Mitochondria-associated niches in health and disease

The endosymbiotic theory, first formulated more than a hundred years ago, posits the origin of mitochondria as descendants of free-living prokaryotes that developed a symbiotic relationship with primitive host cells (Gray et al., 1999). This theory has opened new avenues for our understanding of cells and the evolution of eukaryotic organisms (Lazcano and Peretó, 2017). The term mitochondria-associated membranes (MAMs) was coined after the initial discovery that the endoplasmic reticulum (ER) has a bona fide interaction domain with mitochondria, which was required for proper lipid synthesis (Vance, 1990). In the past decade, accumulating studies have defined MAMs as sites of physical communication between mitochondria and the ER, where molecular exchanges are facilitated through the mediation of tethering molecules (Prudent and McBride, 2017). Moreover, another term to describe these regions of membrane physical closeness is also used in the field; ER–mitochondria contact sites (ERMCSs). With recent advances in high-resolution live-cell microscopy (Sigal et al., 2018; Wu et al., 2021), as well as the implementation of machine-learning algorithms in microscopy (Chen et al., 2021), new mechanisms and interactions have come to light that have helped researchers to better understand the internal organization of the cell and its organelles and metabolic pathways, based on a dynamic view of the intracellular network.

In this Review, we highlight the importance of the interconnectivity between mitochondria and other organelles for overall cell function, and contextualize the underlying mechanisms, which include regulation of membrane dynamics, Ca²⁺ signaling and lipid exchange in both health and disease. In addition, we explore interorganelle responses to stress and how an imbalance in these responses promotes disease development and outline future prospects for therapeutic intervention.

Importantly, we propose here the concept of mitochondria-associated niches (MANs) as a function-oriented term. We believe that the term MAMs does not capture the full scope of organelle interactions, as it is conceptually tied primarily to structural interactions with and/or juxtaposition of mitochondrial membranes. As the highly dynamic nature of these interactions is now well defined, we propose that the concept of MANs might better evoke the true meaning of these intracellular niches between different organelles that together control a large number of highly regulated cellular processes, as detailed herein.

The processes by which mitochondria change their shape and overall structure are known as mitochondrial membrane dynamics and comprise regulated mitochondrial fission and fusion. The balance between fission and fusion contributes to mitochondrial quality control and responds to various metabolic pathways, such as autophagy, cell death and bioenergetics (Herrera-Cruz and Simmen, 2017; Prudent and McBride, 2017), that are closely linked to the maintenance of health or the occurrence of disease (Yapa et al., 2021). Given the independent evolutionary origin of mitochondria, it was initially thought that the dynamics of mitochondrial membranes depended solely on the mitochondria themselves (Chan, 2020), but ample evidence now shows that other organelles, notably the ER, also play a crucial role.

Mammalian mitochondrial fission is responsible for the generation of new mitochondria and is regulated by several processes, such as cell division, mitochondrial localization, mitophagy, bioenergetics and multiple stress pathways (Chan, 2020). The mitochondrial fission machinery has also been extensively studied as an important regulator in other highly coordinated processes, such as cell death (detailed in Box 1).

However, there are aspects of mitochondrial fission that are still not well understood. ERMCSs have been suggested as the site of mitochondrial constriction and fission, providing a conduit for the transport of Ca²⁺ and lipids (Friedman et al., 2011; Kornmann et al., 2009; Rizzuto et al., 1998; Wu et al., 2018). Recent studies have shown that mitochondrial fission occurs at particular sites where the ER contacts the mitochondria, more specifically in mitochondrial DNA (mtDNA)-rich regions (Lewis et al., 2016). At these sites, ER tubules wrap around the mitochondria to create an initial constriction that promotes recruitment of GTPase dynamin-related protein 1 (DRP-1; also known as DNM1L) to its receptors, mitochondrial fission factor (MFF), mitochondrial dynamics protein MID49 (MiD-49; also known as MIEF2) and the mitochondrial dynamics protein MID51 (MiD-51; also known as MIEF1) in the outer mitochondrial membrane (OMM) (Palmer et al., 2013), followed by DRP-1 oligomerization and the assembly of a constriction ring that leads to its scission (Macdonald et al., 2014). Recent extensions of this model further include the ER, components of the cytoskeleton, lysosomes and mitochondria. The regulated integration and coordination of these components make mitochondrial fission a de facto MAN (Fig. 1). Here, the initial tether, comprising inverted formin-2 (INF2) in the ER and Spire1C (also known as SPIRE1) in the OMM, acts as a scaffold for the assembly of actin and myosin IIA filaments, generating a mechanical force that compresses the mitochondrial membrane and initiates the recruitment of DRP-1 (Korobova et al., 2013, 2014; Manor et al., 2015).

In addition, lysosomes have recently been identified as key players in mitochondrial fission. The master regulator of lysosomal dynamics, GTP-bound Ras-related protein Rab7 (herein referring to both Rab7a and Rab7b forms), mediates the tethering of lysosomes and mitochondria, which is counteracted by mitochondrial fission 1 protein (Fis1)-mediated activation of TBC1D15 (a Rab7 GTPase-activating protein) (Onoue et al., 2013). At these contact points, Fis1 also regulates the fission of peripherally damaged mitochondria in an ER-independent manner, suggesting that processes such as mitochondrial fission can be differentially regulated by different organelles (Kleele et al., 2021) (Fig. 1). Moreover, in some cases of Parkinson's disease (PD), inhibition of TBC1D15 by a mutant form of the lysosomal enzyme β-glucocerebrosidase (GBA1) prolonged lysosomal–mitochondrial tethering and disrupted mitochondrial function (Kim et al., 2021b). Additionally, the Ca²⁺-dependent phospholipid-interacting protein annexin A6 (AnxA6), found in endolysosomal membranes, has been shown to promote TBC1D15-mediated Rab7 inactivation (Meneses-Salas et al., 2020) and DRP-1 inhibition in the absence of Ca²⁺ (Chlystun et al., 2013), further underscoring the multi-organelle interconnectivity of the mitochondrial fission niche.

The final steps of mitochondrial fission are not yet well understood. Some studies suggest a role for dynamin 2 (Dyn2) in the final scission of mitochondria (Lee et al., 2016), although this is not fully required (Fonseca et al., 2019; Kamerkar et al., 2018). Other work suggests an involvement of phosphatidylinositol 4-phosphate (PI4P)-containing vesicles from the trans-Golgi network (TGN), as PI4P might be required for mitochondrial cleavage downstream of DRP-1 (Hung et al., 2017). ADP-ribosylation factor-1 (Arf1) or phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) have been associated with increased mitochondrial elongation and interconnectivity (Nagashima et al., 2020) (Fig. 1).

The interaction of mitochondria with other organelles, such as peroxisomes, endosomes, the plasma membrane and lipid droplets, can also affect the fission process. For example, the oxysterol-binding proteins (OSBP)-related proteins ORP5 and ORP8, known to transport phosphatidylserine (PS) from the ER to the plasma membrane, have been shown to be critical in the maintenance of mitochondrial morphology, cristae structure and respiratory activity (Tábara et al., 2021). In addition, the mitochondrial fission machinery also shares some components with other organelles; for example, peroxisomal division is mediated by DRP-1 and its adaptors MFF and Fis1 (Schrader et al., 2016), amassing evidence for the complexity and interconnectivity of multi-organelle membrane dynamics in this MAN.

Mitochondrial fusion increases the overall mitochondrial capacity for oxidative phosphorylation and enables redistribution of mtDNA in response to specific cellular energy demands and stress (Gao and Hu, 2021). In contrast to mitochondrial fission, the fusion machinery appears to be more confined to the mitochondria and less dependent on physical contact with the ER. When two mitochondria come closer together, in a movement mediated by the depolymerisation of actin filaments from the cytoskeletal network (Mattenberger et al., 2003), the GTPases mitofusin 1 and 2 (MFN1 and MFN2) join two adjacent OMMs, forming homotypic and heterotypic complexes (Hoppins et al., 2011). Fusion of inner mitochondrial membranes (IMMs) is mediated by optic atrophy 1 (OPA1) and is dependent on mitochondrial membrane polarization, as the stability of OPA1 is very sensitive to changes in the structural integrity of the IMM (Del Dotto et al., 2018). Furthermore, important mitochondrial fusion factors, such as MFN2, play a fundamental role in the structural connection between the ER and mitochondria (Naon et al., 2016). Similarly, the mitochondria and the ER-resident protein trichoplein (also known as myostatin), promote mitochondrial fusion by tethering ER and mitochondria via MFN2 (Cerqua et al., 2010), suggesting that mitochondrial fusion dynamics might also occur via MANs.

The molecular interplay between the ER and mitochondria has been extensively studied and includes numerous signaling pathways such as Ca²⁺ signaling, lipid metabolism, bioenergetics, inflammation, autophagy and apoptosis (Herrera-Cruz and Simmen, 2017; Scorrano et al., 2019), which have been traditionally associated with ERMCSs. As previously mentioned, we believe that these regions are not just a mere juxtaposition of two or more membranes, but that they function as communication hubs, spatially arranged niches of great importance for cellular function, as highlighted below.

The role of the Ca²⁺ signaling at the MAN consists of crosstalk between the ER and mitochondria, which plays important roles in multiple cellular pathways. The ER is the main intracellular Ca²⁺ store (De La Fuente et al., 2013; Somlyo, 1984), distributing Ca²⁺ to the cytosol via inositol 1,4,5-triphosphate receptors (IP₃Rs) (Streb et al., 1983). Under normal conditions, Ca²⁺ is highly concentrated in the ER lumen by the activity of the (sarco)endoplasmic reticulum Ca²⁺-ATPase (SERCA), which actively pumps Ca²⁺ from the cytosol into the ER. From the ER, Ca²⁺ is transported to the mitochondria through IP₃R channels, taken up to the intermembrane space by the voltage-dependent anion-selective channel protein (VDAC), at the OMM, and finally entering the mitochondrial matrix via the mitochondrial Ca²⁺ uniporter complex (MCU), at the IMM (Marchi and Pinton, 2014; Fig. 2A). In the mitochondria, Ca²⁺ is essential for bioenergetics as it regulates Ca²⁺-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle, ATP synthase and the malate-aspartate shuttle (Giorgi et al., 2018). In addition, constitutive Ca²⁺ transfer through the IP₃Rs is necessary to maintain adequate levels of reduced mitochondrial nicotinamide adenine dinucleotide (NADH), which is essential for oxidative phosphorylation and ATP production (Cárdenas et al., 2010). Furthermore, mitochondrial rapid micro-oxidative bursts that emerge from transient depolarization can stimulate IP₃R-mediated Ca²⁺ release in the context of stress or the presence of some apoptotic stimuli (Booth et al., 2021), suggesting that the regulation of Ca²⁺ transfer between these two organelles is more refined and complex than previously anticipated. Compared with the ER, the mitochondrial Ca²⁺ concentration is quite low under normal conditions (Somlyo, 1984), but can be increased by various stress conditions. For example, changes in the expression of tumor suppressors and oncogenes can modulate mitochondrial Ca²⁺ concentration and trigger specific cellular responses, such as apoptosis and necroptosis (Faizan and Ahmad, 2021). In contrast to the OMM, the IMM is impermeable to Ca²⁺; therefore, the import of Ca²⁺ into the matrix is mainly mediated by the MCU complex (Marchi and Pinton, 2014) (Fig. 2A). In addition to bona fide channels, several ER structural proteins play additional roles in the regulation of Ca²⁺ signaling. For example, phosphofurin acidic cluster sorting protein 2 (PACS2), originally described as an ER–mitochondria tether, also modulates Ca²⁺ signaling (Simmen et al., 2005). The ER–mitochondrial tether MFN2 also enables Ca²⁺ signaling, allowing greater sensitivity to apoptosis by increasing Ca²⁺ influx into the mitochondria (Guo et al., 2007; Wang et al., 2015) (Fig. 2A). Interestingly, other stimuli, such as hydrogen peroxide and superoxide, can also trigger Ca²⁺ influx from the ER into the mitochondria. For instance, MCU activity can be stimulated by the accumulation of reactive oxygen species (ROS) on ERMCSs as a result of increased Ca²⁺ efflux from the ER into the mitochondria (Dong et al., 2017). Finally, lysosomal proteins, such as the transient receptor potential channel mucolipin 1 (TRPML1), have also been found to mediate Ca²⁺ signaling alongside the ER and mitochondria, configuring a possible tripartite organelle Ca²⁺ MAN (see Box 2).

Lipid transfer configures an important role of the MAN as it is one of the main means of ER–mitochondrial crosstalk and one of the first functions reported for ERMCSs, originally identified as a PS-rich fraction (Vance, 2014). PS is synthesized in the ER and then transferred to the mitochondria where it is decarboxylated to phosphatidylethanolamine (PE) (Vance, 2008; Voelker, 1989) (Fig. 2B). Mitoguardin-2, an OMM protein, has been shown to operate in this niche by mediating the transference of PS from lipid droplets to the ER through an interaction with vesicle-associated membrane protein-associated protein B/C (VAPB) (Kim et al., 2022). In addition, enzymes involved in cholesterol and sphingolipid synthesis are also located at ERMCSs (Fujimoto et al., 2012; Vance, 2014). Similarly, the chaperone Sigma-1R (Sig-1R; also known as SIGMAR1), which is implicated in neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), associates with simple sphingolipids, such as ceramides, at the ER to regulate Ca²⁺ release (Hayashi and Fujimoto, 2010). The synthesis of phosphatidic acid (PA) in the ER and its conversion to cardiolipin (CL) in the mitochondria is an excellent example of a lipid exchange MAN. Considered as the signature phospholipid of the mitochondria, CL makes up ∼20% of the total lipid of the IMM and is crucial for important mitochondrial processes, such as respiration, apoptosis, mitophagy and membrane dynamics (Claypool and Koehler, 2012; Herrera-Cruz and Simmen, 2017; Kagan et al., 2005). Interestingly, the accumulation of PA in the mitochondria inhibits DRP-1 activity and thus mitochondrial fission (Adachi et al., 2016), underscoring the importance of these phospholipids not only as structural molecules but also as key players in the regulation of various processes at MANs including lipid exchange, membrane dynamics and cell death (Kameoka et al., 2018). This interconnectivity is further supported by a study showing that, in mammals, Miro (also known as RHOT1), a Ca²⁺-activated GTPase that regulates mitochondrial dynamics and motility via microtubule interactions (Nemani et al., 2018), recruits the lipid transport protein vacuolar protein sorting 13 homolog D (VPS13D) to the ER, creating a lipid conduit to the mitochondria (Guillén-Samander et al., 2021) (Fig. 2B). Although the origin for the concept of MAMs, due to a lack of sensitive tools, the study of the lipid transfer niches in mammals is only recently expanding with the support of new methods such as METALIC, a mass tag mass spectrometry-based technique for tracking specific lipids in the cells (John Peter et al., 2022).

In addition to forming niches of fission and molecular exchanges with the mitochondria, the ER is also responsible for the production of one-third of all the eukaryotic proteins for multiple cellular functions (Hetz et al., 2020). Different physiological and pathological conditions can alter the protein folding process at the ER and increase the level of misfolded proteins, leading to a condition known as ER stress. Cells cope with ER stress through mechanistic adaptations collectively named the unfolded protein response (UPR). In mammals, the UPR is orchestrated by three main signaling cascades that respond to the folding status of proteins in the ER lumen. The three arms of the UPR are initiated by three different ER transmembrane protein sensors: inositol-requiring enzyme 1 α (IRE1α), protein kinase RNA activated-like ER kinase (PERK; also known as EIF2AK3) and activating transcription factor 6 (ATF6) (Kaufman, 2002).

The PERK UPR arm was the first to be described as being regulated at the level of what we consider a MAN, based on studies describing morphological changes in mitochondria induced by PERK deficiency (Carreras-Sureda et al., 2019; Mori et al., 2013). During the UPR, PERK activation results in phosphorylation of the eukaryotic translation initiation factor 2 subunit-α (eIF2α), promoting transient inhibition of protein synthesis, while also inducing the translation of activating transcription factor 4 (ATF4), leading to increased expression of genes involved in stress responses (Kaufman, 2002; Ron and Walter, 2007) (Fig. 3A). Interestingly, PERK ablation has been shown to increase the number of contacts between ER and mitochondria and desensitize cells to ROS-induced cell death (Verfaillie et al., 2012). Furthermore, MFN2 has been shown to modulate the UPR by inhibiting PERK activity (Muñoz et al., 2013) and by controlling the kinetics of ER stress events, thereby preventing ER stress-induced apoptosis (Ngoh et al., 2012). PERK overexpression also induced mitochondrial fragmentation (Muñoz et al., 2013), highlighting the importance of the UPR at the interface of multiple cellular signaling pathways as well as mitochondrial membrane dynamics. PERK has been shown to modulate mitochondrial dynamics by promoting mitochondrial hyperfusion in the initial stages of ER stress, preventing pathological mitochondrial fragmentation (Lebeau et al., 2018). PERK can also increase oxidative phosphorylation (OXPHOS) by regulating the levels of the splicing factor arginine/serine-rich 19 (SCAF1) under conditions of nutrient starvation or ER stress (Balsa et al., 2019). Regulation of OXPHOS by PERK has been shown to promote import of the MICOS complex subunit MIC19 (MIC19; also known as CHCHD3) into the mitochondria, thereby inducing cristae formation upon occurrence of energy-intensive processes, such as cold stress or β-adrenergic stimuli (Latorre-Muro et al., 2021) (Fig. 3A). Similarly, a novel stress pathway termed the mitochondrial UPR (mtUPR), coordinated by OMA1–DELE1–HRI (HRI is also known as EIF2AK1) and induced by imbalances in mitochondrial proteostasis, is mediated by an integrated stress response that is relayed from the mitochondria (OMA1–DELE1) to the cytosol (DELE1–HRI), involving the activation of eIF2α and ATF4 in the cytosol (Fessler et al., 2020; Guo et al., 2020) (Fig. 3A). Collectively, these studies highlight how UPR mediators such as PERK and ATF4 can participate in multiple distinct MANs, having roles in adaptation to bioenergetic demands and stress responses that are not directly related to the canonical UPR, opening new avenues for further investigations on this topic.

The IRE1α arm of the UPR is activated by ER stress, which involves IRE1α autophosphorylation and activation of its RNase domain (Credle et al., 2005; Zhou et al., 2006). Upon activation, IRE1α catalyzes the splicing of XBP1 into XBP1s, which leads to upregulation of genes involved in the translocation, folding and secretion of ER proteins (Calfon et al., 2002; Yoshida et al., 2001). In addition, IRE1α mediates the degradation of selected mRNAs and microRNAs through a process called regulated IRE1-dependent decay (RIDD), which is thought to mitigate ER protein overload by reducing RNA abundance (Hetz et al., 2020) (Fig. 3A). As previously mentioned, IRE1α is highly enriched in some MANs, where it plays an important role in several processes. Recently, our group described that IRE1α regulates Ca²⁺ signaling by acting as a scaffold for IP₃Rs, underscoring the role of IRE1α in mitochondrial bioenergetics at MANs. IRE1α ablation promotes mitochondrial metabolic stress by reducing ATP levels and activating the AMP-activated protein kinase (AMPK) pathway and autophagy (Carreras-Sureda et al., 2019). Sig-1R also brings together distinct niches as it is required for IRE1α-mediated responses to mitochondrial ROS and subsequent XBP1 mRNA splicing (Mori et al., 2013) (Fig. 3A), potentially coupling the UPR to lipid metabolism and linking two different aspects of MANs (Hayashi and Su, 2004).

Interestingly, the loss of MFN2 or PACS2 induces ER stress, while also untethering ER and mitochondria (Sebastián et al., 2012; Simmen et al., 2005). Furthermore, PACS2 has been found to be involved in ER stress by interacting with and retaining phosphorylated calnexin (CNX) on the ER (Myhill et al., 2008), thereby disrupting protein N-glycosylation and folding (Kozlov and Gehring, 2020).

Overall, the UPR requires a tight connection between the ER and the mitochondria to function properly, configuring a bona fide MAN. At the same time, proteins resident in these niches regulate the amplitude and duration of the UPR, thereby playing an important role in influencing cell fate.

Macroautophagy (hereafter autophagy) is an adaptive catabolic process by which intracellular macromolecules are broken down into simpler cellular elements such as amino acids, nucleotides and fatty acids, which are used by the cell to fuel metabolic pathways (Dikic and Elazar, 2018; Prinz et al., 2020). Autophagy is commonly initiated in response to stress conditions such as starvation, hypoxia and ER stress, and it is characterized by the formation and expansion of an isolation membrane named phagophore into a closed double-membrane vesicle called an autophagosome in a sequential process coordinated by the members of the autophagy-related (ATG) proteins (Nishimura and Tooze, 2020) (Fig. 3B). During this process, the autophagosome sequesters various intracellular materials and later fuses with lysosomes to degrade intravesicular products (Levy et al., 2017). In mammals, autophagosome initiation and maturation occurs in close proximity to the so-called omegasome, a specialized subdomain of the ER enriched in phosphatidylinositol 3-phosphate (PI3P) (Hayashi-Nishino et al., 2009; Nishimura et al., 2017). In addition to the omegasome, the growing autophagosome simultaneously forms multiple contact sites with other organelles and membranes, including mitochondria, lysosomes, endosomes, the Golgi and the plasma membrane (Biazik et al., 2015). The role of the interactions between mitochondria and other organelles is of such importance for the execution of autophagy that we propose it configures an important MAN. For example, upon starvation, proteins of the Unc-51-like kinase-1 (ULK1) and the class III PI3K (PI3KC3) complexes, which normally diffuse freely in the ER membrane, relocate to contact sites (Hamasaki et al., 2013). Similarly, autophagy-related 14 (ATG14), Beclin 1, AMBRA1 and vacuolar protein sorting 34 (VPS34; also known as PIK3C3) – PI3KC3 complex components – assemble almost exclusively at this MAN to catalyze local PI3P production at the omegasome, thereby initiating the formation of the isolation membrane (Garofalo et al., 2016). Mitochondria are also involved in the formation of autophagosomes by transferring lipids directly from the OMM to the maturing autophagosome through a mechanism that requires interaction with the ER (Hailey et al., 2010). Furthermore, ATG2 and ATG9 proteins are recruited into mitochondria through interactions with the TOM40–TOM70 mitochondrial complex to induce phagophore maturation (Tang et al., 2019) (Fig. 3B). Interestingly, uncoupling the ER from the mitochondria by disrupting the interaction between the ER-resident protein VAPB and the OMM protein protein tyrosine phosphatase-interacting protein 51 (PTPIP51; also known as RMDN3) results in impaired Ca²⁺ transfer and the activation of survival autophagy (De Vos et al., 2012; Gomez-Suaga et al., 2017) (Fig. 3B). Therefore, proteins of this niche could have a dual, opposite effect on autophagy depending on the condition. On the one hand, this MAN promotes autophagy by providing a site for the assembly of the initiation complexes, and providing lipids and proteins for autophagosome elongation and maturation. On the other hand, a close and stable contact between the ER and the mitochondria is required for effective Ca²⁺ transfer, which maintains mitochondrial energy homeostasis.

Finally, there is a close interplay between mitochondrial dynamics and the induction of autophagy. Under nutrient-rich conditions, the autophagy protein STX17 associates with DRP-1 at sites of mitochondrial fission, where it promotes its activity by decreasing phosphorylation at S637, resulting in increased mitochondrial division. However, upon starvation, STX17 dissociates from DRP-1 and instead associates with ATG14, recruiting the autophagy PI3K3C initiation complex to this niche (Arasaki et al., 2015) (Fig. 3B). As a result, mitochondrial fission is inhibited during autophagy and mitochondrial elongation is favored. In addition, the ER could serve as a scaffold to facilitate autophagosome–lysosome fusion during the final stages of autophagy, as STX17 is required for fusion of the autophagosomes and lysosomes (Itakura et al., 2012). Given the complex interactivity of these processes, MANs act as signaling platforms, integrating information about the nutrient status of the cell to regulate autophagy as well as mitochondrial dynamics and metabolism.

Ca²⁺ is the major signaling metabolite involved in the regulation of cell death in MANs under stress conditions, and the ER is the major intracellular Ca²⁺ reservoir with an approximate intraluminal concentration ([Ca²⁺]_ER) of 400–800 µM (Giorgi et al., 2018). As previously mentioned, mitochondria require a steady influx of Ca²⁺ to maintain their basal metabolism; however, excessive Ca²⁺ influx leads to overloading of the mitochondrial matrix, which increases the activity of cyclophilin D, followed by opening of the mitochondrial permeability transition pore (mPTP), resulting in mitochondrial swelling and the rupture of the inner and outer mitochondrial membranes (Baines et al., 2005) (Fig. 2A). Mitochondrial permeabilization results in the release of apoptogenic factors, including cytochrome c, and an overall breakdown in mitochondrial bioenergetics, leading to cell death (Green, 2019). Additionally, Ca²⁺ has been proposed to disintegrate the mitochondrial respiratory complex II by interfering with its binding to CL, leading to increased ROS production and cell death (Hwang et al., 2014).

The proteins of the B-cell lymphoma 2 (BCL-2) family are the best-studied accessory regulators of the IP₃Rs and Ca²⁺ signaling in the ER (Pihán et al., 2017). BCL-2 members are functionally divided into anti- and pro-apoptotic proteins, with the former inhibiting cell death by interacting with and sequestering proapoptotic proteins in the cytosol, whereas the latter promote mitochondrial outer membrane permeabilization (MOMP) and cell death in response to multiple cytotoxic stimuli including ER stress, DNA damage and viral infections (Kale et al., 2018; Singh et al., 2019). Typically, antiapoptotic BCL-2 family members, such as BCL-2 itself, BCL-X_L (encoded by BCL2L1), and MCL-1 bind to different regions of IP₃Rs on the ERMCS and inhibit Ca²⁺ release, mitochondrial uptake and cell death (Marchi et al., 2018,b93; Prudent and McBride, 2017) (Fig. 2A). The inhibitory function of BCL-2 via IP₃R plays an important role in several pathophysiological processes and can be exploited to increase therapeutic efficacy in cancer (Akl et al., 2013; Bittremieux et al., 2019).

The antiapoptotic BCL-X_L has a similar function in regulating Ca²⁺ to BCL-2 and inhibits sustained Ca²⁺ leakage through IP₃R and cell death (Rosa et al., 2022). However, early studies revealed an additional function, where BCL-X_L interacts with the C-terminal domain of the IP₃R and sensitizes it to small concentrations of IP₃, resulting in the transmission of a small oscillating burst of Ca²⁺ to the mitochondria (Li et al., 2007; White et al., 2005). In the mitochondrial matrix, these oscillating Ca²⁺ bursts increase mitochondrial bioenergetics by making cells more resistant to proapoptotic stimuli (Yang et al., 2016). However, this model has been challenged by a recent study showing that BCL-X_L interacts with the same domains of IP₃R as BCL-2 always resulting in its inhibition, regardless of the concentration of IP₃ used (Rosa et al., 2022). Finally, several proteins can interact with and modulate the function of BCL-2 proteins, thereby indirectly regulating the activity of IP₃Rs. For example, the rhomboid-like superfamily pseudokinases iRhom1 and iRhom2 interact with and sequester BCL-2, releasing its inhibitory effects on the IP₃R (Fig. 2A), resulting in an increased Ca²⁺ release and mitochondrial uptake during ER stress (Dulloo et al., 2022). However, the precise contribution of this mechanism to ER stress-induced cell death has not yet been fully determined, as BCL-2 is also activated during ER stress by IRE1α and CHOP (also known as DDIT3), exerting additional regulatory functions in cell death beyond its role as an IP₃R inhibitor (Pihán et al., 2017).

The proapoptotic BCL-2 ovarian killer (BOK) also localizes in the ER, where it binds to the IP₃R and regulates its activity (Echeverry et al., 2013; Schulman et al., 2013) (Fig. 2A). BOK is a less-studied member of the multidomain BCL-2 family and, unlike BAX and BAK (also known as BAK1), induces MOMP via a BCL-2-independent mechanism governed by its stability (Llambi et al., 2016). Interestingly, BOK has been shown to be required for the proper apposition and integrity of ERMCSs. Indeed, several ERMCS proteins, including the IP₃R, are missing in BOK-knockout cells, making the cell more resistant to stimulus-induced Ca²⁺ transfer and cell death. Furthermore, the correct localization of the IP₃R in ERMCS depends in part on its interaction with BOK, which is required for Ca²⁺ transfer to the mitochondria and apoptosis under ER stress (Carpio et al., 2021).

Members of the TMBIM family also control cell death at this niche. TMBIM6/BI-1 is located at MAMs and inhibits Ca²⁺ release from IP₃R at the ER membrane, preventing mitochondrial Ca²⁺ overload, opening of the mPTP and apoptosis (Rojas-Rivera and Hetz, 2015). By contrast, upon lysosomal perturbation, TMBIM1 promotes lysosomal BAX translocation to induce cell death via lysosomal membrane permeabilization (Pihán et al., 2021; Pihán et al., 2022).

Other molecular signaling pathways in MANs can also trigger cell death. For example, ceramide, an endogenous lipid mediator of apoptosis, triggers ER Ca²⁺ release and accumulation in the mitochondria, leading to mitochondrial fragmentation. BCL-2 and other antiapoptotic proteins inhibit the action of ceramide by lowering the intraluminal Ca²⁺ concentration in ER, thereby decreasing the pool of stimulus-dependent Ca²⁺ released (Pinton et al., 2001). Interestingly, some lysosomal storage diseases, such as GM1 gangliosidosis, which is caused by abnormal accumulation of the glycosphingolipid GM1 ganglioside, show accumulation of ceramide in this niche, where it binds directly to and regulates the IP₃R, increasing ER-to-mitochondria Ca²⁺ transfer and apoptosis (Sano et al., 2009). Further studies are needed to uncover the role of sphingolipids in MANs in regulating mitochondrial cell death.

Finally, PERK promotes the effective transfer of ROS from the ER to the mitochondria under ER stress induced by oxidation. As mentioned above, PERK acts as a tether, increasing both the number and proximity of ERMCSs, leading to the spread of ROS into the mitochondria with subsequent induction of cell death (Verfaillie et al., 2012).

Taken together, these studies illustrate that MANs act as complex hubs for both the regulation of mitochondrial bioenergetics and autophagy through Ca²⁺ signaling, and as sites for the induction of cell death under stress conditions. Ultimately, the outcome for the cell depends on the presence or absence of, or interactions with, various regulatory factors, including members of the BCL-2 protein family, IP₃Rs and components of the UPR, and the number and proximity of ER–mitochondrial junctions. Their presence establishes different signaling niches that strongly influence cell fate.

Dysfunctions of MANs have been implicated in the pathology of several metabolic and neurodegenerative diseases (further expanded in Table S1).

In PD, the functionality of the mitochondrial fission niche is altered by mutations in phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), which promotes DRP-1-mediated mitochondrial fragmentation and reduces mitochondrial motility (Wang et al., 2011a; Yu et al., 2011). Conversely, parkin mutations promote the degradation of DRP-1 through ubiquitylation (Wang et al., 2011b). In this same niche, mutations in MFN2 causes neurodegenerative diseases such as Charcot–Marie–Tooth disease (CMT) type 2A (CMT2A) (Cartoni and Martinou, 2009), which is characterized by axonal degeneration, muscular atrophy and peripheral neuropathy (Züchner et al., 2004). This study was further supported by a recent work showing that DRP-1 ubiquitylation and degradation was increased in CMT2A patients with the R364W MFN2 mutation, which promotes mitochondrial hyperfusion, suggesting that MFN2 regulates DRP-1 function (Das et al., 2021).

A good example of pathologies arising from dysfunctions in the Ca²⁺ signaling niche are mutations in Sig-1R, which can lead to neurodegenerative diseases such as ALS. Owing to its interactions with a variety of voltage and ion channels on this MAN, Sig-1R plays an important role in neuronal function and in promoting proteostasis. Indeed, loss-of-functions in Sig-1R impair its binding to IP₃R, resulting in IP₃R mislocalization, Ca²⁺ signaling disruption, a reduction in ER–mitochondria contacts and, importantly, accumulation of mutant superoxide dismutase 1, a key driver of familial forms of ALS, at ERMCSs (Al-Saif et al., 2011; Watanabe et al., 2016).

Alterations in the lipid exchange niche play an important role in ER stress induction in non-alcoholic steatohepatitis (NASH)-like steatosis by dynamically controlling phosphatidylserine transfer from the ER to mitochondria in hepatocytes (Sood et al., 2014). Interestingly, MFN2 has been shown to be downregulated in NASH models; however, overexpression of MFN2 is sufficient to reverse the effects of NASH, which might be due to MFN2 ability to bind PS and transfer it from the ER into the mitochondria, thereby providing protection against NASH-like phenotypes and liver cancer (Hernández-Alvarez et al., 2019).

Metabolic diseases can also arise from dysfunctions in several MANs. For example, the insulin secretion pathway depends on proper lipid transport from the ER to the mitochondria by enzymes such as diacylglycerol acetyltransferase 2, which converts diacylglycerols into triglycerides. Impaired lipid conversion at the lipid exchange niche leads to the accumulation of diacylglycerols and thus repression of the insulin receptor (Lowell and Shulman, 2005). In addition, disruption of other MAN-associated processes, such as Ca²⁺ exchange and UPR, have also been associated with insulin resistance (Cheng et al., 2020).

Mitochondrial–lysosomal niches have also been implicated in neurodegenerative diseases, such as PD, resulting from PINK1 and Parkin mutations, which result in mitochondrial defects and neuronal loss (Park et al., 2018; Yambire et al., 2020). Metabolic disorders, such as the lysosomal storage disease (LSD) Niemann–Pick type C (NPC), might also result from defects in Ca²⁺ signaling and lipid accumulation in mitochondrial–lysosomal niches (Annunziata et al., 2018). Finally, the neurodegenerative LSD GM1 gangliosidosis has been shown to lead to neuronal degeneration through ER Ca²⁺ depletion and activation of the UPR (Caciotti et al., 2011; Jeyakumar et al., 2003; Ledeen and Wu, 2006). Overall, these studies further underscore the role of various MANs in maintaining health and inducing disease through multiple alterations in the communication between the mitochondria and other organelles, such as the ER and lysosomes.

Recent advances in our understanding of cellular and organelle biology have allowed us to better understand the complexity of the interactions between different intracellular compartments and to consider the importance of studying the biology of the cell as a highly interconnected system. The development of new tools such as proximity-based fluorescent probes and novel super-resolution microscopes have made it possible to unveil the intricacies of the signaling pathways involved in the physical and functional interaction between mitochondria and other organelles, such as the ER and lysosomes. Likewise, these tools might open the door to the development of novel therapeutic strategies that in the future could help in the treatment of cancer, metabolic pathologies and neurological diseases by targeting dysfunctions of interorganelle interfaces (Carreras-Sureda et al., 2017; Christ et al., 2020; Saito and Imaizumi, 2018). We regard each of these highly regulated interfaces between organelles as a niche, a signaling hub with functional relevance for organelle and cell physiology. In this Review, we have attempted to promote the new concept of MANs as a means to reference our understanding of the cellular mechanisms that regulate the functional and structural interactions between mitochondria and other organelles in health and disease. For example, important integrative cellular responses such as the UPR, mitochondrial membrane dynamics, Ca²⁺ signaling, lipid exchange and autophagy are regulated by MANs. In this line, proteins identified biochemically as belonging to a MAM are not necessarily part of a functional signaling pathway and do not necessarily configure a ‘signaling niche’. For example, REEP1, a protein that regulates ER tubular morphology has been found in ERMCSs (Lim et al., 2015; Wang et al., 2021), but so far this localization of REEP1 is not associated with a signaling pathway; it is simply found at these contact sites because of superimposition of different organelles membranes. In contrast, bona fide MAN proteins are part of a functional mechanism that occurs through the association of two or more organelles and associated proteins, lipids and metabolites. Naturally, there is an overlap between the factors that can be categorized under both concepts, and, as new signaling pathways are uncovered, more molecules could be reassigned from being only physically located at a mitochondrial membrane to being part of a functional niche. Therefore, the concept of MANs proposed here emphasizes the functional pathways that emerge from exchanges between two or more organelles, as we believe that proteins belonging to these niches are prime candidates for studies aimed at understanding how these signaling pathways are related to cell physiology, and thus health and disease. Although knowledge in this field has increased in recent years, an integrative understanding that could allow the specific modulation of molecules present in MANs with the aim to determine their impact on the severity of various pathologies is still missing. Therefore, by evolving our perspective of intracellular communication, we are also hopefully improving the landscape for drug discovery and therapeutic interventions.