The human cytomegalovirus protein UL37 exon 1 associates with internal lipid rafts

The human cytomegalovirus (HCMV) protein UL37 exon 1 (pUL37x1), also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), sequentially traffics from the endoplasmic reticulum (ER) through mitochondrion-associated membranes (MAMs) to the outer mitochondrial membrane (OMM), where it robustly inhibits apoptosis. Here, we report the association of pUL37x1/vMIA with internal lipid rafts (LRs) in the ER/MAM. The MAM, which serves as a site for lipid transfer and calcium signaling to mitochondria, is enriched in detergent-resistant membrane (DRM)-forming lipids, including cholesterol and ceramide, which are found in lower concentrations in the bulk ER. Sigma 1 receptor (Sig-1R), a MAM chaperone affecting calcium signaling to mitochondria, is anchored in the MAM by its LR association. Because of its trafficking through the MAM and partial colocalization with Sig-1R, we tested whether pUL37x1/vMIA associates with MAM LRs. Extraction with methyl-β-cyclodextrin (MβCD) removed pUL37x1/vMIA from lysed but not intact cells, indicating its association with internal LRs. Furthermore, the isolation of DRMs from purified intracellular organelles independently verified the localization of pUL37x1/vMIA within ER/MAM LRs. However, pUL37x1/vMIA was not detected in DRMs from mitochondria. pUL37x1/vMIA associated with LRs during all temporal phases of HCMV infection, indicating the likely importance of this location for HCMV growth. Although detected during its sequential trafficking to the OMM, the pUL37x1/vMIA LR association was independent of its mitochondrial targeting signals. Rather, it was dependent upon cholesterol binding. These studies suggest a conserved ability of UL37 proteins to interact with cholesterol and LRs, which is functionally distinguishable from their sequential trafficking to mitochondria.     

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.     

Cell death regulation by MAMs: from molecular mechanisms to therapeutic implications in cardiovascular diseases

The endoplasmic reticulum (ER) and mitochondria are interconnected intracellular organelles with vital roles in the regulation of cell signaling and function. While the ER participates in a number of biological processes including lipid biosynthesis, Ca²⁺ storage and protein folding and processing, mitochondria are highly dynamic organelles governing ATP synthesis, free radical production, innate immunity and apoptosis. Interplay between the ER and mitochondria plays a crucial role in regulating energy metabolism and cell fate control under stress. The mitochondria-associated membranes (MAMs) denote physical contact sites between ER and mitochondria that mediate bidirectional communications between the two organelles. Although Ca²⁺ transport from ER to mitochondria is vital for mitochondrial homeostasis and energy metabolism, unrestrained Ca²⁺ transfer may result in mitochondrial Ca²⁺ overload, mitochondrial damage and cell death. Here we summarize the roles of MAMs in cell physiology and its impact in pathological conditions with a focus on cardiovascular disease. The possibility of manipulating ER-mitochondria contacts as potential therapeutic approaches is also discussed.Subject terms: Cardiovascular diseases, Cardiomyopathies     

Lipid Transport between the Endoplasmic Reticulum and Mitochondria

Mitochondria are partially autonomous organelles that depend on the import of certain proteins and lipids to maintain cell survival and membrane formation. Although phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine are synthesized by mitochondrial enzymes, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sterols need to be imported from other organelles. The origin of most lipids imported into mitochondria is the endoplasmic reticulum, which requires interaction of these two subcellular compartments. Recently, protein complexes that are involved in membrane contact between endoplasmic reticulum and mitochondria were identified, but their role in lipid transport is still unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatory aspects that are important for lipid homeostasis.Biological membranes are major structural components of all cell types. They protect the cell from external influences, organize the interior in distinct compartments and allow balanced flux of components. Besides their specific proteome, organelles exhibit unique lipid compositions, which influence their shape, physical properties, and function. Major lipid classes found in biological membranes are phospholipids, sterols, and sphingolipids.The major “lipid factory” within the cell is the endoplasmic reticulum (ER). It is able to synthesize the bulk of structural phospholipids, sterols, and storage lipids such as triacylglycerols and steryl esters (van Meer et al. 2008). Furthermore, initial steps of ceramide synthesis occur in the ER providing precursors for the formation of complex sphingolipids in other organelles (Futerman 2006). Besides the export of ceramides, the ER supplies a large portion of lipids to other organelles, which cannot produce their own lipids or have a limited capacity to do so. Organelle interaction and transport of lipids require specific carrier proteins, membrane contact sites, tethering complexes, and/or vesicle flux. These processes are highly important for the maintenance of cell structure and survival but are still a matter of dispute. Most prominent organelle interaction partners are the ER and mitochondria. A subfraction of the ER named mitochondria-associated membrane (MAM) (Vance 1990) was described to be involved in lipid translocation to mitochondria. MAM is part of the ER network, which was shown to be in contact or close proximity to the outer mitochondrial membrane (OMM). Contact sites between MAM and mitochondria were assumed to facilitate exchange of components between the two compartments. Interestingly, MAM harbor a number of lipid synthesizing enzymes (Gaigg et al. 1994). Recently, molecular components governing membrane contact between the two organelles were identified (Dolman et al. 2005; Csordás et al. 2006; de Brito and Scorrano 2008; Kornmann et al. 2009; Friedman et al. 2010; Lavieu et al. 2010), although the specific role of these components in lipid translocation is not yet clear.     

The ER-mitochondria interface: the social network of cell death

When cellular organelles communicate bad things can happen. Recent findings uncovered that the junction between the endoplasmic reticulum (ER) and the mitochondria holds a crucial role for cell death regulation. Not only does this locale connect the two best-known organelles in apoptosis, numerous regulators of cell death are concentrated at this spot, providing a terrain for intense signal transfers. Ca2+ is the most prominent signalling factor that is released from the ER and, at high concentration, mediates the transfer of an apoptosis signal to mitochondria as the executioner organelle for cell death. An elaborate array of checks and balances is fine-tuning this process including Bcl-2 family members. Moreover, MAMs, "mitochondria-associated membranes", are distinct membrane sections at the ER that are in close contact with mitochondria and have been found to exchange lipids and lipid-derived molecules such as ceramide for apoptosis induction. Recent work has also described a reverse transfer of apoptosis signals, from mitochondria to the ER, via cytochrome c release and prolonged IP3R opening or through the mitochondrial fission factor Fis1 and Bap31 at the ER, which form the ARCosome, a novel caspase-activation complex.     

Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival

Communication between the endoplasmic reticulum (ER) and mitochondrion is important for bioenergetics and cellular survival. The ER supplies Ca(2+) directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). We found here that the ER protein sigma-1 receptor (Sig-1R), which is implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca(2+)-sensitive and ligand-operated receptor chaperone at MAM. Normally, Sig-1Rs form a complex at MAM with another chaperone, BiP. Upon ER Ca(2+) depletion or via ligand stimulation, Sig-1Rs dissociate from BiP, leading to a prolonged Ca(2+) signaling into mitochondria via IP3Rs. Sig-1Rs can translocate under chronic ER stress. Increasing Sig-1Rs in cells counteracts ER stress response, whereas decreasing them enhances apoptosis. These results reveal that the orchestrated ER chaperone machinery at MAM, by sensing ER Ca(2+) concentrations, regulates ER-mitochondrial interorganellar Ca(2+) signaling and cell survival.     

Mitofusin‐2 knockdown increases ER–mitochondria contact and decreases amyloid β‐peptide production

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria‐associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM‐associated proteins and enhanced ER to mitochondria Ca²⁺ transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β‐peptide (Aβ)‐related neuronal models. Here, we report that siRNA knockdown of mitofusin‐2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca²⁺ transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra‐ and extracellular Aβ₄₀ and Aβ₄₂. Analysis of γ‐secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ‐secretase complex function. Amyloid‐β precursor protein (APP), β‐site APP‐cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER–mitochondria contact affects γ‐secretase activity and Aβ generation. Increased ER–mitochondria contact results in lower γ‐secretase activity suggesting a new mechanism by which Aβ generation can be controlled.     

Evidence for the involvement of lipid rafts localized at the ER-mitochondria associated membranes in autophagosome formation

Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic “brick” of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome.     

Upregulated function of mitochondria-associated ER membranes in Alzheimer disease

Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER–mitochondrial communication—as measured by cholesteryl ester and phospholipid synthesis, respectively—are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER–mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.     

Loss and gain of function of Grp75 or mitofusin 2 distinctly alter cholesterol metabolism,but all promote triglyceride accumulation in hepatocytes

In the liver, contact sites between the endoplasmic reticulum (ER) and mitochondria (named MAMs) may be crucial hubs for the regulation of lipid metabolism, thus contributing to the exacerbation or prevention of fatty liver. We hypothesized that tether proteins located at MAMs could play a key role in preventing triglyceride accumulation in hepatocytes and nonalcoholic fatty liver disease (NAFLD) occurrence. To test this, we explored the role of two key partners in building MAM integrity and functionality, the glucose-regulated protein 75 (Grp75) and mitofusin 2 (Mfn2), which liver contents are altered in obesity and NAFLD. Grp75 or Mfn2 expression was either silenced using siRNA or overexpressed with adenoviruses in Huh7 cells.Silencing of Grp75 and Mfn2 resulted in decreased ER-mitochondria interactions, mitochondrial network fusion state and mitochondrial oxidative capacity, while overexpression of the two proteins induced mirror impacts on these parameters. Furthermore, Grp75 or Mfn2 silencing decreased cellular cholesterol content and enhanced triglyceride secretion in ApoB100 lipoproteins, while their overexpression led to reverse effects. Cellular phosphatidylcholine/phosphatidylethanolamine ratio was decreased only upon overexpression of the proteins, potentially contributing to altered ApoB100 assembly and secretion. Despite the opposite differences, both silencing and overexpression of Grp75 or Mfn2 induced triglyceride storage, although a fatty acid challenge was required to express the alteration upon protein silencing. Among the mechanisms potentially involved in this phenotype, ER stress was closely associated with altered triglyceride metabolism after Grp75 or Mfn2 overexpression, while blunted mitochondrial FA oxidation capacity may be the main defect causing triglyceride accumulation upon Grp75 or Mfn2 silencing. Further studies are required to decipher the link between modulation of Grp75 or Mfn2 expression, change in MAM integrity and alteration of cholesterol content of the cell.In conclusion, Grp75 or Mfn2 silencing and overexpression in Huh7 cells contribute to altering MAM integrity and cholesterol storage in opposite directions, but all promote triglyceride accumulation through distinct cellular pathways. This study also highlights that besides Mfn2, Grp75 could play a central role in hepatic lipid and cholesterol metabolism in obesity and NAFLD.     

Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondrion-associated membranes of human cells

The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent antiapoptotic protein, which prevents cytochrome c release from mitochondria. The UL37x1 NH₂-terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase 1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using subcellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria, and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37_NH2 and gpUL37_COOH were both detected in the ER and MAM fraction, even though only pUL37_NH2 is preferentially imported into mitochondria but gpUL37_COOH is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH₂-terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation.     

Phospholipid Synthesis and Transport in Mammalian Cells

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter‐organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria‐associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter‐organelle phospholipid transport in mammalian cells.     

Palmitoylated TMX and calnexin target to the mitochondria-associated membrane

The mitochondria-associated membrane (MAM) is a domain of the endoplasmic reticulum (ER) that mediates the exchange of ions, lipids and metabolites between the ER and mitochondria. ER chaperones and oxidoreductases are critical components of the MAM. However, the localization motifs and mechanisms for most MAM proteins have remained elusive. Using two highly related ER oxidoreductases as a model system, we now show that palmitoylation enriches ER-localized proteins on the MAM. We demonstrate that palmitoylation of cysteine residue(s) adjacent to the membrane-spanning domain promotes MAM enrichment of the transmembrane thioredoxin family protein TMX. In addition to TMX, our results also show that calnexin shuttles between the rough ER and the MAM depending on its palmitoylation status. Mutation of the TMX and calnexin palmitoylation sites and chemical interference with palmitoylation disrupt their MAM enrichment. Since ER-localized heme oxygenase-1, but not cytosolic GRP75 require palmitoylation to reside on the MAM, our findings identify palmitoylation as key for MAM enrichment of ER membrane proteins.     

MAM (mitochondria-associated membranes) in mammalian cells: Lipids and beyond

One mechanism by which communication between the endoplasmic reticulum (ER) and mitochondria is achieved is by close juxtaposition between these organelles via mitochondria-associated membranes (MAM). The MAM consist of a region of the ER that is enriched in several lipid biosynthetic enzyme activities and becomes reversibly tethered to mitochondria. Specific proteins are localized, sometimes transiently, in the MAM. Several of these proteins have been implicated in tethering the MAM to mitochondria. In mammalian cells, formation of these contact sites between MAM and mitochondria appears to be required for key cellular events including the transport of calcium from the ER to mitochondria, the import of phosphatidylserine into mitochondria from the ER for decarboxylation to phosphatidylethanolamine, the formation of autophagosomes, regulation of the morphology, dynamics and functions of mitochondria, and cell survival. This review focuses on the functions proposed for MAM in mediating these events in mammalian cells. In light of the apparent involvement of MAM in multiple fundamental cellular processes, recent studies indicate that impaired contact between MAM and mitochondria might underlie the pathology of several human neurodegenerative diseases, including Alzheimer's disease. Moreover, MAM has been implicated in modulating glucose homeostasis and insulin resistance, as well as in some viral infections.     

Early Presymptomatic Changes in the Proteome of Mitochondria-Associated Membrane in the APP/PS1 Mouse Model of Alzheimer’s Disease

Intracellular β-amyloid (Aβ) accumulation is an early event in Alzheimer’s disease (AD) progression. Recently, it has been uncovered that presenilins (PSs), the key components of the amyloid precursor protein (APP) processing and the β-amyloid producing γ-secretase complex, are highly enriched in a special sub-compartment of the endoplasmic reticulum (ER) functionally connected to mitochondria, called mitochondria-associated ER membrane (MAM). A current hypothesis of pathogenesis of Alzheimer’s diseases (AD) suggests that MAM is involved in the initial phase of AD. Since MAM supplies mitochondria with essential proteins, the increasing level of PSs and β-amyloid could lead to metabolic dysfunction because of the impairment of ER-mitochondrion crosstalk. To reveal the early molecular changes of this subcellular compartment in AD development MAM fraction was isolated from the cerebral cortex of 3 months old APP/PS1 mouse model of AD and age-matched C57BL/6 control mice, then mass spectrometry-based quantitative proteome analysis was performed. The enrichment and purity of MAM preparations were validated with EM, LC-MS/MS and protein enrichment analysis. Label-free LC-MS/MS was used to reveal the differences between the proteome of the transgenic and control mice. We obtained 77 increased and 49 decreased protein level changes in the range of ??6.365 to +?2.988, which have mitochondrial, ER or ribosomal localization according to Gene Ontology database. The highest degree of difference between the two groups was shown by the ATP-binding cassette G1 (Abcg1) which plays a crucial role in cholesterol metabolism and suppresses Aβ accumulation. Most of the other protein changes were associated with increased protein synthesis, endoplasmic-reticulum-associated protein degradation (ERAD), oxidative stress response, decreased mitochondrial protein transport and ATP production. The interaction network analysis revealed a strong relationship between the detected MAM protein changes and AD. Moreover, it explored several MAM proteins with hub position suggesting their importance in Aβ induced early MAM dysregulation. Our identified MAM protein changes precede the onset of dementia-like symptoms in the APP/PS1 model, suggesting their importance in the development of AD.     

Proteomic Analysis of Mitochondrial-Associated ER Membranes (MAM) during RNA Virus Infection Reveals Dynamic Changes in Protein and Organelle Trafficking

RIG-I pathway signaling of innate immunity against RNA virus infection is organized between the ER and mitochondria on a subdomain of the ER called the mitochondrial-associated ER membrane (MAM). The RIG-I adaptor protein MAVS transmits downstream signaling of antiviral immunity, with signaling complexes assembling on the MAM in association with mitochondria and peroxisomes. To identify components that regulate MAVS signalosome assembly on the MAM, we characterized the proteome of MAM, ER, and cytosol from cells infected with either chronic (hepatitis C) or acute (Sendai) RNA virus infections, as well as mock-infected cells. Comparative analysis of protein trafficking dynamics during both chronic and acute viral infection reveals differential protein profiles in the MAM during RIG-I pathway activation. We identified proteins and biochemical pathways recruited into and out of the MAM in both chronic and acute RNA viral infections, representing proteins that drive immunity and/or regulate viral replication. In addition, by using this comparative proteomics approach, we identified 3 new MAVS-interacting proteins, RAB1B, VTN, and LONP1, and defined LONP1 as a positive regulator of the RIG-I pathway. Our proteomic analysis also reveals a dynamic cross-talk between subcellular compartments during both acute and chronic RNA virus infection, and demonstrates the importance of the MAM as a central platform that coordinates innate immune signaling to initiate immunity against RNA virus infection.     

Structural and functional link between the mitochondrial network and the endoplasmic reticulum

Mitochondrial and endoplasmic reticulum (ER) networks are fundamental for the maintenance of cellular homeostasis and for determination of cell fate under stress conditions. Recent structural and functional studies revealed the interaction of these networks. These zones of close contact between ER and mitochondria called MAM (mitochondria associated membranes) support communication between the two organelles including bioenergetics and cell survival. The existence of macromolecular complexes in these contact sites has also been revealed. In this contribution, we will review: (i) the ER and mitochondria structure and their dynamics, (ii) the basic principles of ER mitochondrial Ca²⁺ transport, (iii) the physiological/pathological role of this cross-talk.     

New functions of mitochondria associated membranes in cellular signaling

In all eukaryotic cells, the endoplasmic reticulum (ER) and the mitochondria establish a tight interplay, which is structurally and functionally modulated through a proteinaceous tether formed at specific subdomains of the ER membrane, designated mitochondria-associated membranes or MAMs. The tethering function of the MAMs allows the regulation of lipid synthesis and rapid transmission of calcium (Ca^2 +) signals between the ER and mitochondria, which is crucial to shape intracellular Ca^2 + signaling and regulate mitochondrial bioenergetics. Research on the molecular characterization and function of MAMs has boomed in the last few years and the list of signaling and structural proteins dynamically associated with the ER–mitochondria contact sites in physiological and pathological conditions, is rapidly increasing along with the realization of an unprecedented complexity underlying the functional role of MAMs. Besides their established role as a signaling hub for Ca^2 + and lipid transfer between ER and mitochondria, MAMs have been recently shown to regulate mitochondrial shape and motility, energy metabolism and redox status and to be central to the modulation of various key processes like ER stress, autophagy and inflammasome signaling. In this review we will discuss some emerging cell-autonomous and cell non-autonomous roles of the MAMs in mammalian cells and their relevance for important human diseases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Cholesterol depletion induces ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell

Cholesterol is a critical component of lipid rafts implicated in regulating multiple signal transduction. The anthrax toxin receptor 2 (ANTXR2) is a type I membrane protein acting as the second receptor for the anthrax toxin. In this study, we first investigated the association between cholesterol and ANTXR2. We provided the evidence that cholesterol depletion by methyl-beta-cyclodextrin (MβCD) promoted ANTXR2 expression in U251 neuroglioma cell, which was reversed by cholesterol supplement. MβCD-induced ANTXR2 up-regulation contributed to ERK1/2 phosphorylation, which was responsible for MT1-MMP and MMP-2 activation. Our data suggested that cellular cholesterol regulated ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell.