ER-mitochondria distance is a critical parameter for efficient mitochondrial Ca2+ uptake and oxidative metabolism.

IP3 receptor (IP3R)-mediated Ca2+ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca2+ uptake and oxidative metabolism and is linked to different pathologies, including Parkinson's disease (PD). The dependence of Ca2+ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically encoded Ca2+ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of ~20 nm is optimal for Ca2+ transfer and mitochondrial oxidative metabolism due to enrichment of IP3R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced, which correlated with a reduction of mitochondrial Ca2+ uptake. Stabilization of the ER-mitochondrial interaction at 20 nm, but not at 10 nm, fully rescued mitochondrial Ca2+ uptake in PD astrocytes. Our work determines with precision the optimal distance for Ca2+ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function.

Mitochondria Dysfunction and Neuroinflammation in Neurodegeneration: Who Comes First?

Neurodegenerative diseases (NDs) encompass an assorted array of disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, each characterised by distinct clinical manifestations and underlying pathological mechanisms. While some cases have a genetic basis, many NDs occur sporadically. Despite their differences, these diseases commonly feature chronic neuroinflammation as a hallmark. Consensus has recently been reached on the possibility that mitochondria dysfunction and protein aggregation can mutually contribute to the activation of neuroinflammatory response and thus to the onset and progression of these disorders. In the present review, we discuss the contribution of mitochondria dysfunction and neuroinflammation to the aetiology and progression of NDs, highlighting the possibility that new potential therapeutic targets can be identified to tackle neurodegenerative processes and alleviate the progression of these pathologies.

SEPN1-related myopathy depends on the oxidoreductase ERO1A and is druggable with the chemical chaperone TUDCA.

Selenoprotein N (SEPN1) is a protein of the endoplasmic reticulum (ER) whose inherited defects originate SEPN1-related myopathy (SEPN1-RM). Here, we identify an interaction between SEPN1 and the ER-stress-induced oxidoreductase ERO1A. SEPN1 and ERO1A, both enriched in mitochondria-associated membranes (MAMs), are involved in the redox regulation of proteins. ERO1A depletion in SEPN1 knockout cells restores ER redox, re-equilibrates short-range MAMs, and rescues mitochondrial bioenergetics. ERO1A knockout in a mouse background of SEPN1 loss blunts ER stress and improves multiple MAM functions, including Ca2+ levels and bioenergetics, thus reversing diaphragmatic weakness. The treatment of SEPN1 knockout mice with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) mirrors the results of ERO1A loss. Importantly, muscle biopsies from patients with SEPN1-RM exhibit ERO1A overexpression, and TUDCA-treated SEPN1-RM patient-derived primary myoblasts show improvement in bioenergetics. These findings point to ERO1A as a biomarker and a viable target for intervention and to TUDCA as a pharmacological treatment for SEPN1-RM.

A SPLICS reporter reveals [Formula: see text]-synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation.

Mitochondrial and lysosomal activities are crucial to maintain cellular homeostasis: optimal coordination is achieved at their membrane contact sites where distinct protein machineries regulate organelle network dynamics, ions and metabolites exchange. Here we describe a genetically encoded SPLICS reporter for short- and long- juxtapositions between mitochondria and lysosomes. We report the existence of narrow and wide lysosome-mitochondria contacts differently modulated by mitophagy, autophagy and genetic manipulation of tethering factors. The overexpression of α-synuclein (α-syn) reduces the apposition of mitochondria/lysosomes membranes and affects their privileged Ca2+ transfer, impinging on TFEB nuclear translocation. We observe enhanced TFEB nuclear translocation in α-syn-overexpressing cells. We propose that α-syn, by interfering with mitochondria/lysosomes tethering impacts on local Ca2+ regulated pathways, among which TFEB mediated signaling, and in turn mitochondrial and lysosomal function. Defects in mitochondria and lysosome represent a common hallmark of neurodegenerative diseases: targeting their communication could open therapeutic avenues.

Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

ATP2B2 de novo variants as a cause of variable neurodevelopmental disorders that feature dystonia, ataxia, intellectual disability, behavioral symptoms, and seizures.

PURPOSE: ATP2B2 encodes the variant-constrained plasma-membrane calcium-transporting ATPase-2, expressed in sensory ear cells and specialized neurons. ATP2B2/Atp2b2 variants were previously linked to isolated hearing loss in patients and neurodevelopmental deficits with ataxia in mice. We aimed to establish the association between ATP2B2 and human neurological disorders. METHODS: Multinational case recruitment, scrutiny of trio-based genomics data, in silico analyses, and functional variant characterization were performed. RESULTS: We assembled 7 individuals harboring rare, predicted deleterious heterozygous ATP2B2 variants. The alleles comprised 5 missense substitutions that affected evolutionarily conserved sites and 2 frameshift variants in the penultimate exon. For 6 variants, a de novo status was confirmed. Unlike described patients with hearing loss, the individuals displayed a spectrum of neurological abnormalities, ranging from ataxia with dystonic features to complex neurodevelopmental manifestations with intellectual disability, autism, and seizures. Two cases with recurrent amino-acid variation showed distinctive overlap with cerebellar atrophy-associated ataxia and epilepsy. In cell-based studies, all variants caused significant alterations in cytosolic calcium handling with both loss- and gain-of-function effects. CONCLUSION: Presentations in our series recapitulate key phenotypic aspects of Atp2b2-mouse models and underline the importance of precise calcium regulation for neurodevelopment and cerebellar function. Our study documents a role for ATP2B2 variants in causing heterogeneous neurodevelopmental and movement-disorder syndromes.

SERCA2 phosphorylation at the heart of the disease.

Gonnot et al. [1] thoroughly investigated the regulatory role of glycogen synthase kinase 3 beta (GSK3ß) in modulating cardiac isoform 2 of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2) activity. They have found that in ischemic hearts of patients and mouse-GSK3ß -mediated SERCA2 phosphorylation at serine 663 dampens the SERCA2 pump activity and induces Ca2+ overload which sensitizes towards myocardial ischemia-reperfusion (I/R) injury. The inhibition of serine 663 phosphorylation significantly increases SERCA2 activity and, by preventing cytosolic and mitochondrial Ca2+ overload, reduces cell death during reperfusion. Augmented SERCA2 activity also substantially improves excitation-contraction coupling in cardiomyocytes upon recovery from reperfusion injury. This study provides valuable insights into pathophysiological relevance of GSK3ß -mediated SERCA2 phosphorylation in the context of heart diseases and paves the way for designing novel clinical therapeutic approaches to alleviate post infartion heart failure.

Ca2+ signalling: A common language for organelles crosstalk in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease caused by multifactorial pathogenic mechanisms. Familial PD is linked with genetic mutations in genes whose products are either associated with mitochondrial function or endo-lysosomal pathways. Of note, mitochondria are essential to sustain high energy demanding synaptic activity of neurons and alterations in mitochondrial Ca2+ signaling have been proposed as causal events for neurodegenerative process, although the mechanisms responsible for the selective loss of specific neuronal populations in the different neurodegenerative diseases is still not clear. Here, we specifically discuss the importance of a correct mitochondrial communication with the other organelles occurring at regions where their membranes become in close contact. We discuss the nature and the role of contact sites that mitochondria establish with ER, lysosomes, and peroxisomes, and how PD related proteins participate in the regulation/dysregulation of the tethering complexes. Unravelling molecular details of mitochondria tethering could contribute to identify specific therapeutic targets and develop new strategies to counteract the progression of the disease.

CaMKII inhibition due to TRIC-B loss-of-function dysregulates SMAD signaling in osteogenesis imperfecta.

Ca2+ is a second messenger that regulates a variety of cellular responses in bone, including osteoblast differentiation. Mutations in trimeric intracellular cation channel B (TRIC-B), an endoplasmic reticulum channel specific for K+, a counter ion for Ca2+flux, affect bone and cause a recessive form of osteogenesis imperfecta (OI) with a still puzzling mechanism. Using a conditional Tmem38b knock out mouse, we demonstrated that lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. At the cellular level, delayed osteoblast differentiation and decreased collagen synthesis were found consequent to the Ca2+ imbalance and associated with reduced collagen incorporation in the extracellular matrix and poor mineralization. The impaired SMAD signaling detected in mutant mice, and validated in OI patient osteoblasts, explained the osteoblast malfunction. The reduced SMAD phosphorylation and nuclear translocation were mainly caused by alteration in Ca2+ calmodulin kinase II (CaMKII)-mediated signaling and to a less extend by a lower TGF-ß reservoir. SMAD signaling, osteoblast differentiation and matrix mineralization were only partially rescued by TGF-ß treatment, strengthening the impact of CaMKII-SMAD axes on osteoblast function. Our data established the TRIC-B role in osteoblasts and deepened the contribution of the CaMKII-SMAD signaling in bone.

Perturbation of the host cell Ca2+ homeostasis and ER-mitochondria contact sites by the SARS-CoV-2 structural proteins E and M.

Coronavirus disease (COVID-19) is a contagious respiratory disease caused by the SARS-CoV-2 virus. The clinical phenotypes are variable, ranging from spontaneous recovery to serious illness and death. On March 2020, a global COVID-19 pandemic was declared by the World Health Organization (WHO). As of February 2023, almost 670 million cases and 6,8 million deaths have been confirmed worldwide. Coronaviruses, including SARS-CoV-2, contain a single-stranded RNA genome enclosed in a viral capsid consisting of four structural proteins: the nucleocapsid (N) protein, in the ribonucleoprotein core, the spike (S) protein, the envelope (E) protein, and the membrane (M) protein, embedded in the surface envelope. In particular, the E protein is a poorly characterized viroporin with high identity amongst all the ß-coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43) and a low mutation rate. Here, we focused our attention on the study of SARS-CoV-2 E and M proteins, and we found a general perturbation of the host cell calcium (Ca2+) homeostasis and a selective rearrangement of the interorganelle contact sites. In vitro and in vivo biochemical analyses revealed that the binding of specific nanobodies to soluble regions of SARS-CoV-2 E protein reversed the observed phenotypes, suggesting that the E protein might be an important therapeutic candidate not only for vaccine development, but also for the clinical management of COVID designing drug regimens that, so far, are very limited.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca2+-microdomains by tuning PMCA3 activity.

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca2+ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

The PLEKHA7-PDZD11 complex regulates the localization of the calcium pump PMCA and calcium handling in cultured cells.

The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell-cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7-PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.

Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic.

The study of organelle contact sites has received a great impulse due to increased interest in the understanding of their involvement in many disease conditions. Split-GFP-based contact sites (SPLICS) reporters emerged as essential tools to easily detect changes in a wide range of organelle contact sites in cultured cells and in vivo, e.g., in zebrafish larvae. We report here on the generation of a new vector library of SPLICS cloned into a piggyBac system for stable and inducible expression of the reporters in a cell line of interest to overcome any potential weakness due to variable protein expression in transient transfection studies. Stable HeLa cell lines expressing SPLICS between the endoplasmic reticulum (ER) and mitochondria (MT), the ER and plasma membrane (PM), peroxisomes (PO) and ER, and PO and MT, were generated and tested for their ability to express the reporters upon treatment with doxycycline. Moreover, to take advantage of these cellular models, we decided to follow the behavior of different membrane contact sites upon modulating cholesterol traffic. Interestingly, we found that the acute pharmacological inhibition of the intracellular cholesterol transporter 1 (NPC1) differently affects membrane contact sites, highlighting the importance of different interfaces for cholesterol sensing and distribution within the cell.

Angiotensin II Promotes SARS-CoV-2 Infection via Upregulation of ACE2 in Human Bronchial Cells.

Blockers of the renin-angiotensin system (RAS) have been reported to increase the angiotensin converting enzyme (ACE)2, the cellular receptor of SARS-CoV-2, and thus the risk and course of COVID-19. Therefore, we investigated if angiotensin (Ang) II and RAS blockers affected ACE2 expression and SARS-CoV-2 infectivity in human epithelial bronchial Calu-3 cells. By infectivity and spike-mediated cell-cell fusion assays, we showed that Ang II acting on the angiotensin type 1 receptor markedly increased ACE2 at mRNA and protein levels, resulting in enhanced SARS-CoV-2 cell entry. These effects were abolished by irbesartan and not affected by the blockade of ACE-1-mediated Ang II formation with ramipril, and of ACE2- mediated Ang II conversion into Ang 1-7 with MLN-4760. Thus, enhanced Ang II production in patients with an activated RAS might expose to a greater spread of COVID-19 infection in lung cells. The protective action of Angiotensin type 1 receptor antagonists (ARBs) documented in these studies provides a mechanistic explanation for the lack of worse outcomes in high-risk COVID-19 patients on RAS blockers.

Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca2+ -affinity targeted aequorin reporter.

Precise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca2+ -affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non-cleavable N-terminal ER signal peptide (fl2). The correct targeting of fl2-AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well-established in animal cells - consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling - was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+ ]ER ) were recorded in response to different environmental stresses, displaying stimulus-specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+ ]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set-up of an ER-targeted aequorin chimera extends and complements the currently available toolkit of organelle-targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system.

Get Closer to the World of Contact Sites: A Beginner's Guide to Proximity-Driven Fluorescent Probes.

To maintain cellular homeostasis and to coordinate the proper response to a specific stimulus, information must be integrated throughout the cell in a well-organized network, in which organelles are the crucial nodes and membrane contact sites are the main edges. Membrane contact sites are the cellular subdomains where two or more organelles come into close apposition and interact with each other. Even though many inter-organelle contacts have been identified, most of them are still not fully characterized, therefore their study is an appealing and expanding field of research. Thanks to significant technological progress, many tools are now available or are in rapid development, making it difficult to choose which one is the most suitable for answering a specific biological question. Here we distinguish two different experimental approaches for studying inter-organelle contact sites. The first one aims to morphologically characterize the sites of membrane contact and to identify the molecular players involved, relying mainly on the application of biochemical and electron microscopy (EM)-related methods. The second approach aims to understand the functional importance of a specific contact, focusing on spatio-temporal details. For this purpose, proximity-driven fluorescent probes are the experimental tools of choice, since they allow the monitoring and quantification of membrane contact sites and their dynamics in living cells under different cellular conditions or upon different stimuli. In this review, we focus on these tools with the purpose of highlighting their great versatility and how they can be applied in the study of membrane contacts. We will extensively describe all the different types of proximity-driven fluorescent tools, discussing their benefits and drawbacks, ultimately providing some suggestions to choose and apply the appropriate methods on a case-to-case basis and to obtain the best experimental outcomes.

Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells.

Membrane contact sites between organelles are essential for maintaining cellular homeostasis, which requires the continuous exchange of signaling molecules, ions, nutrients and lipids. Alterations of different contact sites are associated with a wide spectrum of human diseases. However, visualizing and quantifying these contact sites remains a challenge. This protocol describes the use of split-GFP-based contact site sensors (SPLICS) in microscopy applications for mapping organelle contact sites both in fixed and living cells. SPLICS sensors are engineered to express equimolar amounts of the organelle-targeted nonfluorescent ß11 and GFP1-10 portions of the split-GFP protein in a single vector, and are capable of reconstituting fluorescence when two opposing membranes come into proximity. Reconstitution will occur only over the cell volume at defined contact sites resulting in a bright signal that can be detected easily and quantified automatically with specific custom-made plugins. The use of minimal targeting sequences facilitates targeting specificity and membrane coverage, avoiding artifacts due to full-length fusion protein overexpression and, thus, possible perturbations of the cell's physiology. SPLICS sensors engineered to simultaneously detect multiple contact sites within the same cell have been generated by exploiting the ability of the ß11 GFP fragment to reconstitute different color-shifted variants of the GFP1-10 fragment. Here, we describe a detailed protocol to set up SPLICS expression in living cells (2-3 d), detection and acquisition (1 d), and automated quantification with custom plugins (1-2 d). We also advise on construct design and characterization for novel organelle contacts.

Ca2+ handling at the mitochondria-ER contact sites in neurodegeneration.

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are morpho-functional units, formed at the loci of close apposition of the ER-forming endomembrane and outer mitochondrial membrane (OMM). These sites contribute to fundamental cellular processes including lipid biosynthesis, autophagy, apoptosis, ER-stress and calcium (Ca2+) signalling. At MERCS, Ca2+ ions are transferred from the ER directly to mitochondria through a core protein complex composed of inositol-1,4,5 trisphosphate receptor (InsP3R), voltage-gated anion channel 1 (VDAC1), mitochondrial calcium uniporter (MCU) and adaptor protein glucose-regulated protein 75 (Grp75); this complex is regulated by several associated proteins. Deregulation of ER-mitochondria Ca2+ transfer contributes to pathogenesis of neurodegenerative and other diseases. The efficacy of Ca2+ transfer between ER and mitochondria depends on the protein composition of MERCS, which controls ER-mitochondria interaction regulating, for example, the transversal distance between ER membrane and OMM and the extension of the longitudinal interface between ER and mitochondria. These parameters are altered in neurodegeneration. Here we overview the ER and mitochondrial Ca2+ homeostasis, the composition of ER-mitochondrial Ca2+ transfer machinery and alterations of the ER-mitochondria Ca2+ transfer in three major neurodegenerative diseases: motor neurone diseases, Parkinson disease and Alzheimer's disease.