries of specific domains such as structural biology or single-organ pathology [ 12 – 15 ], the distinct contribution of this work lies in its comprehensive, cross-disciplinary synthesis. By bridging diverse pathological contexts—from neurodegeneration to nephrology—we derive a unified “spatiotemporal paradox” framework that reconciles conflicting structural data. Furthermore, we extend the discussion beyond the cell to the organismal level, integrating systemic signaling axes (mitokines, microbiome) and establishing a holistic roadmap for clinical translation and drug repurposing. Historical context and discovery As early as the 1950s, electron microscopy first revealed the close apposition between the ER and mitochondrial membranes [ 2 ]. For a long time, however, these structures were considered artifacts of cell fixation and their physiological significance unrecognized [ 2 ]. It was not until 1990 that Jean Vance, using sophisticated subcellular fractionation techniques, successfully isolated and purified this unique membrane component from liver cells, officially naming them MAMs [ 2 ]. This pioneering work not only provided conclusive biochemical evidence for the existence of MAMs but also revealed their critical role in cellular lipid metabolism, thus heralding a new era in the study of organelle crosstalk [ 16 ]. Subsequent research, particularly in the late 1990s, utilized fluorescent protein technology to achieve dynamic visualization of ER–mitochondria interactions in living cells, further confirming that these contact points are real, active structures [ 2 ]. In 2009, the isolation of MAM from rat liver cells confirmed that it is composed of membrane fragments derived from both the ER and the outer mitochondrial membrane (OMM) [ 17 ]. MAMs: a dynamic signaling hub Modern cell biology has elevated the understanding of MAMs from static structural bridges to highly dynamic and plastic signal integration platforms [ 18 ]. These contact sites cover approximately 5–20% of the mitochondrial outer membrane surface, forming an extensive communication network. The structure of MAMs is not static; the tightness, extent, and duration of contact are rapidly remodeled in response to the cell’s metabolic state, stress levels, and signaling stimuli [ 19 ]. This dynamic plasticity allows MAMs to function as an efficient communication hub, enabling the rapid and precise transfer of signaling molecules (such as calcium ions) and metabolites (such as lipids) between the ER and mitochondria—two functionally distinct yet interdependent organelles [ 18 ]. In this way, MAMs coordinately regulate a series of core life processes, including cellular energy metabolism, protein folding, lipid homeostasis, apoptosis, and autophagy, making them a key node in maintaining cellular homeostasis [ 20 ]. Therefore, a deep understanding of MAM regulatory mechanisms and their targeting for therapy not only provides a new perspective for elucidating the pathogenesis of these complex diseases but also holds the promise of creating a novel therapeutic paradigm that can fundamentally correct cellular dysfunction, thereby paving a new pathway for the development of precision medicine. Molecular architecture and physiological functions of MAMs To fully comprehend the role of MAMs in disease, it is essential to first elucidate their intricate molecular structure and diverse physiological functions. MAMs are not merely physical connection points but are highly specialized subcellular regions composed of specific proteins and lipids. A mammalian proteomic analysis employing ingenuity pathway analysis (IPA) revealed a diverse array of proteins within MAMs, including enzymes (e.g., kinases and phosphatases), transporters, and ion channels. These protein classes are implicated in established MAM functions, such as energy metabolism, small molecule transport, ion translocation, and cell signaling [ 21 ]. Ultrastructural features The characteristic ultrastructure of MAMs is a precisely regulated gap between the ER membrane and the outer mitochondrial membrane (OMM), typically ranging from 10 to 30 nm. To visualize this architecture, Fig. 1 presents a multiscale analysis: conventional transmission electron microscopy (TEM, Fig. 1 A) definitively confirms the physical electron-dense tethers (indicated by the yellow arrow) and the narrow interorganelle cleft. Complementing this, super-resolution stimulated emission depletion microscopy (STED) microscopy (Fig. 1 B) overcomes the diffraction limit to reveal the precise spatial organization of these contact sites at the molecular level. This distance is close enough to ensure efficient transmembrane transport of signaling molecules like yet far enough to prevent unwanted membrane fusion, thus maintaining the identity of each organelle [ 18 ]. Studies have found that contact sites formed by smooth endoplasmic reticulum typically exhibit narrower intermembrane distances (approximately 10–15 nm) compared with those formed by rough endoplasmic reticulum (approximately 20–30 nm), which may be attributed to the steric hindrance of ribosomes [ 22 ]. Proteomic and lipidomic analyses have confirmed that MAMs are biochemically distinct from purified ER or mitochondrial fractions, being enriched with a host of proteins and lipids that create a unique biochemical microenvironment [ 22 , 23 ]. Proteins associated with MAMs can be primarily categorized into three groups on the basis of their subcellular localization: the first group consists of proteins exclusively localized to MAMs, the second includes proteins present in both MAMs and other organelles, and the third comprises proteins that transiently localize to MAMs under specific conditions [ 22 ]. Fig. 1. Open in a new tab Multiscale visualization of MAM architecture. A Ultrastructural view via transmission electron microscopy (TEM). Representative micrograph displaying the close physical apposition between the endoplasmic reticulum (ER) and mitochondria. The yellow arrow indicates the characteristic electron-dense tethering structures that bridge the organelle interface, maintaining a physiological gap of approximately 10–30 nm. Scale bar: 200 nm. B Nanoscopic view via STED microscopy. Super-resolution imaging reveals the fine spatial organization of MAMs with nanometer-scale precision. The enhanced lateral resolution distinguishes the ER network (labeled in green) from the mitochondrial network (labeled in red), highlighting the intricate entanglement and specific contact sites that are typically obscured in conventional confocal microscopy owing to the diffraction limit. Scale bar, 2 μm Key protein complexes mediating ER–mitochondria tethering Stable connections between the ER and mitochondria are maintained by a series of proteins or protein complexes termed tethering proteins. These proteins either directly span both membranes or are anchored separately on each membrane surface before forming linkages. Current research reveals significant differences in MAM composition between yeast and mammalian cells: the ERMES complex plays a critical role in yeast, while mammalian systems rely on more sophisticated protein complexes [ 24 ]. These molecular tethers not only ensure the structural integrity of MAMs but also directly participate in functional regulation (Table 1 ). Table 1. Key tethering and regulatory proteins of MAM Protein/complex Location (ER, OMM, cytosol) Interacting partners Primary function Associated disease contexts IP3R–GRP75–VDAC1 ER (IP3R), OMM (VDAC1), cytosol (GRP75) IP3R, GRP75, VDAC1 Mediates rapid ER-to-mitochondria transfer, forming microdomains Diabetes, Cardiovascular Disease, Cancer [ 25 ] VAPB–PTPIP51 ER (VAPB), OMM (PTPIP51) VAPB, PTPIP51 Maintains ER–mitochondria tethering; regulates homeostasis, ATP production, autophagy, synaptic function Neurodegenerative diseases [ 26 ] MFN2 ER, OMM MFN1, MFN2 Acts as a tether (forming homo/heterodimers) or a spacer, regulating mitochondrial fusion and ER–mitochondria distance Charcot–Marie–Tooth disease, diabetic kidney disease, cardiovascular disease [ 18 ] ERMES / PDZD8 ER (Mmm1/PDZD8), OMM (Mdm10/34), cytosol (Mdm12) ERMES complex members Maintains structural ER–mitochondria tethering (classic yeast model and its mammalian homolog) Neurodegenerative diseases [ 27 ] ORP5/8 ER PTPIP51 (OMM) Lipid (phosphatidylserine) transport; regulates mitochondrial morphology and respiration Metabolic diseases [ 18 ] BAP31-FIS1 ER (BAP31), OMM (FIS1) BAP31, FIS1, PACS-2 Regulates ER stress-induced apoptosis X-linked syndrome (deafness, dystonia) [ 1 ] PACS-2 ER BAP31, calnexin Regulates MAM integrity, protein sorting to MAMs, and mediates apoptosis and lipid metabolism Metabolic syndrome, diabetic kidney disease [ 20 ] Sig-1R ER (MAMs) BIP/GRP78, IP3R Modulates IP3R stability, buffers ER stress, regulates signaling and cell survival Neurodegenerative diseases, cardiovascular disease, cancer [ 28 ] Open in a new tab The IP3R–GRP75–VDAC calcium channel complex This is the most well-characterized tethering complex, primarily responsible for mediating rapid transfer from the ER to mitochondria [ 25 ]. It consists of the inositol 1,4,5-trisphosphate receptor (IP3R) on the ER, the voltage-dependent anion channel (VDAC) on the OMM, and the molecular chaperone GRP75, which physically bridges the two channels [ 29 ]. This tight coupling creates localized microdomains, ensuring efficient mitochondrial uptake [ 30 ]. In addition to these three major components, studies have demonstrated that the DJ-1 protein localized at MAMs also participates in complex formation. As an antioxidant protein, DJ-1 regulates calcium signaling by scavenging reactive oxygen species (ROS) to mitigate oxidative stress-induced damage to the IP3R–GRP75–VDAC axis [ 31 , 32 ]. The IP3R–GRP75–VDAC complex has been identified to have a prominent role in regulating calcium homeostasis [ 33 ]. Dysregulation of the IP3R–GRP75–VDAC1 complex disrupts calcium homeostasis, leading to mitochondrial calcium overload. This triggers the opening of the mitochondrial permeability transition pore and the subsequent release of reactive oxygen species and cytochrome C, ultimately resulting in skeletal muscle atrophy. Additionally, this complex can regulate dietary palmitic acid-induced de novo lipogenesis in yellow catfish by recruiting Seipin [ 33 ]. The VAPB–PTPIP51 tethering complex As type II transmembrane proteins, the vesicle-associated membrane protein (VAMP) associated proteins (VAPs) are ubiquitously expressed and can be found in the ER and pre-Golgi intermediates [ 34 ]. VAPs function as scaffold proteins, facilitating communication between these organelles [ 35 ]. PTPIP51 (also known as RMDN3) is a mitochondrial protein, depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. As revealed by an in vitro liposome assay, suggests that PTPIP51 transfers lipid radicals from mitochondria to the ER [ 36 ]. VAPB–PTPIP51 tethering complex is formed by the interaction between the ER-resident VAPB and the OMM-localized PTPIP51 [ 37 ]. It is another crucial molecular tether, particularly important in neurons [ 38 ]. VAPB–PTPIP51 ER–mitochondria tethering corrects Ca 2+ and synaptic defects may have therapeutic value for frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) [ 39 ]. Structurally, VAPB and PTPIP51 form a complex through their FFAT and coiled-coil domains to maintain MAM stability [ 40 ]. Furthermore, the VAPB–PTPIP51 complex is involved in regulating homeostasis, ATP production, autophagy, synaptic function, mediate lipid radical transfer and mitochondrial oxidative stress [ 26 , 40 ]. The multifaceted role of Mitofusin 2 (MFN2) MFN2, a crucial GTPase involved in mitochondrial dynamics, regulates key mitochondrial-associated biological processes. The role of MFN2 in MAMs is complex. On the one hand, ER-localized MFN2 can form homodimers or heterodimers with mitochondrial MFN1/2 to tether the organelles [ 18 ]. On the other hand, some studies suggest MFN2 may act as a spacer, preventing excessive contact [ 18 ]. This dual role suggests that cells can fine-tune the ER–mitochondria distance by modulating MFN2’s function to meet different physiological needs. MFN2 and its endogenous interactions play a critical role in interorganelle communication and autophagy. Current research has identified RAB5C (a member of the RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 member 2) as a novel interacting partner of MFN2 that is relevant in autophagy [ 41 ]. However, the precise molecular mechanisms involved require further elucidation. Furthermore, MFN2 is specifically localized at melanosome–mitochondria contact sites. Its knockdown significantly reduces interorganelle connections and impairs melanogenesis [ 42 ]. Beyond influencing autophagy and organelle biogenesis through membrane interactions, MFN2 also functionally promotes ER–mitochondria calcium transfer to maintain calcium homeostasis. In Mfn2 -deficient cells, the transfer of Ca 2+ from the ER to mitochondria is impaired, while the mitochondrial Ca 2+ uptake machinery itself remains intrinsically intact [ 43 , 44 ]. The BAP31–FIS1 tethering complex The mitochondrial fission protein 1 (Fis1) on the outer mitochondrial membrane (OMM) and B-cell receptor-associated protein 31 (BAP31) on the endoplasmic reticulum (ER) interact at MAMs to form a critical platform for transmitting apoptotic signals [ 45 – 47 ]. During apoptosis induction, procaspase-8 accumulates near the BAP31–FIS1 complex and, upon activation, cleaves BAP31 to generate the pro-apoptotic fragment p20-BAP31. This fragment subsequently enhances ER calcium release, leading to mitochondrial calcium overload, altered mitochondrial membrane permeability, cytochrome c release, and ultimately apoptosis [ 47 ]. Furthermore, cytosolic region of BAP31 and Tom40 or NDUFS4 endogenously associated, BAP31 could stimulates translocation of NDUFS4 from cytosol to mitochondria, while ER stress-dependent modification of BAP31 localization and interaction with Bcl-2 induces disruption of the BAP31–Tom40 complex. ER–mitochondria communication via the BAP31- translocase of the outer mitochondrial membrane 40 (Tom40) complex is essential for control of mitochondrial homeostasis [ 48 ]. Other tethering and regulatory proteins Numerous other proteins, including the yeast ERMES complex and its mammalian homolog PDZD8, lipid transfer proteins such as ORP5/8 [ 49 ], and signaling proteins such as the Sigma-1 receptor (Sig-1R) [ 50 ] and PACS-2, are localized to MAMs and contribute to their diverse functions [ 18 , 38 ]. This molecular diversity and functional redundancy ensure the stability and adaptability of MAMs as a crucial cellular structure. MAMs: master regulators of cellular physiology As a highly integrated platform, MAMs coordinate a series of vital physiological processes, where the extent of contact must be maintained within an optimal range [ 13 ]. Under physiological conditions, MAMs maintain a flexible and transient interaction to facilitate metabolic adaptations. However, this balance is precarious. Crucially, the functional outcome of MAMs adheres to a Goldilocks principle. Pathological tightening of these contacts creates a toxic bridge, facilitating massive Ca 2+ transfer that overwhelms mitochondrial buffering capacity, triggers mPTP opening, and precipitates necrotic cell death. Conversely, excessive dissociation isolates mitochondria from ER-derived lipid and calcium support, leading to bioenergetic collapse and failure of membrane repair mechanisms. Loss of contact disrupts the flow of calcium and lipids required for oxidative phosphorylation and membrane repair. Conversely, hyper-stabilized or permanent MAM formation is a hallmark of pathological states (such as Alzheimer’s disease or acute organ injury), creating a toxic bridge that transmits death signals including calcium toxicity and pro-inflammatory triggers. Therefore, maintaining the contact extent within an optimal window is paramount for cellular resilience. Calcium signaling Calcium ions (Ca 2+ ) are universal second messengers that regulate a broad spectrum of cellular processes, including proliferation, differentiation, and programmed cell death. In eukaryotic cells, Ca 2+ is highly concentrated in the ER lumen and in the extracellular space, whereas cytosolic Ca 2+ is maintained at low nanomolar to submicromolar levels through tightly regulated buffering and transport mechanisms [ 51 ]. The ER serves as the principal intracellular Ca 2+ store, coordinating Ca 2+ storage and release in a manner that profoundly influences mitochondrial function (Fig. 2 ). A substantial fraction of this Ca 2+ signaling occurs at MAMs, where close apposition between the ER and the outer mitochondrial membrane (OMM) enables highly efficient, vectorial Ca 2+ transfer from ER to mitochondria [ 52 , 53 ]. Under physiological conditions, controlled mitochondrial Ca 2+ uptake at MAMs is essential for the fine-tuning of oxidative metabolism. Ca 2+ in the mitochondrial matrix activates multiple Ca 2+ -dependent dehydrogenases within the tricarboxylic acid (TCA) cycle, thereby stimulating oxidative phosphorylation and ATP synthesis [ 54 ]. This feedforward loop couples cytosolic stimulation (for example, hormone- or receptor-driven IP3 production) to a rapid increase in mitochondrial ATP output, ensuring that energy supply is matched to cellular demand [ 55 ]. Fig. 2. Open in a new tab The molecular architecture and functional machinery of MAMs However, the same machinery that supports adaptive bioenergetics can become deleterious when Ca 2+ transfer is excessive or dysregulated. Sustained or repeated Ca 2+ spikes at MAMs cause mitochondrial Ca 2+ overload [ 56 ], triggering opening of the mitochondrial permeability transition pore, collapse of the mitochondrial membrane potential, inhibition of ATP production and activation of apoptotic or necrotic cell death pathways [ 13 , 54 , 57 ]. Thus, ER–mitochondria Ca 2+ transfer must be maintained within a narrow range to support cell survival and prevent catastrophic mitochondrial injury. The central conduit for ER-to-mitochondria Ca 2+ transfer is the macromolecular complex formed by inositol 1,4,5-trisphosphate receptors (IP3Rs) on the ER, voltage-dependent anion channels (VDACs) on the OMM, and the molecular chaperone glucose-regulated protein 75 (GRP75), which physically bridges IP3Rs and VDAC1 at MAMs [ 58 – 60 ]. Among the three IP3R isoforms, IP3R2 appears to be the most effective for mitochondrial Ca 2+ delivery. GRP75 stabilizes the juxtaposition of IP3Rs and VDAC1, thereby maintaining MAM architecture and creating Ca 2+ “microdomains” at the ER–mitochondria interface. Within these microdomains, Ca 2+ can be transferred directly from IP3Rs to VDAC1 and then to the inner mitochondrial membrane (IMM) through the mitochondrial calcium uniporter (MCU), often without a large increase in bulk cytosolic Ca 2+ [ 60 ]. IP3R activity is tightly regulated by IP3, Ca 2+ itself, and ATP [ 61 ]. ATP exerts a dual effect: it increases the sensitivity of IP3Rs to IP3 and Ca 2+ , thereby facilitating channel opening and ER Ca 2+ release, but it also stabilizes closed, inactive conformations of IP3Rs, preventing uncontrolled Ca 2+ leakage. Through these opposing actions, ATP contributes to the temporal and spatial precision of Ca 2+ signaling and protects cells from Ca 2+ dysregulation. On the mitochondrial side, Ca 2+ entry through VDAC1 and MCU not only supports ATP generation but also feeds back on ROS production, mitochondrial dynamics, and MAM integrity (Fig. 2 ). Excessive mitochondrial Ca 2+ favors increased production of reactive oxygen species, promotes cytochrome c release and can destabilize MAMs, creating a vicious cycle of Ca 2+ overload, oxidative stress, and organelle dysfunction [ 62 , 63 ]. Lipid metabolism Phospholipids are essential structural components of cellular membranes and are synthesized primarily in the ER before being distributed to other organelles through both vesicular and nonvesicular pathways [ 64 ]. Among the various membrane contact sites, MAMs serve as major hubs for lipid metabolism, lipid trafficking, and phospholipid biogenesis (Fig. 2 ). Multiple tethering factors and lipid transfer proteins (LTPs) operate at MAMs to ensure efficient interorganellar lipid exchange, enabling mitochondria to maintain membrane composition, oxidative metabolism, and organelle integrity. Recent evidence shows that Mic19, a core subunit of the MICOS complex, is essential for maintaining these contacts. Loss of Mic19 reduces ER–mitochondria coupling and impairs mitochondrial lipid handling, leading to defective fatty-acid β-oxidation, crista disorganization, and mitochondrial stress [ 4 ]. A fundamental phospholipid synthesis pathway occurs at MAMs and begins with the conversion of phosphatidic acid to phosphatidylserine (PS) by phosphatidylserine synthase-1/2 (PSS1/2) in the ER membrane. Newly synthesized PS is then transported to mitochondria, where phosphatidylserine decarboxylase (PISD) in the inner mitochondrial membrane converts PS into phosphatidylethanolamine (PE). PE is subsequently shuttled back to the ER and methylated by phosphatidylethanolamine N -methyltransferase 2 (PEMT2) to form phosphatidylcholine (PC) [ 65 , 66 ]. This ER–mitochondria shuttling process constitutes a rate-limiting step in phospholipid biogenesis and is indispensable for maintaining MAM integrity and global lipid homeostasis. Efficient flux through this pathway depends heavily on lipid trafficking mediated by specialized LTPs positioned at MAMs [ 67 ]. Key lipid synthases—including phosphatidylserine synthase 1 (PSS1), phosphatidylserine synthase 2 (PSS2), and acyl-CoA cholesterol acyltransferase (ACAT1)—are enriched here, regulating the synthesis of relevant lipids [ 14 , 68 ]. Beyond synthesizing essential cellular lipids, MAMs also mediate the nonvesicular transport of critical molecules such as phospholipids and cholesterol between the ER and mitochondria, which is vital for maintaining membrane integrity and function [ 13 ]. Recent evidence highlights MAMs as crucial platforms for regulating ferroptosis, an iron-dependent form of regulated necrosis driven by lethal lipid peroxidation [ 69 ]. The long-chain acyl-CoA synthetase 4 (ACSL4), a key ferroptosis executioner, is predominantly localized at MAMs. Here, ACSL4 facilitates the incorporation of polyunsaturated fatty acids (PUFAs) into membrane phospholipids, providing the essential substrates for subsequent iron-catalyzed lipid peroxidation. Pathological conditions that intensify lipid synthesis and oxidation at MAMs, coupled with reduced antioxidant defenses, significantly accelerate ferroptosis in highly metabolic cells. In addition, ferroptosis is also shaped by spatially organized signaling at MAMs. During ferroptosis, lipid peroxidation accumulates in the endoplasmic reticulum and activates protein kinase A (PKA) in a cyclic adenosine monophosphate (cAMP)-dependent manner. The mitochondrial outer membrane A-kinase anchoring protein 1 (AKAP1) anchors PKA and enables local phosphorylation of glucose-regulated protein 75 (GRP75) at S148 within MAMs [ 8 ]. This modification retains GRP75 outside mitochondria, where it binds Kelch-like ECH-associated protein 1 (Keap1) through its ETGE motif and blocks Keap1-mediated suppression of nuclear factor erythroid 2-related factor 2 (Nrf2). Stabilized Nrf2 then activates anti-ferroptotic gene expression. Studies show that the AKAP1–PKA–GRP75 axis is a key regulator of ferroptosis sensitivity in colorectal cancer. This diagram illustrates the critical role of MAMs in cellular homeostasis, highlighting lipid exchange and calcium Ca 2+ signaling: (1) lipid metabolism: MAMs facilitate the transport of phospholipids (PA, PS, PE, PC) and the synthesis of cardiolipin (CL), essential for mitochondrial membrane integrity; (2) calcium signaling: the IP3R–GRP75–VDAC complex mediates Ca 2+ transfer from the ER to mitochondria. While physiological Ca 2+ uptake drives ATP production via the TCA cycle, Ca 2+ overload triggers MPTP opening, ROS production, and apoptosis. Structural tethers such as Mfn2 and PERK maintain this interface; (3) regulation of IP3R: the inset highlights the intricate regulation of the IP3R channel by the PML–PP2A–AKT axis and BCL-2 family proteins, balancing cell survival and death. Schematic diagram created with Figdraw. Mitochondrial dynamics and mitophagy Mitochondria are highly dynamic organelles whose morphology, distribution, and functional integrity depend on a finely regulated balance between fusion, fission, and selective removal of damaged organelles through mitophagy [ 70 ]. MAMs serve as the structural and signaling platform orchestrating these processes, thereby ensuring mitochondrial quality control and cellular stress resilience. Disruption of MAM integrity can therefore profoundly reshape mitochondrial dynamics, influence cell fate, and contribute to the pathogenesis of diseases [ 71 ]. Mfn1 and Mfn2 mediate the fusion of the mitochondrial outer membrane, whereas Opa1 is responsible for the fusion of the inner membrane. The key proteins involved in mitochondrial fission primarily include dynamin-related protein (DRP1/DLP1), mitochondrial dynamics proteins 49/51 (MiD49/51), mitochondrial fission factor (MFF), and fission protein 1 (Fis1) [ 72 ]. Studies have shown that the tubular structures of the ER mark mitochondrial fission sites at MAMs and recruit core fission proteins such as Drp1 (Fig. 2 ), thereby initiating mitochondrial fission [ 13 , 73 , 74 ]. Emerging evidence indicates that MAMs coordinate autophagy and mitophagy through distinct structural mechanisms. They serve as ER cradles for autophagosome initiation. The enrichment of the PI3K complex III subunit ATG14L at these contact sites generates a localized pool of phosphatidylinositol 3-phosphate (PI3P). This PI3P pool recruits the effector protein DFCP1, which nucleates omegasomes—the precursors of autophagosomes—directly from the ER–mitochondria interface.They serve as platforms for autophagosome initiation and function as mitochondrial marking sites for selective clearance of damaged mitochondria [ 75 ]. The VAPB–PTPIP51 complex plays a significant role in autophagy. Under pathological conditions, its overexpression promotes MAM formation, thereby suppressing the autophagic process [ 76 ]. During mitophagy, the PINK1/Parkin pathway serves as one of the most critical pathways [ 77 ]. Upon mitochondrial damage, stabilized PINK1 on the OMM recruits cytosolic Parkin specifically to the MAM interface. This spatial confinement allows the ER to rapidly supply the lipid membrane required to engulf the damaged organelle, thereby ensuring efficient isolation and degradation. PINK1 and Beclin-1 accumulate at MAMs, promoting ER–mitochondria contact and autophagosome formation [ 78 , 79 ]. Furthermore, MAMs function as a key activation hub for innate immune signaling, directly linking metabolic states with immune responses [ 13 ]. The unfolded protein response (UPR) The unfolded protein response (UPR) is activated when misfolded proteins accumulate in the ER, a process monitored by three stress sensors—protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) [ 80 ]. Under basal conditions, these sensors remain inactive by binding immunoglobulin protein (BiP). During ER stress, BiP dissociates to bind misfolded proteins, thereby releasing and activating the three sensors [ 81 ]. Early UPR signaling promotes ER adaptation, whereas prolonged stress shifts the response toward inflammation and apoptosis [ 82 ]. MAMs also function as essential platforms for coordinating both canonical and noncanonical UPR signaling. PERK is highly enriched at MAMs, where it not only phosphorylates eIF2α to induce ATF4–CHOP-dependent apoptosis but also strengthens ER–mitochondria tethering and amplifies reactive oxygen species signaling by recruiting BAX to mitochondria [ 83 ]. PERK furthermore mediates phospholipid transfer by interacting with extended synaptotagmin-1 (E-Syt1), a noncanonical mechanism supporting mitochondrial stability [ 84 ]. Mitofusin-2 (MFN2) directly restrains PERK activation, establishing a bidirectional feedback loop between MAM architecture and UPR activity [ 85 ]. IRE1α also accumulates at MAMs, where it performs functions beyond its classical role in X-box binding protein 1 (XBP1) mRNA splicing [ 86 ]. At these sites, IRE1α stabilizes inositol 1,4,5-trisphosphate receptors (IP3Rs), sustaining mitochondrial Ca 2+ uptake and ATP production [ 87 , 88 ]. This enrichment of UPR sensors at MAMs represents an adaptive bioenergetic strategy. Protein refolding is an ATP-intensive process. By localizing to MAMs, activated PERK and IRE1α can immediately stabilize IP3Rs to enhance ER-to-mitochondria Ca 2+ transfer. This signal boosts TCA cycle activity and mitochondrial ATP production, thereby fueling the high energy demands of chaperone-mediated refolding. This mechanism ensures that the UPR initially functions as a protective metabolic boost before switching to apoptotic signaling if the stress remains unresolved. Several MAM-resident proteins modulate IRE1α signaling: sigma-1 receptor (SIG1R) enhances IRE1α dimer stability during stress [ 89 ], while the mitochondrial E3 ligase MARCH5/MITOL prevents excessive IRE1α activation through ubiquitination [ 90 ]. Vesicle-associated membrane protein-associated protein B (VAPB) also facilitates IRE1–XBP1 signaling, and its loss dampens this branch of the UPR [ 91 ]. ATF6 activity is similarly influenced by ER–mitochondria communication. After ER stress, ATF6 migrates to the Golgi for proteolytic activation into ATF6p50 [ 85 , 92 ]. ER–mitochondria calcium flux regulates ATF6 output, and studies in Caenorhabditis elegans have shown that reducing ATF6 activity improves organismal lifespan by rebalancing ER Ca 2+ handling and IP3R-mediated transfer [ 93 ]. Under stress, VAPB binds and restrains ATF6, adding another layer of MAM-dependent regulation [ 94 , 95 ]. Beyond the canonical stress sensors, the ER membrane hosts a specialized class of “protective proteins” known as selenoproteins, which play a pivotal role in buffering ER stress and maintaining MAM homeostasis. Selenoproteins (such as selenoprotein T, S, and K) are enriched in the ER and are essential for redox regulation and the ER-associated degradation (ERAD) pathway. By facilitating the removal of misfolded proteins and scavenging ROS, they prevent the chronic hyperactivation of the UPR that typically leads to MAM dysregulation and apoptosis. Recent studies highlight the systemic importance of selenoproteins for neuro-cardiac protection. In the cardiac system, selenoproteins have been shown to protect cardiomyocytes from ischemia-induced ER stress by stabilizing calcium handling proteins at the MAM interface [ 96 ]. Similarly, in the nervous system, they act as neuroprotective agents. Evidence indicates that selenoprotein deficiency exacerbates neuroinflammation and ER stress, while their upregulation preserves neuronal viability and synaptic function in neurodegenerative models [ 97 ]. Thus, selenoproteins represent intrinsic “MAM stabilizers,” suggesting that dietary selenium supplementation or pharmacological mimetics of these proteins could serve as a viable strategy to mitigate organelle stress in both heart failure and neurodegeneration. Innate immunity Studies have revealed that MAMs participate in the activation of inflammation and provide a signaling platform for the NLRP3 inflammasome, promoting its assembly and activation [ 98 ]. Prolonged ER stress can turn the normally protective UPR into an inflammatory response. Sustained UPR activity can activate nuclear factor kappa B (NF-κB) and the NLR family pyrin domain-containing 3 (NLRP3) inflammasome. These pathways increase the production of inflammatory cytokines such as interleukin-1β (IL-1β) [ 99 , 100 ]. ER stress can also damage mitochondria. Damaged mitochondria can release mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA can activate the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway [ 101 ]. cGAS detects DNA in the cytosol. The DNA can come from pathogens or from the cell itself, such as mtDNA or nuclear DNA [ 102 ]. When cGAS binds DNA, it changes shape, forms a dimer, and produces 2′3’-cyclic GMP–AMP (cGAMP). cGAMP then binds to STING. STING moves from the ER to the ER–Golgi intermediate compartment and then to the Golgi. At the Golgi, STING activates TANK-binding kinase 1 (TBK1). TBK1 phosphorylates interferon regulatory factor 3 (IRF3) [ 102 ]. Phosphorylated IRF3 forms a dimer and enters the nucleus, where it starts the transcription of type I interferons (MLA 145). STING can also activate NF-κB through TBK1 or IκB kinase ε (IKKε), which increases other inflammatory cytokines and chemokines [ 103 ]. MAMs play a key role here because they help trigger STING activation and regulate its activity [ 104 ]. STING mainly sits on the ER and is enriched at MAMs. Its ligand-binding domain faces the cytosol [ 105 ]. This position helps STING sense stress signals inside the cell. Oxidative stress, inflammation, and mitochondrial injury can cause mtDNA release, which activates the cGAS–STING pathway (Fig. 3 ). Other MAM-related proteins may also influence STING signaling. Loss of disulfide-bond A-like protein (DsbA-L) damaged mitochondria, increased mtDNA release, and activated cGAS–STING. This drove inflammation and metabolic problems in high-fat diet models [ 106 ]. Overexpressing DsbA-L reduced this pathway and protected against inflammation [ 107 ]. STING knockout also protected mice from diet-induced obesity by increasing thermogenesis [ 108 ]. Fig. 3. Open in a new tab MAMs serve as a pivotal platform for sensing mitochondrial stress and orchestrating innate immune responses The NLRP3 inflammasome is another major inflammatory mechanism [ 109 ]. In resting cells, NLRP3 sits on the ER membrane and in the cytosol. When the cell senses triggers such as potassium (K + ) efflux, mitochondrial injury, reactive oxygen species (ROS), or calcium (Ca 2+ ) rise [ 108 ], and NLRP3 forms oligomers. It then recruits apoptosis-associated speck-like protein containing a CARD (ASC) and procaspase-1. Together they form the NLRP3 inflammasome [ 110 ]. This complex activates caspase-1. Caspase-1 processes IL-1β and IL-18 into their active forms. Activated NLRP3 moves from the ER to MAMs. At MAMs, it senses mitochondrial ROS and mtDNA from damaged mitochondria [ 111 ]. NLRP3 also interacts with mitochondrial antiviral signaling protein (MAVS), which increases cytokine production [ 112 ]. Many stressors, such as ER stress and Ca 2+ overload, can start NLRP3 inflammasome activation [ 113 ]. VDAC1 also helps activate NLRP3 by releasing mtDNA [ 114 ]. Reducing VDAC1 lowered caspase-1 activation, IL-1β secretion, and mitochondrial ROS [ 98 ]. STING also acts upstream of NLRP3 [ 115 ]. In APOL1-associated kidney disease, both STING and NLRP3 were increased in podocytes [ 116 ]. (Table 2 ). Table 2. Comparative analysis of tissue-specific MAM heterogeneity and regulatory functions Tissue/cell type Regulatory proteins Physiological priority Mechanism of regulation Pathological consequence of dysfunction Cardiomyocytes heart) RyR2 IP3R2-GRP75-VDAC1 Excitation–bioenergetic coupling Nanodomains sync mechanical beat with TCA cycle activation Arrhythmia Uncoupling leads to Ca 2+ leak and bioenergetic failure Hepatocytes (liver) IP3R1-GRP75-VDAC1 MFN2 CNPY2 Metabolic flexibility Regulates insulin signaling via Akt/mTORC2; VLDL secretion NASH/insulin resistance ER stress drives inflammation and lipid accumulation Neurons (brain) VAPB–PTPIP51, Sig-1R Synaptic transmission Sig-1R buffer stress; lipid transfer AD/ALS Synaptic loss Skeletal muscle VDAC IP3R1 Excitation–contraction coupling TBC1D15 regulates fission during exercise Insulin resistance glucose uptake Adipocytes (fat tissue) Seipin FSP27 Lipid droplet (LD) biogenesis Seipin concentrates at ER–Mito–LD junctions Lipodystrophy/obesity Ectopic lipid deposition Renal tubular cells (kidney) MFN2, PACS-2, DsbA-L, FUNDC1 Bioenergetics and repair MFN2 maintains mitochondrial morphology AKI & DKD Disruption leads to ATP depletion, ROS burst Immune cells (macrophages) NLRP3–MAVS STING Innate immunity MAMs recruit NLRP3 for inflammasome assembly Sepsis: constitutive assembly drives chronic inflammation Open in a new tab This diagram illustrates: (1) Sensing mitochondrial damage: stress conditions lead to mitochondrial dysfunction, ROS accumulation, and the leakage of mitochondrial DNA (mtDNA) into the cytosol; (2) The cGAS-STING axis: cytosolic mtDNA is sensed by cGAS, generating cGAMP. This second messenger activates STING, which resides at the ER–mitochondria interface (MAMs). STING recruits TBK1, leading to the phosphorylation and nuclear translocation of IRF3 and NF-kappaB, thereby triggering the production of type I interferons and pro-inflammatory cytokines; (3) Mitochondrial quality control: the diagram also highlights the protective roles of mitophagy (clearing damaged mitochondria) and mitochondrial dynamics (fission via DRP1 and fusion via MFN2), which collectively maintain mitochondrial integrity and prevent excessive immune activation. Schematic diagram created with Figdraw. MAM dysregulation in the human disease spectrum MAM imbalance represents a form of systemic metabolic maladaptation at the cellular level, constituting a common cross-pathological mechanism [ 117 ]. Under physiological conditions, these dynamic contacts orchestrate a delicate balance of energy production and quality control. However, when this coupling is compromised, it triggers a cascade of deleterious events—including mitochondrial calcium overload, oxidative stress bursts, and sterile inflammation—that propagate cellular injury. This imbalance represents a fundamental form of systemic metabolic maladaptation. As detailed below, perturbations in MAM architecture and function have now been identified as a common cross-pathological mechanism linking seemingly distinct etiologies, ranging from neurodegeneration and cardiovascular failure to metabolic syndrome and kidney disease (Table 3 ). Table 3. Key tethering complexes and regulatory proteins of MAMs and their pathological relevance Disease Specific pathology MAM status Key molecular mechanism Therapeutic strategy Kidney IR-induced AKI [ 118 ] Hyperconnectivity (< 10 nm) IP3R–GRP75–VDAC1 tightening Block/truncate (e.g., GA-Cu) Kidney Cisplatin-induced AKI [ 119 ] Disruption (> 30 nm) MFN2 Stabilize Kidney Cisplatin-induced AKI [ 72 ] Hyperconnectivity (< 15 nm) TRPA1 Disruption (e.g., HC-030031) Kidney IR-induced AKI [ 120 ] Disruption (> 30 nm) MFN2 Stabilize (e.g., MFN2 overexpression) Kidney IR-induced AKI-CKD [ 121 ] Hyperconnectivity (< 15 nm) Pannexin1 Disruption Kidney Chronic/diabetic nephropathy [ 122 ] Disruption (> 50 nm) PACS-2, CNPY2 anomaly Rebuild/stabilize (e.g., PACS-2) Kidney Diabetic nephropathy [ 123 ] Disruption (> 50 nm) MAPK1 Stabilize (e.g., VX-11e) Kidney Diabetic nephropathy [ 124 ] Disruption (> 50 nm) Reticulon-1A Stabilize Kidney Diabetic nephropathy [ 125 ] Hyperconnectivity (< 10 nm) VAPB–PTPIP51 Disruption (Yiqi Huoxue recipe) Kidney Diabetic nephropathy [ 126 ] Disruption (> 50 nm) MFN2, FUNDC1 Enhance connection (e.g., VDR agonist) Neuro Alzheimer’s Hyperconnectivity (< 10 nm) PS1/2 mutation, C1q accumulation Downregulate Neuro Alzheimer’s (late/Tau) Parkinson’s [ 37 , 40 ] amyotrophic lateral sclerosis, ischemic stroke [ 127 ] Disruption (> 30 nm) Tau blocks VAPB–PTPIP51 Stabilize/repair Neuro Alzheimer’s [ 128 ] Hyperconnectivity (< 10 nm) IP3R, VDAC1 Disruption Neuro Parkinson’s (early-onset familial) [ 129 ] Disruption (> 30 nm) DJ-1 Stabilize/repair Neuro Parkinson’s [ 130 ] Hyperconnectivity (< 10 nm) PINK1, Parkin Disruption Neuro Amyotrophic lateral sclerosis [ 131 ] Disruption (> 30 nm) MFN2 Stabilize (e.g., MFN2) Neuro Amyotrophic lateral sclerosis [ 132 ] Disruption (> 30 nm) Sigma 1 Enhance (e.g., PRE-084, SA4503 receptor agonist) Neuro Subarachnoid hemorrhage [ 133 ] Hyperconnectivity (< 10 nm) DRP1 Disruption (e.g., P110) Cardio I/R Hyperconnectivity (< 10 nm) Ca 2+ burst Inhibit connection Cardio Heart failure/hypertrophy Disruption (> 40 nm) Mfn2 loss Rebuild connection (e.g., linker peptide) Cardio Cardiac hypertrophy [ 134 ] Disruption (> 30 nm) FMO2 Enhance connection Cardio Myocardial infarction [ 6 ] Disruption (> 30 nm) Mtus1 Enhance connection Mtus1A overexpression Cardio Cardiac aging [ 9 ] Disruption (> 30 nm) Phosphatidylethanolamine (PE) Rebuild connection (e.g., linker peptide) Cardio Cardiac hypertrophy [ 135 ] Hyper-connectivity (< 15 nm) SIRT3–ATAD3A Enhance connection (e.g., SZC-6) Cardio Myocardial ischemia/reperfusion injury [ 136 ] Disruption (> 30 nm) ATAD3A Stabilize/repair (e.g., thymoquinone) Cardio Heart failure [ 137 ] Disruption (> 30 nm) STX17 Stabilize/repair Cardio Diabetic cardiomyopathy [ 138 ] Hyperconnectivity (< 10–15 nm) VDAC1, PACS2, IP3R2 Disruption Cardio Pulmonary artery hypertension [ 139 ] Disruption (> 30 nm) Mfn2, IP3R3 Stabilize/repair Oncology Ovarian cancer [ 140 ] Hyperconnectivity (< 15 nm) GRP75 Inhibit connection Oncology Tumor angiogenesis [ 141 ] Hyperconnectivity (< 10–15 nm) FUNDC1 Disruption Oncology Hepatocellular carcinoma [ 142 ] Hyperconnectivity (< 10–15 nm) VDAC1 Disruption (e.g., metformin) Oncology Breast cancer [ 143 ] Disruption (> 30 nm) Drp1, MFN2 Enhance connection (e.g., flubendazole) Oncology Hepatocellular carcinoma [ 144 ] Hyperconnectivity (< 10) Sigma 1 Disruption (e.g., rimcazole) Liver Obesity/fatty liver Hyperconnectivity (< 10) IP3R1 Moderate interference Liver Hepatic ischemia/reperfusion injury [ 145 ] Disruption (> 30 nm) PINK1 Stabilize/repair Liver Obesity/fatty liver [ 146 ] Hyperconnectivity (< 10) PACS-2, IP3R1 Disruption Liver Diabetic hepatopathy [ 147 ] Hyperconnectivity (< 10) VDAC1 Disruption (e.g., lncRNA H19 inhibition) Liver NASH [ 148 ] Disruption (> 30 nm) Cds2, IP3R-VDAC1 Enhance connection (e.g., PPARα agonist) Liver NAFLD [ 149 ] Hyperconnectivity (< 10) IP3R1–GRP75–VDAC1 tightening Disruption (e.g., SIRT1 inhibitor) Liver Mechanical trauma [ 150 ] Hyperconnectivity IP3R1–GRP75–VDAC1 tightening Disruption (e.g., melatonin) Liver Acute liver failure [ 151 ] Disruption (> 30 nm) MFN2 Enhance connection (e.g., AGK2) Liver NASH [ 152 ] Disruption (> 30 nm) MFN2 Rebuild connection (e.g., ñinker peptide) Lung Ventilator-induced lung Injury [ 153 ] Hyperconnectivity (< 10) IP3R Disruption (e.g., 2-APB) Open in a new tab Neurodegenerative diseases MAM dysfunction is a key pathological hallmark in neurodegenerative diseases [ 154 , 155 ]. Controlling mitochondria-associated endoplasmic reticulum membranes via protein S -palmitoylation may represent a novel therapeutic target for neurodegenerative diseases [ 156 ]. Alzheimer’s disease (AD) Alzheimer’s disease is defined by widespread loss of neurons and synapses, along with the pathological buildup of β-amyloid plaques and hyperphosphorylated Tau protein [ 157 ]. MAMs play multiple roles in AD pathogenesis; they serve as the primary site for Aβ generation, where amyloid precursor protein (APP) and its processing enzymes (like γ-secretase) are enriched [ 158 ]. Increased MAM activity promotes Aβ production and its translocation to mitochondria, exacerbating mitochondrial dysfunction [ 158 ]. Altered MAMfunction also disrupts lipid metabolism and calcium signaling [ 159 ]. The high-risk APOE4 genotype is associated with enhanced MAM function, further disturbing lipid and cholesterol metabolism [ 160 ]. In AD, the interaction between the ER and mitochondria gets stronger. This raises the levels of IP3R and VDAC1, and causes more calcium transport [ 128 ]. Excessive calcium release from MAMs impairs synaptic plasticity and promotes Aβ deposition and neurofibrillary tangle formation, leading to cognitive deficits [ 161 ]. Pathological Tau disrupts the VAPB–PTPIP51 tether, reducing ER–mitochondria contacts and impairing mitochondrial cholesterol metabolism and neurosteroid synthesis [ 162 ]. This Tau-mediated MAM dissociation is reversible by GSK3β inhibition [ 162 ]. Parkinson’s disease (PD) In Parkinson’s disease, degeneration of substantia nigra neurons progressively impairs motor control, giving rise to tremors, gait abnormalities, and muscle stiffness [ 163 ]. α-Synuclein aggregation is a key pathological event in PD. It directly binds to VAPB on the ER, competitively inhibiting the VAPB–PTPIP51 interaction and disrupting ER–mitochondria contacts [ 37 ]. Additionally, α-synuclein impairs the IP3R–GRP75–VDAC complex [ 28 ]. In early-onset familial PD, DJ-1 plays an important role. The loss or mutation of DJ-1 leads to the accumulation of α-Syn [ 129 ]. These mechanisms collectively lead to deficient mitochondrial calcium uptake and reduced ATP production, compromising neuronal energy supply and survival [ 28 ]. Furthermore, mutations in PINK1 and Parkin localized to MAMs have been implicated in familial Parkinson’s disease (PD) [ 164 ]. Mutations in PINK1 or Parkin increase mitochondrial–ER contact, enhancing calcium exchange and ATP production [ 130 , 165 ]. Huntington’s disease (HD) HD exhibits disrupted mitochondrial fission–fusion balance, leading to mitochondrial fragmentation [ 166 , 167 ]. Increased Drp1 activity impairs ER–mitochondria contacts, causing MAM dysfunction and calcium signaling defects [ 168 ]. Furthermore, mutant huntingtin protein interacts with Sigma-1 receptor, reducing its association with the IP3R–GRP75–VDAC complex and further compromising ER–mitochondria tethering, ultimately disrupting calcium transfer and mitochondrial function [ 169 ]. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) In ALS/FTD, multiple pathogenic proteins (including TDP-43, FUS, and dipeptide repeat proteins from C9orf72 repeat expansions) disrupt the VAPB–PTPIP51 tether by activating GSK3β kinase [ 37 ]. Mutations in the VAPB gene (such as P56S), which directly impair its binding to PTPIP51, are also a cause of familial ALS [ 37 ]. The disruption of this molecular tether is an early and critical event in ALS/FTD pathology, leading to disrupted neuronal calcium homeostasis, impaired autophagy, and synaptic dysfunction [ 26 ]. Moreover, in ALS, MFN2 inhibits the axonal transport of calpastatin, which increases calpain activity in neurons and causes neuromuscular synapse degeneration [ 131 ]. Metabolic syndrome MAMs are critical for integrating nutrient and hormonal signals, and their dysfunction is closely linked to metabolic syndrome [ 170 ]. Insulin resistance and type 2 diabetes Diabetes represents a metabolic condition defined by reduced insulin sensitivity, insufficient insulin production, and disturbances in glucose homeostasis [ 171 ]. MAMs serve as crucial hubs for insulin signaling [ 172 ]. In type 2 diabetes, insulin resistance disrupts gluconeogenesis and glycogenolysis, while MAM alterations impair insulin-mediated glucose uptake [ 173 ]. The mTORC2–Akt signaling axis at MAMs phosphorylates and regulates MAM proteins such as IP3R, HK2, and PACS2) upon insulin stimulation, maintaining MAM integrity and glucose metabolism [ 174 ]. Under insulin resistance, MAM structural integrity is compromised, impairing insulin signal transduction and exacerbating hyperglycemia [ 20 ]. In pancreatic β-cells, aberrant expression of proteins such as PDZD8 causes MAM hyperconnectivity, leading to calcium overload, cell death, and impaired insulin secretion [ 2 , 175 ]. Obesity and non-alcoholic fatty liver disease (NAFLD/NASH) NAFLD is a multifactorial metabolic syndrome of still unclear origin, largely associated with pathological lipid accumulation in hepatocytes [ 176 ]. Recent findings highlight that MAM-dependent modulation of mitochondrial structure and bioenergetic function is a key driver of NAFLD pathogenesis [ 2 , 177 ]. MAM dysfunction plays a critical role in NAFLD progression to NASH. Studies show that ER–mitochondria contact density significantly increases during NAFL-to-NASH transition, correlating with metabolic syndrome severity [ 178 ]. Liver-specific MFN2 knockout impairs phosphatidylserine transfer from ER to mitochondria, promoting ER stress, inflammation, and ultimately NASH-like phenotype and hepatocellular carcinoma [ 2 ]. High-fat diet-induced overactivation of the IP3R–GRP75–VDAC complex exacerbates hepatic lipid accumulation and inflammatory response [ 29 ]. Mechanistically, at the ER–mitochondria contact sites, the IP3R–GRP75–VDAC complex recruits Seipin to regulate hepatic lipid generation and promote lipid droplet biogenesis [ 179 ]. The gut microbiota Recent evidence highlights that this crosstalk is mediated by a dichotomy of signaling molecules: while beneficial gut-derived metabolites such as short-chain fatty acids (SCFAs) upregulate PGC-1α and its downstream target MFN2 to actively promote the physiological stabilization of MAMs, dysbiosis-induced leaky gut triggers a pathogenic cascade [ 180 ]. Specifically, the influx of lipopolysaccharide (LPS) induces ER stress that drives the pathological tightening of the IP3R–GRP75–VDAC tether; this structural alteration not only directly triggers NLRP3 inflammasome assembly and activation at the interface to drive chronic inflammation but also interferes with ATG14L-mediated autophagosome nucleation, thereby establishing MAMs as the critical intracellular relay station that translates gut-derived environmental signals into cellular metabolic or inflammatory fates. Cardiovascular diseases Cardiomyocytes have extremely high demands for energy and calcium ions, making MAM homeostasis crucial for their function [ 18 ]. Sigma-1 receptor activation inhibits microglial M1 polarization in stress-induced hypertensive rats by modulating ER–mitochondria contacts and mitochondrial function [ 181 ]. Heart failure and cardiomyopathy Calcium homeostasis and mitochondrial function are critical in heart failure (HF), with MAMs playing a key regulatory role. Structural and functional abnormalities in MAMs have been observed in both reduced (HFrEF) and preserved ejection fraction (HFpEF) heart failure. In HF, enhanced IP3R-2 activity at cardiomyocyte MAMs induces calcium overload and apoptosis [ 182 ]. Downregulation of FUNDC1 impairs calcium transfer from sarcoplasmic reticulum to mitochondria, disrupting the CREB/Fis1 pathway and compromising cardiac function [ 183 ]. The downregulation of STX17 impairs DRP1 phosphorylation, thereby inhibiting mitophagy and ultimately exacerbating the damage [ 137 ]. In obese cardiomyopathy, upregulated SNARE protein STX17 at MAMs causes excessive ER–mitochondria tethering, leading to mitochondrial calcium overload, ROS burst, and myocardial injury [ 184 ]. The absence of SIRT3 affects downstream ATAD3A, and this also causes excessive MAM formation [ 135 ]. Conversely, loss of CLIC4 reduces MAM connectivity and is associated with increased myocardial infarction risk, reaffirming the “Goldilocks principle” of MAM homeostasis [ 185 ]. In diabetic cardiomyopathy, MAMs also play a critical role. Hyperglycemia increases MAMs and causes aberrant expression of the PACS2/IP3R2/FUNDC1/VDAC1 pathway, thereby activating downstream apoptotic signaling and resulting in myocardial injury [ 138 ]. Myocardial ischemia–reperfusion injury (MIRI) MIRI pathogenesis centers on calcium overload, oxidative stress, and cell death –processes converging at MAMs. During MIRI, ER stress and MAM-mediated organelle communication disruption are key events [ 186 ]. Specifically, DIAPH1 interaction with MFN2 increases MAM tightness, exacerbating injury [ 187 ]. However, reduced ATAD3A disrupts MAM integrity, causing abnormal calcium signaling and myocardial injury [ 136 ]. Targeting MAMs (e.g., by inhibiting DRP1-mediated mitochondrial fission) demonstrates cardioprotective effects [ 28 ]. Glycogen synthase kinase-3β (GSK-3β), localized to SR/ER, regulates cardiomyocyte calcium homeostasis via IP3R complexes [ 18 ]. GSK-3β inhibition attenuates calcium overload and apoptosis, reducing MIRI [ 18 , 188 ]. Oncology Cancer cells remodel MAMs to meet their unique metabolic demands and evade cell death [ 22 ]. Metabolic reprogramming and proliferation Many cancer cells enhance ER–mitochondria connectivity (e.g., by upregulating the IP3R–GRP75–VDAC complex) to increase Ca 2+ flux [ 189 ], thereby overdriving mitochondrial metabolism to supply ample ATP and biosynthetic precursors for rapid proliferation [ 28 ]. MAMs also regulate angiogenesis; for instance, FUNDC1-mediated MAM formation promotes VEGFR2 expression, supporting tumor vasculogenesis [ 141 ]. Mitochondrial dynamics and autophagy Tumor cells utilize MAMs to regulate mitochondrial fission, aiding survival under stress. During hypoxia, increased FUNDC1 activity recruits and oligomerizes Drp1 at MAMs to initiate mitochondrial constriction. AMPK persistently binds and activates MFF, adapting mitochondrial function to metabolic demands [ 190 ]. MAMs also regulate autophagy; under stress, AMPK interacts with MFN2 to promote energy–stress-induced autophagy [ 190 ]. Recent studies show MAMs influence mitophagy; abnormally enhanced MAM function induces mitochondrial fragmentation and defects, triggering mitophagy that facilitates tumorigenesis [ 191 ]. Calcium and ROS signaling Tumor suppressors such as p53, PTEN, and BAP1 significantly impact tumor cell survival by regulating mitochondrial Ca 2+ uptake at MAMs [ 192 , 193 ]. Redox enzymes at MAMs (e.g., ERO1α, TMX1, and TMX3) are upregulated in cancer cells, enhancing ER oxidative folding, increasing ROS production, and inducing oxidative stress [ 194 , 195 ]. Apoptosis resistance and chemoresistance Cancer cells manipulate MAMs to evade apoptosis. Tumor suppressors p53 and PTEN exert pro-apoptotic effects at MAMs—p53 by interacting with SERCA, and PTEN by inhibiting Akt-mediated IP3R phosphorylation, jointly promoting Ca 2+ transfer to mitochondria to induce cell death [ 19 ]. However, many cancers counteract this by upregulating Bcl-2 to inhibit IP3R or downregulating PTEN [ 19 ]. Reduced MAM connectivity is also linked to multidrug resistance in neuroblastoma, indicating that MAM structural changes directly influence chemosensitivity [ 196 ]. Lipid peroxidation and ferroptosis Acyl-CoA synthetase long-chain family member 4 (ACSL4) is the executioner enzyme of ferroptosis [ 197 ]. It dictates sensitivity by enriching cellular membranes with long-chain polyunsaturated fatty acids (PUFAs), the primary substrates for peroxidation. Crucially, ACSL4 is enriched at MAMs. This localization is not coincidental; the MAM provides the unique lipid environment—rich in newly synthesized phospholipids—necessary for ACSL4 to generate the death signal. Upon ferroptotic stress (e.g., GPX4 inhibition), ACSL4 at the MAM drives the rapid accumulation of lipid peroxides. This accumulation is confined initially to the ER–mitochondria interface before spreading to the rest of the cell [ 69 ]. This compartmentalization explains why general antioxidants are often less effective than targeted lipophilic antioxidants. Kidney diseases Given the kidney’s susceptibility to metabolic fluctuation and hypoxic stress, MAM dysfunction plays a central role in the pathogenesis of kidney diseases, particularly diabetic kidney disease (DKD) and acute kidney injury (AKI). Perturbations in MAM composition and dynamics directly compromise mitochondrial bioenergetics and ER proteostasis. In AKI, this manifests as hyperactive ER–mitochondria coupling that drives oxidative stress and insulin resistance in podocytes. In contrast, DKD is characterized by acute disruption of MAM-mediated regulatory networks, leading to energy depletion and necroinflammation in proximal tubules (Table 3 ). Thus, preserving the physiological architecture of MAMs is essential for maintaining renal cell survival under stress conditions [ 198 ]. Role in diabetic kidney disease (DKD) In DKD, high-glucose environments disrupt MAM integrity in renal cells such as podocytes and tubular cells [ 16 ]. Hyperglycemia disrupts MAM homeostasis through multiple pathways. In podocytes, high-glucose-induced MFN2 downregulation impairs its interaction with PERK, leading to excessive PERK pathway activation, mitochondrial dysfunction, and apoptosis [ 199 ]. Concurrently, upregulated expression of the ER protein Reticulon-1A (RTN1A) in DKD promotes its interaction with VDAC1, causing hexokinase-1 dissociation from mitochondria and triggering apoptosis and inflammation [ 124 ]. Furthermore, downregulation of MAM proteins such as PACS-2 and DsbA-L exacerbates mitochondrial fission, ER stress, and lipid accumulation, promoting tubular injury [ 123 , 200 ]. The downregulation of FUNDC1 suppresses mitophagy, leading to ROS burst and further exacerbating MAM disruption [ 126 ]. Role in acute kidney injury (AKI) and its transition to chronic kidney disease (CKD) MAMs serve as a critical link connecting ER stress and mitochondrial dysfunction in AKI pathogenesis [ 201 ]. Similar to the structural disruption observed in DKD, models of AKI such as renal ischemia–reperfusion injury (IRI) and cisplatin nephrotoxicity are characterized by a loss of MAM integrity. This reduction in ER–mitochondria contacts is closely linked to excessive mitochondrial fission and tubular cell apoptosis [ 202 ]. In cisplatin-induced AKI, MAM disruption increases mitochondrial ROS production and apoptosis, whereas MFN2 overexpression preserves MAM integrity and ameliorates injury [ 16 , 119 ]. Persistent MAM dysfunction may impair cellular repair mechanisms, promoting inflammation and fibrosis, thereby acting as a key driver of AKI to CKD progression [ 121 , 203 ]. Across multiple disease models, MAM dysfunction emerges prior to mitochondrial fragmentation and apoptosis, indicating its role as an early pathological driver rather than a secondary consequence. Whether triggered by proteotoxicity in neurodegenerative diseases, nutrient toxicity in metabolic disorders, oncogenic signaling in cancer, or glucolipotoxicity in kidney injury, these divergent upstream insults converge at the disruption of ER–mitochondria interface homeostasis to exert their pathogenic effects. Sex differences: the estrogen–mitochondria connection Epidemiological data consistently show that pre-menopausal women are protected against cardiovascular and renal metabolic diseases compared with men [ 204 ]. A significant portion of this protection is mediated by rstrogen acting directly on mitochondrial dynamics. E2 binds to estrogen receptors (ERα/β), which act as transcription factors to directly upregulate the expression of MFN2 and antioxidant enzymes (such as SOD2) [ 205 ]. Higher MFN2 levels in females promote robust but regulated MAM integrity. This supports efficient mitochondrial fusion (quality control) and bioenergetics without the toxic tightening seen in males under stress. Loss of E2 during menopause leads to a precipitous drop in MFN2, correlating with the onset of frail mitochondria and increased cardiovascular risk [ 206 ]. This suggests that MAM-targeted therapies may need to be sex-stratified or that estrogen receptor modulators could serve as MAM stabilizers. Tissue-specific heterogeneity: the functional priority principle While the core molecular machinery of MAMs is conserved, its composition and functional prioritization exhibit profound tissue-specific heterogeneity. This cellular context dictates how different organs adapt to stress. As detailed in Table 2 , the architecture of MAMs is not uniform but is customized to meet the specific physiological mandate of each tissue. For instance, in excitable tissues such as cardiomyocytes, the MAM architecture prioritizes excitation–bioenergetic coupling via RyR2 nanodomains to synchronize ATP production with contraction [ 207 , 208 ]. Similarly, in neurons, the interface is tailored for synaptic maintenance, where VAPB–PTPIP51 tethers facilitate the massive lipid trafficking required for continuous synaptic vesicle cycling [ 209 , 210 ]. In contrast, Hepatocytes and adipocytes utilize MAMs primarily as hubs for metabolic flexibility, governing lipid droplet biogenesis and glucose sensing. The Kidney presents a unique duality, where podocytes prioritize nutrient sensing while tubular cells prioritize bioenergetics [ 123 , 124 , 200 , 211 ]. This heterogeneity explains the distinct pathological trajectories observed across diseases and highlights why “one-size-fits-all” MAM stabilizers may fail. A detailed comparison of these tissue-specific profiles is provided below. MAMs as systemic integrators: mechanisms of interorgan crosstalk MAMs do not merely regulate the cell that houses them; they function as broadcasting stations. Dysfunction at the MAM interface triggers the release of soluble and vesicular signals that alter the physiology of remote tissues, establishing critical axes of interorgan communication. The MAM–mitokine axis: endocrine regulation of metabolism Mitochondrial stress signals are integrated at MAMs to regulate the secretion of mitokines, cytokines derived from mitochondrial stress responses that act as systemic hormones. The ER stress sensor PERK is enriched at MAMs. Disruption of MAM integrity (e.g., MFN2 downregulation) activates the PERK–ATF4 pathway [ 85 ]. ATF4 acts as a transcriptional activator for Fgf21 and Gdf15. Fibroblast growth factor 21 (FGF21): Primarily secreted by the liver upon MAM stress, FGF21 acts on adipose tissue to promote browning and lipolysis, and on the brain to suppress sugar intake [ 212 ] .This represents a liver-to-adipose/brain axis to mobilize energy reserves during hepatic stress. Growth differentiation factor 15 (GDF15): A biomarker of mitochondrial dysfunction and aging. MAM dysfunction triggers GDF15 secretion, which acts on the hindbrain (GFRAL receptors) to induce anorexia (appetite suppression) [ 213 ]. This systemic metabolic brake protects the organism from nutrient overload when mitochondrial processing capacity is compromised. Pathological interorgan crosstalk Liver–kidney axis: In NASH, hepatocyte MAM disruption alters the secretome (fetuin-A, lipid-rich EVs), which travels to the kidney, inducing podocyte MAM remodeling and insulin resistance, linking liver disease to diabetic nephropathy [ 16 ]. Gut–brain axis: Gut microbiota metabolites (e.g., SCFAs) regulate MAM integrity in enterocytes and potentially vagal afferents. Dysbiosis leads to leaky MAMs, promoting the release of neurotoxic EVs that may propagate alpha-synuclein pathology to the brain [ 180 , 214 ]. MAM dysfunction: a spatiotemporal paradox A synthesis of current evidence reveals a complex paradox: both pathological hyperconnectivity and disruptive dissociation of MAMs can drive disease progression (Table 3 ). This suggests that MAM function follows an inverted U-shaped curve, where cellular homeostasis relies on maintaining ER–mitochondria crosstalk within an optimal physiological window. To define this window quantitatively, we integrated ultrastructural data from TEM and super-resolution studies across multiple pathologies. The physiological baseline (10–30 nm) This is the optimal range for functional tethering. It allows for the accumulation of large protein complexes (IP3R–GRP75–VDAC1) while creating a calcium microdomain where Ca 2+ can reach 10–20 μM, essential for driving bioenergetics via the low-affinity MCU. Pathological tightening (< 10 nm) Here, membranes are too close, causing steric hindrance that blocks large regulatory complexes (e.g., IP3R tetramers). This “tight” contact is often toxic, leading to membrane hemifusion or lipid toxicity. Functional dissociation (> 30–50 nm) Communication becomes inefficient. Calcium ions diffusing from the ER are diluted into the bulk cytosol before reaching the mitochondrial surface, failing to trigger bioenergetic upregulation. On one end of the spectrum, conditions characterized by acute metabolic oversupply or early-stage stress—such as obesity-related nephropathy and the very initial phase of reperfusion—may trigger maladaptive tightening. This toxic bridge facilitates excessive Ca 2+ transfer from the ER to mitochondria, triggering mitochondrial Ca 2+ overload, ROS bursts, and the opening of the mPTP. Here, the therapeutic goal is to normalize these contacts to block apoptotic signal transduction. Conversely, in established renal pathologies such as advanced DKD and chemically induced AKI (e.g., cisplatin), the structural tethering of MAMs is profoundly compromised. As described above, the downregulation of tethering proteins (e.g., MFN2, PACS-2) leads to metabolic isolation. This prevents essential lipid exchange and impairs Ca 2+ -driven stimulation of the TCA cycle, leading to bioenergetic collapse. In these scenarios, therapeutic strategies must aim to stabilize or rebuild the tethering complexes to restore metabolic flux. This dichotomy highlights that MAM dysfunction is not a static binary state but a dynamic spectrum that evolves during disease progression. Therefore, future MAM-targeted therapies must act as smart modulators—capable of rewiring connectivity back to the physiological set-point—rather than merely inhibiting or enhancing it indiscriminately. Understanding MAM dysfunction requires a temporal perspective, distinguishing between adaptive compensation and terminal failure: Early phase (compensatory tightening): In the initial stages of metabolic stress (e.g., early obesity or acute ischemia), cells often undergo “adaptive tightening” of MAMs (via IP3R–GRP75–VDAC upregulation). This is a compensatory attempt to boost mitochondrial calcium uptake and ATP production to meet increased metabolic demand. Late phase (decompensatory dissociation): However, chronic stress leads to the collapse of this compensatory mechanism. The accumulation of ROS and lipotoxicity eventually degrades tethering proteins (such as MFN2 or VAPB), leading to“decompensatory dissociation.” This temporal shift explains the conflicting reports of “too tight” versus “too loose” contacts and highlights that therapeutic strategies must be staged according to disease progression. Frontier technologies for exploring the ER–mitochondria interface While substantial evidence establishes the causality of MAM dysregulation, resolving its spatiotemporal dynamics and cellular heterogeneity in complex pathological contexts remains a major challenge. This has significantly driven the innovation and application of multiscale, high-resolution technologies. Research on MAMs is shifting from static structural characterization to dynamic functional analysis, driven by cutting-edge technologies. These advances provide unprecedented tools for examining this complex subcellular structure with nanoscale resolution, systematic perspectives, and dynamic dimensions. Methodological limitations of current techniques Electron microscopy (EM) While EM provides the gold standard for spatial resolution (< 1 nm), it is limited to fixed, non-living samples. It captures a static snapshot that fails to reflect the rapid, dynamic remodeling of MAMs (which occurs on the order of seconds) [ 215 ]. Furthermore, fixation artifacts can distort membrane morphology, leading to inconsistent measurements of cleft width. Confocal microscopy Standard fluorescence microscopy is diffraction-limited (~200 nm resolution), making it impossible to accurately resolve the 10–30 nm MAM cleft. Co-localization analysis (using Pearson’s coefficient) often yields false positives, overestimating the extent of functional contact by conflating proximity with interaction. Split-GFP probes The use of split-GFP (where half of GFP is on the ER and half on the mitochondria) is a common detection method. However, the reassembly of GFP is often irreversible and thermodynamically stable. This locks the organelles together, artificially stabilizing MAMs and preventing the observation of dissociation events. This observer effect can severely distort data, making dynamic contacts appear static and potentially inducing stress itself [ 216 ]. Next-generation tools for the Goldilocks zone Conventional optical microscopy, limited by the diffraction barrier, cannot clearly resolve the fine nanostructure of MAMs. The emergence of super-resolution microscopy (SRM) has revolutionized this field. In addition, fluorescence-based techniques enable visualization of MAMs. Crucially, these imaging technologies provide the quantitative metrics required to distinguish the pathological tightening versus disruption described in “ MAM dysfunction: a spatiotemporal paradox ” section. Experimentally, tightening is quantified as a significant reduction in the ER–mitochondria distance (typically < 10–15 nm by EM) or an increase in the density of tethering complexes (via PLA or split-GFP). In contrast, disruption is characterized by an expansion of the cleft (> 30 nm) and a loss of proximity signals. Super-resolution microscopy Techniques such as stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM) can break the diffraction limit. These methods enable researchers to observe the dynamic remodeling of MAMs in live cells at a resolution of several tens of nanometers—for example, visualizing how ER tubules wrap around mitochondria and monitoring the recruitment and dissociation of specific proteins at contact sites [ 1 ]. Furthermore, fluorescent probes originally developed for observing other membrane contact sites (e.g., ER–plasma membrane contacts), such as MAPPER, also provide insights for designing and developing tools to specifically label MAMs [ 1 , 217 ]. Computational challenges and solutions for nanoscale quantification While super-resolution microscopy (SRM) can resolve the nanostructure of MAMs with high clarity, the precise quantification of spatial interaction—specifically the intermembrane distance and the aggregation state of tethering proteins (such as the IP3R–GRP75–VDAC complex) within the 10–30 nm gaps —still presents a significant computational challenge. The complexity of multichannel SRM data, coupled with inherent limitations such as signal sparsity and uneven labeling distribution, often prevents traditional measurement techniques from accurately capturing the true nature of dynamic interactions at MAMs. To address this multichannel gap, the field is increasingly adopting advanced computational reconstruction methods. These algorithms employ mathematical modeling to compute and reconstruct interaction maps between molecules tagged across different fluorescent channels. By integrating SRM with these powerful computational tools, researchers can more accurately quantify the specific structural features of MAMs (e.g., tethering protein stoichiometry, distance distribution) and precisely correlate these nanoscale geometries with functional states and disease phenotypes [ 218 ]. Cryo-electron tomography (cryo-ET) Cryo-ET enables three-dimensional reconstruction of cells in a near-native physiological state, achieving molecular-level resolution. By reconstructing the interface in a near-native state, cryo-ET revealed that MAMs are not fused membranes but are separated by a specific 10–30-nm gap. This specific distance is a critical biological insight, as it is narrow enough to allow Ca 2+ “tunneling” between IP3R and VDAC without cytoplasmic dilution, yet wide enough to prevent membrane fusion. Cryo-ET essentially confirmed the “quasi-synaptic” nature of ER–mitochondria communication [ 1 ]. In situ proximity ligation assay (PLA) This technique enables the visualization of protein–protein interactions within MAMs [ 219 , 220 ]. PLA utilizes a probe system where primary antibodies target endogenous proteins of interest, followed by binding with secondary antibodies conjugated to specific oligonucleotides. If the two target proteins are in close proximity, exogenously added connector oligonucleotides facilitate the formation of a circular DNA structure. This circularized DNA is then amplified and detected using fluorescently labeled probes, allowing precise visualization of protein interactions at MAM interfaces [ 221 ]. FRET- and BRET-based systems FRET-based fluorescent proteins fused to ER and mitochondrial markers can detect interactions between MAM proteins [ 43 , 222 ]. FRET enables energy transfer from an excited donor fluorophore to a suitable acceptor fluorophore within a limited distance. When the two proteins are not in close proximity, the FRET donor (e.g., CFP) absorbs light energy but cannot transfer it to the FRET acceptor (e.g., YFP), resulting in blue fluorescence without FRET [ 223 ]. In contrast, BRET is more stable and sensitive, as its donor fluorophore is provided by luciferase, eliminating the need for external light sources and reducing phototoxicity. However, BRET probes targeting MAM proteins remain limited and require further development [ 224 ]. Split-GFP-based contact site sensor (SPLICS) and NanoLuc binary technology (NanoBiT) Split-GFP reconstituted systems are less reversible, making it challenging to analyze the dynamic formation of mitochondria–ER contacts in real time [ 225 ]. In contrast, NanoBiT can be used to monitor the reversible dynamics of MAMs in living cells [ 225 ]. The NanoBiT system comprises two subunits, LgBiT (18 kDa) and SmBiT (1.3 kDa), which are split fragments of a deep-sea shrimp luciferase. These subunits are optimized for reversible protein–protein interaction analysis (with K D = 190 µM) and produce extremely high-intensity luminescence, enabling clear visualization of MAMs in live cells. These tools allow for high-throughput detection of MAMs and facilitate the analysis of their dynamic behavior under more physiological conditions [ 226 ]. These tools provided a key biological insight: MAMs are highly plastic. Unlike biochemical fractionation that captures an average state, these sensors revealed that ER–mitochondria contacts are transient and reversible under physiological conditions but become rigid and permanently “tightened” under pathological stress (e.g., in Alzheimer’s models). This distinction has been crucial for identifying “MAM loosening” or “MAM tightening” as specific therapeutic targets. Systematic component analysis: multi-omics integration To comprehensively understand the molecular composition of MAMs, researchers are increasingly adopting multi-omics strategies. Proteomics and lipidomics High-precision mass spectrometry has been used to perform in-depth proteomic and lipidomic analyses of purified MAM fractions, identifying hundreds of enriched proteins and a unique lipid profile [ 21 ]. These studies have created a “molecular blueprint” of MAMs and revealed their dynamic compositional changes under stimuli such as viral infections [ 227 ]. Multi-omics integrated analysis Single-omics data only provide one-dimensional information. To construct a more comprehensive regulatory network, researchers are increasingly adopting advanced bioinformatic frameworks such as multi-omics factor analysis (MOFA) [ 228 ]. These approaches integrate data from different layers (e.g., proteomics, metabolomics, and transcriptomics) to uncover core regulatory axes and hidden molecular networks driving MAM functional changes, thereby achieving a transition from a parts list to a system function map [ 228 ]. Resolving heterogeneity: single-cell and spatial omics Even within the same tissue, significant heterogeneity may exist in the function and composition of MAMs among different cells. For instance, distinct segments of renal tubules in the kidney perform specialized functions, and their MAM characteristics are likely to be markedly different [ 16 ]. scRNA-seq data from human tissues reveal that core tethering genes (e.g., MFN2 and VAPB) are not housekeeping genes with constant expression. Instead, they exhibit high variability correlated with cell state. Stem cells versus differentiated cells: Pluripotent stem cells maintain loose, plastic MAMs. Upon differentiation (e.g., into cardiomyocytes or neurons), expression of MFN2 and MCU surges, tightening MAMs to support the metabolic shift from glycolysis to oxidative phosphorylation [ 229 ]. Plasticity regulators: In glioblastoma, scRNA-seq has identified transcriptional repressors such as MYT1L that restrict cellular plasticity. Loss of MYT1L leads to a “progenitor-like” state with altered organelle connectivity, driving therapeutic resistance. Single-cell omics Single-cell sequencing allows for the analysis of MAM-related gene expression at the individual cell level, helping to understand how MAMs are specialized in different cell subpopulations [ 1 ]. Different cell subpopulations may possess MAM-dependent metabolic signatures, and this spatial heterogeneity provides a theoretical basis for precision targeting. Spatial omics By integrating histological imaging with omics sequencing, spatial omics technologies enable the acquisition of high-throughput molecular data while preserving spatial tissue context. Applying this technology to MAM research holds promise for mapping the “functional atlas” of MAMs within their native tissue microenvironment, thereby revealing their regulatory mechanisms in specific physiological and pathological contexts. Mapping dynamic flows: metabolic flux analysis To understand the true activity of metabolic enzymes at MAMs, metabolic flux analysis is employed. Metabolic flux analysis This technique utilizes stable isotope-labeled substrates to trace the transformation pathways and rates of metabolites within cells. Applying this approach to MAM research enables quantitative measurement of the exchange flux of key molecules—such as lipids and calcium ion precursors—between the ER and mitochondria [ 170 ]. This shifts the research paradigm from static descriptions of “what is present” to dynamic functional assessments of what is being done and how fast it is occurring, providing direct evidence for understanding the central role of MAMs in cellular metabolic regulation. The rapid advancement in MAM research is fundamentally driven by a technology-empowered discovery cycle. Initial electron microscopy provided static structural snapshots. Subsequent biochemical isolation techniques coupled with proteomics revealed MAMs as a complex molecular hub housing hundreds of proteins. Super-resolution live-cell imaging then revolutionized our understanding by demonstrating MAMs as a dynamically remodeling structure. Currently, emerging multi-omics and single-cell technologies are propelling research into the realm of systems biology, aiming to uncover cell-type-specific molecular signatures and intricate regulatory networks of MAMs. This process operates not linearly but as a positive feedback loop: for instance, proteomic identification of a novel MAM protein immediately sparks the need to visualize its dynamic behavior via super-resolution microscopy and to investigate its functional impact through metabolic flux analysis. It is this continuously accelerating technology-discovery cycle that constitutes the core engine of progress in the field. Methodological limitations and the in vivo gap Despite the substantial technological advances outlined above, a fundamental gap persists between high-resolution in vitro structural characterization and the dynamic functional behavior of MAMs in vivo. Current methodologies share several intrinsic limitations that must be carefully considered when interpreting mechanistic conclusions. Cryo-electron tomography (Cryo-ET) and electron microscopy (EM) achieve near-atomic spatial resolution but rely on chemical or cryogenic fixation, thereby capturing only static structural snapshots. Such approaches are inherently blind to the rapid and reversible remodeling of MAMs, which occurs on millisecond to second timescales. In contrast, live-cell reporters such as split-GFP-based sensors enable dynamic visualization but are often irreversible upon complementation, precluding accurate tracking of MAM dissociation during adaptive or pathological remodeling. The tissue penetration barrier Super-resolution microscopy (SRM) performs robustly in monolayer cell cultures yet remains fundamentally constrained by light scattering, limited depth penetration, and phototoxicity in thick or highly dynamic tissues. As a result, direct visualization of MAM dynamics in intact organs—such as the beating heart or a functioning kidney—remains largely inaccessible. Consequently, many purported in vivo insights are still extrapolated from post-mortem or fixed tissue analyses rather than derived from real-time observation of living systems. The probe-induced observer effect A further concern arises from the use of genetically encoded proximity sensors, including FRET- or BRET-based linkers, which typically require overexpression of fusion proteins. Elevated levels of these artificial tethers can themselves enhance ER–mitochondria coupling, inadvertently tightening the MAM interface and thereby perturbing the native physiology they are intended to report. Loss of spatial context in biochemical approaches Subcellular fractionation remains a cornerstone technique for MAM proteomic profiling. However, its disruptive nature collapses spatial and cellular heterogeneity by averaging signals across millions of cells. This limitation is particularly problematic in complex organs such as the kidney, where MAM composition and function differ substantially between cell types (e.g., podocytes versus tubular epithelial cells), as discussed in “ Tissue-specific heterogeneity: the functional priority principle ” section. Addressing these methodological blind spots will require the development of nonperturbing, reversible proximity sensors coupled with advanced deep-tissue imaging strategies, such as adaptive optics and motion-corrected intravital microscopy. Only through such innovations can the field begin to resolve the spatiotemporal paradox of MAM regulation within living organisms. Potential therapeutic targets on MAMs Growing recognition of MAMs’ central role in diverse diseases has positioned them as an attractive frontier for therapeutic intervention in translational medicine (Table 4 ). Strategies targeting MAMs aim to fundamentally correct cellular dysfunction by restoring homeostasis in ER–mitochondria communication, offering promising solutions for numerous diseases that currently lack effective treatments. Table 4. Pharmacological agents modulating the ER–mitochondria interface Drug/compound class Primary molecular target (known) Effect on MAMs Key downstream effects Disease context/therapeutic potential SGLT2 Inhibitors (e.g., empagliflozin) SGLT2 Reduces excessive MAM formation via AMPK pathway Restores podocyte homeostasis, mitigates mitochondrial stress and apoptosis Diabetic kidney disease (DKD), heart failure [ 211 ] Metformin (AMPK agonist) Mitochondrial complex I Mediates integrated stress response via PERK at MAMs Activates AMPK, modulates cell metabolism and autophagy, reduces ER stress Type 2 diabetes, metabolic syndrome, cancer [ 230 ] mTOR inhibitors (e.g., rapamycin) mTORC1/mTORC2 Interferes with the mTORC2–Akt signaling hub at MAMs Inhibits cell growth and proliferation, induces autophagy Cancer, organ transplantation, autoimmune diseases [ 174 ] GSK3β inhibitors GSK3β Enhances VAPB–PTPIP51 interaction Restores ER–mitochondria tethering, improves homeostasis and synaptic function Neurodegenerative diseases (ALS, FTD, AD) [ 37 ] DRP1 inhibitors (e.g., Mdivi-1) DRP1 (mitochondrial fission protein) Indirectly affects MAM-mediated mitochondrial fission Inhibits excessive mitochondrial fission, reduces cell death Neurodegenerative diseases (AD, PD), myocardial ischemia–reperfusion injury, cancer [ 28 ] Novel VAPB–PTPIP51 modulators VAPB–PTPIP51 interaction interface Enhance or stabilize ER–mitochondria tethering Restore neuronal signaling and energy metabolism Neurodegenerative diseases (ALS, FTD) [ 167 ] Natural compounds (e.g., ginsenosides) Multiple targets Modulate mitochondrial dynamics, reduce oxidative stress Exert neuroprotective and antidepressant effects Neurological disorders, cardiovascular diseases [ 231 ] Open in a new tab Potential therapeutic targets for neurodegenerative diseases ALS development is linked to Sig-1R mutations at MAMs [ 167 ]. Dysfunction of Sig-1R disrupts ER–mitochondria contacts, lipid raft stability, and calcium signaling, thereby promoting ALS pathogenesis [ 232 ]. Administration of targeted agonists enhances the chaperone activity of Sig-1R, thereby reducing protein aggregation and slowing disease progression, which has been validated in various ALS models [ 233 , 234 ]. MAMs are central to Alzheimer’s disease pathogenesis. GSK3β acts as a negative regulator of MAM signaling, influencing cellular metabolism and stress responses, making it a potential therapeutic target [ 235 – 237 ]. Studies demonstrate that N , N -dimethyltryptamine (DMT) activates Sig-1R, thereby reducing Aβ plaque formation and restoring mitochondrial function [ 238 ]. The pathogenesis of Huntington’s disease (HD) is closely linked to PGC-1α dysfunction at MAMs [ 154 ]. Activation of the PGC-1α pathway improves mitochondrial function and enhances antioxidant enzyme expression, exerting neuroprotective effects [ 239 ]. Potential therapeutic targets for cardiovascular diseases In myocardial ischemia–reperfusion injury (MIRI), inhibition of the GSK3β interaction with the IP3R1–GRP75–VDAC1 complex reduces calcium transfer, thereby alleviating pathological obstruction [ 188 , 240 ]. Similarly, inhibition of the fusion proteins MFN1 and MFN2 also affects mPTP channel opening, thereby protecting cardiomyocytes [ 241 ]. Modulation of DRP1 function can regulate the progression of heart failure. For example, inhibiting DRP1 prevents mitochondrial network damage and alleviates norepinephrine-induced cardiomyocyte hypertrophy [ 242 ]. MAMs also play a significant role in cardiovascular inflammatory diseases [ 243 ]. The microtubule inhibitor nocodazole disrupts coupling between MAMs, thereby blocking subsequent mitochondrial metabolic reprogramming and T-cell activation [ 244 ]. Potential therapeutic targets for tumor diseases MAM plays a significant role in tumor cell metabolism, susceptibility to death, as well as invasion and metastasis [ 245 ]. Numerous studies have demonstrated that MAM-associated signaling pathways are critically involved in the development and progression of various types of tumors. Targeting MAM-related signaling pathways provides novel strategies for cancer therapy. For instance, in cisplatin-resistant ovarian cancer, MAM is markedly enriched. Targeting GRP75 can disrupt MAM integrity, reduce ER-to-mitochondria calcium flux, and further promote cisplatin-induced mitochondrial dysfunction and apoptosis [ 140 ]. Additionally, targeting the BH4 domain of Bcl-2 to enhance IP3R-mediated Ca 2+ release has been shown to increase the sensitivity of ovarian cancer to cisplatin treatment [ 246 ]. Beyond targeting calcium signaling pathways, modulating relevant metabolic targets is also a viable approach. For example, TMX1, a thioredoxin-related transmembrane protein located at MAM, mediates contact between the ER and mitochondria, thereby influencing energy metabolism processes in tumor cells such as mitochondrial oxygen consumption and ATP production, and promoting apoptosis [ 247 ]. Inhibition of GSK3β promotes the binding of HK-I to VDAC, thereby enhancing mitochondrial metabolite uptake and respiration, influencing the metabolic reprogramming of memory CD8 + T cells, and serving as a potential target for cancer immunotherapy [ 248 ]. Potential therapeutic targets for metabolic diseases Dysfunction of the endoplasmic reticulum and mitochondria is a key pathogenic mechanism in metabolic diseases, and MAMs serve as an interactive platform between these two organelles, thus playing a crucial role [ 249 , 250 ]. In obesity-related diseases, inhibition of IP3R1 and PACS-2 reduces mitochondrial calcium overload, thereby improving cellular metabolic status and insulin sensitivity [ 146 ]. Targeting CypD in MAMs modulates Ca 2+ transfer between the ER and mitochondria, thereby regulating insulin resistance and representing a potential therapeutic target for type 2 diabetes [ 251 ]. Treatment of CypD-knockout mice with metformin also improves hepatic insulin sensitivity [ 252 ]. Potential therapeutic targets for kidney disease MAM plays a significant role in renal injury diseases, and the development of related targeted therapies holds considerable potential. In diabetic nephropathy, targeted modulation of MAMs can regulate biological processes such as glucose and lipid metabolism and autophagy. For instance, AMPK—a key energy-sensing kinase—interacts with MFN2 to regulate the structure and function of MAMs [ 253 ]. However, curcumin activates the AMPK pathway to inhibit renal lipid accumulation and alleviate kidney injury in DKD [ 254 ]. Wogonin targets Bcl-2 to modulate its interactions with key MAM proteins, thereby balancing autophagy and apoptosis processes to protect glomerular podocytes [ 255 , 256 ]. Beyond traditional small molecules, emerging nanomedicine offers precise tools for physically editing MAM structures [ 257 ]. A recent study demonstrated the use of gallic acid-modified polyphenol–copper nanodots (GA-Cu) for AKI therapy. These ultrasmall nanodots preferentially accumulate in renal tubular mitochondria and act as dual-function agents: they scavenge excessive ROS and, crucially, physically disrupt the assembly of the IP3R–GRP75–VDAC1 tethering complex. By selectively truncating this pathological ER–mitochondria crosstalk, GA-Cu nanodots effectively blocked mitochondrial calcium overload and suppressed tubular cell death, providing the first proof-of-concept for using inorganic nanomaterials to strictly regulate MAM plasticity in kidney diseases [ 118 ]. Strategic comparison: direct structural editing versus indirect metabolic modulation Current therapeutic strategies targeting MAMs fall into two distinct paradigms: direct targeting of the physical tethers and indirect modulation via upstream metabolic sensors. Understanding the trade-offs between these approaches is critical for clinical translation. Direct targeting: precision structural editing This strategy involves designing small molecules or peptides to physically disrupt or stabilize specific tethering complexes. Mechanism: It acts directly on the structural components of MAMs, offering high specificity for the ER–mitochondria interface. Advantages: Ideally suited for acute conditions where rapid, decisive intervention is needed to truncate a lethal signal. For example, in AKI, the immediate disruption of the IP3R–GRP75–VDAC1 complex effectively blocks calcium overload and cell death [ 122 ]. Limitations: The major risk is overcorrection. Given that MAMs are essential for physiology, completely blocking these tethers can lead to pathological organelle dissociation and bioenergetic collapse. Furthermore, targeting protein–protein interactions (PPIs) remains a formidable pharmacological challenge. indirect modulation: homeostatic functional tuning This strategy utilizes established metabolic modulators (e.g., SGLT2 inhibitors, metformin, mTOR inhibitors) to regulate the signaling pathways (AMPK, mTORC2) that control MAM dynamics. Mechanism: It acts on the signaling networks, hijacking the cell’s intrinsic nutrient-sensing machinery to fine-tune the extent of ER–mitochondria contact. Advantages: Instead of forcibly breaking or gluing contacts, these agents tend to normalize MAMs back to a physiological set-point. For instance, SGLT2 inhibitors activate AMPK to reduce maladaptive MAM hyperconnectivity in diabetic podocytes without causing dissociation. This homeostatic restoration likely underlies their superior safety profile in chronic treatment [ 120 ]. Limitations: These drugs are pleiotropic, affecting numerous cellular pathways beyond MAMs, which complicates the isolation of their specific interface-mediated effects. Conclusion: While direct targeting offers precision for acute insults, indirect modulation via metabolic sensors represents a safer, more translatable strategy for managing chronic metabolic and neurodegenerative diseases. Direct targeting of tethering proteins: precision interventions Given the spatiotemporal paradox described in “ MAMs as systemic integrators: mechanisms of interorgan crosstalk ” section, direct targeting strategies must be bifurcated into two opposing approaches: molecular stabilizers to rebuild dissociated contacts and physical disruptors to truncate pathological hyperconnectivity. Molecular stabilizers for rebuilding connectivity For diseases characterized by the loss of MAM integrity (e.g., ALS/FTD, advanced DKD), the therapeutic objective is to structurally reinforce the failing interface. Small molecule stabilizers: A leading example is the targeting of the VAPB–PTPIP51 complex. In ALS models, where GSK3β-mediated phosphorylation dissociates this tether, screening is underway for small molecules that can allosterically enhance the VAPB–PTPIP51 interaction [ 169 ]. Agonist-mediated tethering: The Sigma-1 receptor (Sig-1R) agonists (e.g., PRE-084) function as chaperone glues, promoting the stability of the IP3R–GRP75–VDAC1 complex to rescue mitochondrial bioenergetics in neurodegeneration [ 133 ]. Physical disruptors for truncating hyperconnectivity For conditions driven by acute MAM tightening (e.g., AKI, ischemia–reperfusion, certain cancers), the strategy shifts to physically blocking the tethering machinery to prevent calcium overload. Blocking peptides: Synthetic peptides mimicking the binding domains of tethering proteins can competitively inhibit assembly. For instance, peptides derived from the GRP75 interface have been used to disrupt IP3R–GRP75–VDAC1 coupling in cisplatin-resistant ovarian cancer, effectively restoring chemosensitivity [ 143 ]. Nanomedicine interventions: Emerging inorganic nanomaterials offer a novel disruption modality. As discussed in “ Potential therapeutic targets for kidney disease ” section, gallic acid–copper (GA-Cu) nanodots have been engineered to accumulate in renal mitochondria, where they physically interfere with the assembly of the IP3R–GRP75–VDAC1 complex. This steric hindrance strategy effectively blocks the toxic Ca 2+ bridge in AKI without relying on traditional receptor antagonism, providing a more translatable strategy for managing chronic metabolic and neurodegenerative diseases. Drug repurposing: unveiling novel mechanisms at MAMs SGLT2 inhibitors Sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g., empagliflozin) were initially developed as glucose-lowering agents, primarily by inhibiting renal glucose reabsorption in proximal tubules [ 258 ]. However, large-scale clinical trials unexpectedly revealed their robust cardioprotective and renoprotective effects, extending far beyond glucose control [ 259 ]. A pivotal recent study uncovered a novel mechanism: in diabetic kidney disease models, SGLT2 inhibitors correct aberrantly increased MAM formation in podocytes by activating the AMPK pathway, thereby restoring ER–mitochondria homeostasis [ 211 ]. This discovery directly links a major class of cardiorenal protective drugs to the regulation of MAMs, not only providing a solid mechanistic explanation for their broad clinical benefits but also offering strong clinical validation for MAMs as a promising therapeutic target [ 258 ]. Metformin and AMPK agonists Metformin, a first-line therapy for type 2 diabetes, primarily targets mitochondrial complex I [ 230 ]. Its inhibition lowers the cellular ATP/AMP ratio, thereby activating the energy sensor AMPK [ 230 ]. Further research reveals that this mitochondrially initiated stress signal is transmitted via the ER stress sensor PERK—localized at MAMs—triggering the integrated stress response (ISR) [ 230 ]. Thus, metformin’s mechanism of action exemplifies the role of MAMs as an interface integrating mitochondrial and ER stress signaling, effectively positioning the drug as a modulator of the ER–mitochondria stress axis [ 260 ]. mTOR inhibitors The mammalian target of rapamycin (mTOR), a central kinase regulating cell growth and metabolism, forms two distinct complexes—mTORC1 and mTORC2 [ 261 ]. Research demonstrates that the mTORC2 complex specifically localizes to MAMs, where it functions as a critical signaling node [ 174 ]. Upon stimulation by growth factors such as insulin, mTORC2 at MAMs becomes activated and phosphorylates its downstream kinase Akt. Activated Akt subsequently phosphorylates a series of substrate proteins at MAMs—including IP3R, hexokinase 2 (HK2), and PACS2—thereby coordinately regulating MAMs structural integrity, mitochondrial metabolism, and cell survival [ 174 , 262 ]. This mechanism explains how mTOR inhibitors such as rapamycin exert their broad biological effects (e.g., inhibiting tumor growth, immunosuppression), as they likely function by disrupting this MAM-centered signaling hub. Current clinical research and future outlook While most novel drugs specifically designed to target MAMs remain in the preclinical stage, the discovery of MAM-modulating effects in existing pharmaceuticals has injected strong momentum into this field. The development of MAM-targeting therapies is undergoing a repurposing revolution. The major breakthroughs in this area have not originated from designing new drugs from scratch, but rather from recognizing that several highly successful blockbuster drugs (such as metformin and SGLT2 inhibitors) have actually been functioning as MAM modulators all along. For instance, while SGLT2 inhibitors were initially developed for glucose lowering, their powerful cardiorenal protective effects have long eluded complete explanation by any single mechanism [ 258 ]. Recent research has directly demonstrated their ability to rebalance MAMs in diabetic podocytes via the AMPK pathway, providing a novel and compelling molecular mechanism to explain their pleiotropic benefits [ 211 ]. Similarly, metformin’s primary target is mitochondrial complex I, but its downstream effects are now proven to be transmitted through PERK kinase localized at MAMs [ 230 ]. This retrospective discovery of successful drugs’ mechanisms provides the most compelling validation for MAMs as a viable therapeutic target. This insight suggests that systematically screening existing drug libraries for compounds with similar MAM-modulating activity represents a highly efficient drug development pathway—one that could significantly shorten development timelines compared with de novo drug discovery. This not only reshapes our understanding of these classic drugs’ mechanisms but also opens new possibilities for their application across broader disease areas. Conclusions and future perspectives Summary The MAM is a fundamental principle of eukaryotic cell organization, functioning as a highly integrated signaling hub that coordinates cellular metabolism, stress responses, and survival decisions. This review has systematically outlined the molecular composition and multidimensional physiological functions of MAMs, and delved into how their dysfunction serves as a common pathological basis for a wide range of major human diseases. The dysregulation of MAMs represents a convergence point for various downstream pathogenic factors, highlighting their fundamental importance in maintaining cellular homeostasis. Unresolved scientific challenges and translational barriers Despite the compelling evidence linking MAM dysfunction to diverse pathologies, several intrinsic limitations and unresolved challenges warrant cautious interpretation of the current literature: The in vitro to in vivo gap Current understanding relies heavily on monolayer cell cultures, which lack the complex hormonal and mechanical cues of living organs. Validating the spatiotemporal paradox (e.g., dynamic remodeling during a heartbeat) in deep, living tissues remains a critical frontier that static imaging cannot address. Distinguishing causality from consequence Establishing whether MAM disruption is the primary driver of pathogenesis or merely a secondary bystander of cellular stress (e.g., lipid toxicity) remains challenging. Developing tools to selectively perturb MAMs without altering global organelle physiology is essential to prove causality. Therapeutic specificity As highlighted in “ Potential therapeutic targets on MAMs ” section, current MAM-modulating agents (e.g., metformin, SGLT2i) are inherently pleiotropic. Attributing their clinical efficacy solely to MAM rewiring is currently speculative. The field urgently needs highly specific modulators that target the interface machinery without off-target metabolic effects to validate MAMs as druggable targets. Prioritized research roadmap Resolving the in vivo dynamic paradox: Most insights on MAM tightening versus loosening are derived from static EM snapshots or monolayer cell cultures. We critically lack real-time data in living tissues. The field must prioritize the development of nonperturbing, reversible biosensors (building on next-generation SPLICS/NanoBiT) compatible with intravital microscopy. Visualizing MAM dynamics in a beating heart or a functioning kidney is essential to confirm if the “biphasic” remodeling observed in diabetes models holds true in vivo. Defining the MAM interactome in cancer: While we know that specific players such as ACSL4 and STING operate at MAMs, the full proteomic landscape of the tumor MAM remains undefined. Researchers should employ spatially resolved proteomics (e.g., APEX2 mapping) specifically comparing tumor versus adjacent normal tissue. Identifying tumor-specific tethers could yield targets that selectively kill cancer cells without harming healthy tissue—the “Holy Grail” of toxicity sparing. Precision stratification via liquid biopsy: “MAM stabilizers” and “MAM disruptors” are currently in development. However, applying them indiscriminately poses a risk (e.g., stabilizing a MAM in a cell that needs to loosen contacts to survive). Clinical translation requires the development of MAM-specific biomarkers (likely circulating miRNAs or exosomes carrying MAM proteins) that indicate the real-time state of a patient’s organelle contacts (i.e., “too tight” versus “too loose”). This would allow for precision prescription: stabilizers for ALS (loose MAMs) versus disruptors for ischemia (tight MAMs). Conclusions In the era of precision medicine, MAMs have evolved from structural curiosities to actionable high-value targets. By resolving the spatiotemporal paradox of their regulation, we are poised to develop a new generation of therapeutics that fundamentally rewire cellular homeostasis. Acknowledgements The authors thank colleagues for helpful discussions. Abbreviations MAM Mitochondria-associated membranes ER Endoplasmic reticulum ER stress Endoplasmic reticulum stress MCS Membrane contact sites OMM Outer mitochondrial membrane PERK RNA-dependent protein kinase (PKR)-like ER kinase ROS Reactive oxygen species UPR Unfolded protein responses PINK1 PTEN induced kinase 1 GSK-3β Glycogen synthase kinase‐3β ALS Amyotrophic lateral sclerosis AD Alzheimer’s disease CVDs Cardiovascular diseases PD Parkinson’s disease NAFLD Non-alcoholic fatty liver disease AKI Acute kidney injury CKD Chronic kidney injury DKD Diabetic kidney disease Mfn2 Mitofusin 2 BiP Binding immunoglobulin protein PACS2 Phosphofurin acidic cluster sorting protein 2 Fis1 Fission protein 1 homolog BAP31 B-cell receptor-associated protein 31 PI3K Phosphoinositide 3-kinase PML Promyelocytic leukemia BECN1 Beclin-1 Drp1 Dynamin-related protein 1 VAPB Vesicle-associated membrane protein-associated protein B IP3R Inositol 1,4,5-triphosphate receptors Grp75 Glucose-regulated protein 75 VDAC1 Voltage-dependent anion channel 1 Sig-1R Sigma-1 receptor FUNDC1 FUN14 domain containing protein 1 IRE1α Inositol-requiring kinase 1 Xbp1 X-box binding protein 1 CASP1 Caspase-1 JNK C-Jun N-terminal kinase eIF2α Eukaryotic transcriptional initiation factor-α subunit CHOP C/EBP homologous protein AMPK Adenosine monophosphate-activated protein kinase Author contributions D.X. and Y.H. contributed to the conceptualization and wrote the original draft of the manuscript. X.Z. participated in the literature data collection and analysis. B.L. and M.W. were responsible for visualization and prepared the figures. Y.L. and Z.P. reviewed and edited the manuscript, supervised the project, and acquired funding. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the National Natural Science Foundation of China (no. 82272208 to Zhiyong Peng, no. 82102273 to Yiming Li). Data availability No datasets were generated or analyzed during the current study. Declarations Ethics approval and consent to participate Not applicable. This article is a review and does not involve any studies with human participants or animals. Consent for publication Not available. Competing interests The authors declare no competing interests. Footnotes Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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