Oxidative bursts of single mitochondria mediate retrograde signaling toward the ER.

The emerging role of mitochondria as signaling organelles raises the question of whether individual mitochondria can initiate heterotypic communication with neighboring organelles. Using fluorescent probes targeted to the endoplasmic-reticulum-mitochondrial interface, we demonstrate that single mitochondria generate oxidative bursts, rapid redox oscillations, confined to the nanoscale environment of the interorganellar contact sites. Using probes fused to inositol 1,4,5-trisphosphate receptors (IP3Rs), we show that Ca2+ channels directly sense oxidative bursts and respond with Ca2+ transients adjacent to active mitochondria. Application of specific mitochondrial stressors or apoptotic stimuli dramatically increases the frequency and amplitude of the oxidative bursts by enhancing transient permeability transition pore openings. Conversely, blocking interface Ca2+ transport via elimination of IP3Rs or mitochondrial calcium uniporter channels suppresses ER-mitochondrial Ca2+ feedback and cell death. Thus, single mitochondria initiate local retrograde signaling by miniature oxidative bursts and, upon metabolic or apoptotic stress, may also amplify signals to the rest of the cell.

Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance.

Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug-resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER-mitochondria-associated membranes (MAMs; ER-mitochondria contacts, ERMCs) in therapy-resistant cells, and genetically or biochemically reducing MAMs in therapy-sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER-mitochondria-associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.

A novel bacterial effector protein mediates ER-LD membrane contacts to regulate host lipid droplets.

Effective intracellular communication between cellular organelles occurs at dedicated membrane contact sites (MCSs). Tether proteins are responsible for the establishment of MCSs, enabling direct communication between organelles to ensure organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). Here, we identify a novel bacterial effector protein, CbEPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii, an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein CbEPF1 which localizes to host lipid droplets (LDs). CbEPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, CbEPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

A three-organelle complex made by wrappER contacts with peroxisomes and mitochondria responds to liver lipid flux changes.

Hepatic lipid homeostasis depends on intracellular pathways that respire fatty acid in peroxisomes and mitochondria, and on systemic pathways that secrete fatty acid into the bloodstream, either free or condensed in very-low-density lipoprotein (VLDL) triglycerides. These systemic and intracellular pathways are interdependent, but it is unclear whether and how they integrate into a single cellular circuit. Here, we report that mouse liver wrappER, a distinct endoplasmic reticulum (ER) compartment with apparent fatty acid- and VLDL-secretion functions, connects peroxisomes and mitochondria. Correlative light electron microscopy, quantitative serial section electron tomography and three-dimensional organelle reconstruction analysis show that the number of peroxisome-wrappER-mitochondria complexes changes throughout fasting-to-feeding transitions and doubles when VLDL synthesis stops following acute genetic ablation of Mttp in the liver. Quantitative proteomic analysis of peroxisome-wrappER-mitochondria complex-enriched fractions indicates that the loss of Mttp upregulates global fatty acid ß-oxidation, thereby integrating the dynamics of this three-organelle association into hepatic fatty acid flux responses. Therefore, liver lipid homeostasis occurs through the convergence of systemic and intracellular fatty acid-elimination pathways in the peroxisome-wrappER-mitochondria complex.

Are endoplasmic reticulum subdomains shaped by asymmetric distribution of phospholipids? Evidence from a C. elegans model system.

Physical contact between organelles are widespread, in part to facilitate the shuttling of protein and lipid cargoes for cellular homeostasis. How do protein-protein and protein-lipid interactions shape organelle subdomains that constitute contact sites? The endoplasmic reticulum (ER) forms extensive contacts with multiple organelles, including lipid droplets (LDs) that are central to cellular fat storage and mobilization. Here, we focus on ER-LD contacts that are highlighted by the conserved protein seipin, which promotes LD biogenesis and expansion. Seipin is enriched in ER tubules that form cage-like structures around a subset of LDs. Such enrichment is strongly dependent on polyunsaturated and cyclopropane fatty acids. Based on these findings, we speculate on molecular events that lead to the formation of seipin-positive peri-LD cages in which protein movement is restricted. We hypothesize that asymmetric distribution of specific phospholipids distinguishes cage membrane tubules from the bulk ER.

Mesoscale Metabolic Channeling Revealed by Multimodal Microscopy.

Metabolic homeostasis within cells and tissues requires engagement of catabolic and anabolic pathways consuming nutrients needed to generate energy to drive these and other subcellular processes. However, the current understanding of cell homeostasis and metabolism, including how cells utilize nutrients, comes largely from tissue and cell models analyzed after fractionation. These bulk strategies do not reveal the spatial characteristics of cell metabolism at the single cell level, and how these aspects relate to the location of cells and organelles within the complexity of the tissue they reside within. Here we pioneer the use of high-resolution electron and stable isotope microscopy (MIMS-EM) to quantitatively map the fate of nutrient-derived 13C atoms at subcellular scale. When combined with machine-learning image segmentation, our approach allows us to establish the cellular and organellar spatial pattern of glucose 13C flux in hepatocytes in situ. We applied network analysis algorithms to chart the landscape of organelle-organelle contact networks and identified subpopulations of mitochondria and lipid droplets that have distinct organelle interactions and 13C enrichment levels. In addition, we revealed a new relationship between the initiation of glycogenesis and proximity of lipid droplets. Our results establish MIMS-EM as a new tool for tracking and quantifying nutrient metabolism at the subcellular scale, and to identify the spatial channeling of nutrient-derived atoms in the context of organelle-organelle interactions in situ.

Mitochondrial heterogeneity and crosstalk in aging: Time for a paradigm shift?

The hallmarks of aging have been influential in guiding the biology of aging research, with more recent and growing recognition of the interdependence of these hallmarks on age-related health outcomes. However, a current challenge is personalizing aging trajectories to promote healthy aging, given the diversity of genotypes and lived experience. We suggest that incorporating heterogeneity-including intrinsic (e.g., genetic and structural) and extrinsic (e.g., environmental and exposome) factors and their interdependence of hallmarks-may move the dial. This editorial perspective will focus on one hallmark, namely mitochondrial dysfunction, to exemplify how consideration of heterogeneity and interdependence or crosstalk may reveal new perspectives and opportunities for personalizing aging research. To this end, we highlight heterogeneity within mitochondria as a model.

The novel bacterial effector protein CbEPF1 mediates ER-LD membrane contacts to regulate host lipid droplet metabolism.

Effective intracellular communication between cellular organelles is pivotal for maintaining cellular homeostasis. Tether proteins, which are responsible for establishing membrane contact sites between cell organelles, enable direct communication between organelles and ultimately influence organelle function and host cell homeostasis. While recent research has identified tether proteins in several bacterial pathogens, their functions have predominantly been associated with mediating inter-organelle communication specifically between the bacteria containing vacuole (BCV) and the host endoplasmic reticulum (ER). However, this study reveals a novel bacterial effector protein, CbEPF1, which acts as a molecular tether beyond the confines of the BCV and facilitates interactions between host cell organelles. Coxiella burnetii, an obligate intracellular bacterial pathogen, encodes the FFAT motif-containing protein CbEPF1 which localizes to host lipid droplets (LDs). CbEPF1 establishes inter-organelle contact sites between host LDs and the ER through its interactions with VAP family proteins. Intriguingly, CbEPF1 modulates growth of host LDs in a FFAT motif-dependent manner. These findings highlight the potential for bacterial effector proteins to impact host cellular homeostasis by manipulating inter-organelle communication beyond conventional BCVs.

ATG2 and VPS13 proteins: molecular highways transporting lipids to drive membrane expansion and organelle communication.

Communication between organelles is an essential process that helps maintain cellular homeostasis and organelle contact sites have recently emerged as crucial mediators of this communication. The emergence of a class of molecular bridges that span the inter-organelle gaps has now been shown to direct the flow of lipid traffic from one lipid bilayer to another. One of the key components of these molecular bridges is the presence of an N-terminal Chorein/VPS13 domain. This is an evolutionarily conserved domain present in multiple proteins within the endocytic and autophagy trafficking pathways. Herein, we discuss the current state-of-the-art of this class of proteins, focusing on the role of these lipid transporters in the autophagy and endocytic pathways. We discuss the recent biochemical and structural advances that have highlighted the essential role Chorein-N domain containing ATG2 proteins play in driving the formation of the autophagosome and how lipids are transported from the endoplasmic reticulum to the growing phagophore. We also consider the VPS13 proteins, their role in organelle contacts and the endocytic pathway and highlight how disease-causing mutations disrupt these contact sites. Finally, we open the door to discuss other Chorein_N domain containing proteins, for instance, UHRF1BP1/1L, their role in disease and look towards prokaryote examples of Chorein_N-like domains. Taken together, recent advances have highlighted an exciting opportunity to delve deeper into inter-organelle communication and understand how lipids are transported between membrane bilayers and how this process is disrupted in multiple diseases.

Optogenetic Tools for Manipulating Protein Subcellular Localization and Intracellular Signaling at Organelle Contact Sites.

Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein-protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light-activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light-tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Genetic engineering strategy for the generation of modular light-activated protein dimerization units Support Protocol 1: Molecular cloning Basic Protocol 2: Cell culture and transfection Support Protocol 2: Production of dark containers for optogenetic samples Basic Protocol 3: Confocal microscopy and light-dependent activation of the dimerization system Alternate Protocol 1: Protein recruitment to intracellular compartments Alternate Protocol 2: Induction of organelles' membrane tethering Alternate Protocol 3: Optogenetic reconstitution of protein function Basic Protocol 4: Image analysis Support Protocol 3: Analysis of apparent on- and off-kinetics Support Protocol 4: Analysis of changes in organelle overlap over time.

Loosening ER-Mitochondria Coupling by the Expression of the Presenilin 2 Loop Domain.

Presenilin 2 (PS2), one of the three proteins in which mutations are linked to familial Alzheimer's disease (FAD), exerts different functions within the cell independently of being part of the γ-secretase complex, thus unrelated to toxic amyloid peptide formation. In particular, its enrichment in endoplasmic reticulum (ER) membrane domains close to mitochondria (i.e., mitochondria-associated membranes, MAM) enables PS2 to modulate multiple processes taking place on these signaling hubs, such as Ca2+ handling and lipid synthesis. Importantly, upregulated MAM function appears to be critical in AD pathogenesis. We previously showed that FAD-PS2 mutants reinforce ER-mitochondria tethering, by interfering with the activity of mitofusin 2, favoring their Ca2+ crosstalk. Here, we deepened the molecular mechanism underlying PS2 activity on ER-mitochondria tethering, identifying its protein loop as an essential domain to mediate the reinforced ER-mitochondria connection in FAD-PS2 models. Moreover, we introduced a novel tool, the PS2 loop domain targeted to the outer mitochondrial membrane, Mit-PS2-LOOP, that is able to counteract the activity of FAD-PS2 on organelle tethering, which possibly helps in recovering the FAD-PS2-associated cellular alterations linked to an increased organelle coupling.

Better to keep in touch: investigating inter-organelle cross-talk.

The strategic importance for cellular organelles of being in contact with each other, exchanging messenger molecules, is nowadays well established. Different inter-organelle cross-talk pathways finely regulate multiple physiological cellular mechanisms, and their dysregulation has been found to underlie different pathological conditions. In the last years, a great effort has been made to study such organelle interactions, to understand their functional roles within the cell and the molecules involved in their formation and/or modulation. In this contribution, some examples of organelle cross-talk and their contributions in regulating physiological processes are presented. Moreover, the pro and cons of the available methods for a proper, reliable investigation of membrane contact sites are described.

Organelle distribution in neurons: Logistics behind polarized transport.

Highly polarized neurons need to carefully regulate the distribution of organelles and other cargoes into their two morphologically and functionally distinct domains, the somatodendritic and axonal compartments, to maintain proper neuron homeostasis. An outstanding question in the field is how organelles reach their correct destination. Long-range transport along microtubules, driven by motors, ensures a fast and controlled availability of organelles in axons and dendrites, but it remains largely unclear what rules govern their transport into the correct compartment. Here, we review the emerging concepts of polarized cargo trafficking in neurons, highlighting the role of microtubule organization, microtubule-associated proteins, and motor proteins and discuss compartment-specific inclusion and exclusion mechanisms as well as the regulation of correct coupling of cargoes to motor proteins.

Forcing contacts between mitochondria and the endoplasmic reticulum extends lifespan in a Drosophila model of Alzheimer's disease.

Eukaryotic cells are complex systems containing internal compartments with specialised functions. Among these compartments, the endoplasmic reticulum (ER) plays a major role in processing proteins for modification and delivery to other organelles, whereas mitochondria generate energy in the form of ATP. Mitochondria and the ER form physical interactions, defined as mitochondria-ER contact sites (MERCs) to exchange metabolites such as calcium ions (Ca2+) and lipids. Sites of contact between mitochondria and the ER can regulate biological processes such as ATP generation and mitochondrial division. The interactions between mitochondria and the ER are dynamic and respond to the metabolic state of cells. Changes in MERCs have been linked to metabolic pathologies such as diabetes, neurodegenerative diseases and sleep disruption. Here we explored the consequences of increasing contacts between mitochondria and the ER in flies using a synthetic linker. We showed that enhancing MERCs increases locomotion and extends lifespan. We also showed that, in a Drosophila model of Alzheimer's disease linked to toxic amyloid beta (Aß), linker expression can suppress motor impairment and extend lifespan. We conclude that strategies for increasing contacts between mitochondria and the ER may improve symptoms of diseases associated with mitochondria dysfunction. A video abstract for this article is available at https://youtu.be/_YWA4oKZkes.This article has an associated First Person interview with the first author of the paper.

Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

The cell relies on direct interactions among proteins and compartments called organelles to stay alive. Manipulating these interactions allows researchers to control a wide variety of cell behaviors. A system called 'Magnets' uses light to trigger interactions between proteins. Magnets uses a segment of a protein called Vivid from a common bread mold that responds to light. When light shines on two of these segments, it causes them to bind together, in a process known as dimerization. In the Magnets system, Vivid segments are attached to specific proteins or organelles. By using light, researchers can force their target molecules to come together and trigger signals that can change cell behavior. However, the Magnets system has limitations: its stability and low efficiency mean that the cells need to be kept at low temperatures and that several copies of Vivid are needed. These conditions can interfere with the activity of the target proteins. To expand the technique, Benedetti et al. added mutations to make the Vivid protein more similar to proteins found in fungi that thrive at temperatures around 50°C. These changes meant that the enhanced system could work at body temperature in mammals. Further mutations at the interface between the two Vivid segments improved the efficiency of dimerization. This enhanced version was put to the test in different applications, including delivering proteins to different organelles and bringing organelles together. The enhanced Magnets system should enable researchers to control a greater variety of signaling events in the cell. In addition, the methodology established for improving the efficiency of the Magnets system could be useful to researchers working on other proteins.

Complex Interactions Between Membrane-Bound Organelles, Biomolecular Condensates and the Cytoskeleton.

Membrane-bound and membraneless organelles/biomolecular condensates ensure compartmentalization into functionally distinct units enabling proper organization of cellular processes. Membrane-bound organelles form dynamic contacts with each other to enable the exchange of molecules and to regulate organelle division and positioning in coordination with the cytoskeleton. Crosstalk between the cytoskeleton and dynamic membrane-bound organelles has more recently also been found to regulate cytoskeletal organization. Interestingly, recent work has revealed that, in addition, the cytoskeleton and membrane-bound organelles interact with cytoplasmic biomolecular condensates. The extent and relevance of these complex interactions are just beginning to emerge but may be important for cytoskeletal organization and organelle transport and remodeling. In this review, we highlight these emerging functions and emphasize the complex interplay of the cytoskeleton with these organelles. The crosstalk between membrane-bound organelles, biomolecular condensates and the cytoskeleton in highly polarized cells such as neurons could play essential roles in neuronal development, function and maintenance.

Sarcoplasmic Reticulum-Mitochondria Kissing in Cardiomyocytes: Ca2+, ATP, and Undisclosed Secrets.

In cardiomyocytes, to carry out cell contraction, the distribution, morphology, and dynamic interaction of different cellular organelles are tightly regulated. For instance, the repetitive close apposition between junctional sarcoplasmic reticulum (jSR) and specialized sarcolemma invaginations, called transverse-tubules (TTs), is essential for an efficient excitation-contraction coupling (ECC). Upon an action potential, Ca2+ microdomains, generated in synchrony at the interface between TTs and jSR, underlie the prompt increase in cytosolic Ca2+ concentration, ultimately responsible for cell contraction during systole. This process requires a considerable amount of energy and the active participation of mitochondria, which encompass ∼30% of the cell volume and represent the major source of ATP in the heart. Importantly, in adult cardiomyocytes, mitochondria are distributed in a highly orderly fashion and strategically juxtaposed with SR. By taking advantage of the vicinity to Ca2+ releasing sites, they take up Ca2+ and modulate ATP synthesis according to the specific cardiac workload. Interestingly, with respect to SR, a biased, polarized positioning of mitochondrial Ca2+ uptake/efflux machineries has been reported, hinting the importance of a strictly regulated mitochondrial Ca2+ handling for heart activity. This notion, however, has been questioned by the observation that, in some mouse models, the deficiency of specific molecules, modulating mitochondrial Ca2+ dynamics, triggers non-obvious cardiac phenotypes. This review will briefly summarize the physiological significance of SR-mitochondria apposition in cardiomyocytes, as well as the pathological consequences of an altered organelle communication, focusing on Ca2+ signaling. We will discuss ongoing debates and propose future research directions.

In Close Proximity: The Lipid Droplet Proteome and Crosstalk With the Endoplasmic Reticulum.

Lipid droplets (LDs) are conserved, endoplasmic reticulum (ER)-derived organelles that act as a dynamic cellular repository for neutral lipids. Numerous studies have examined the composition of LD proteomes by using mass spectrometry to identify proteins present in biochemically isolated buoyant fractions that are enriched in LDs. Although many bona fide LD proteins were identified, high levels of non-LD proteins that contaminate buoyant fractions complicate the detection of true LD proteins. To overcome this problem, we recently developed a proximity-labeling proteomic method to define high-confidence LD proteomes. Moreover, employing this approach, we discovered that ER-associated degradation impacts the composition of LD proteomes by targeting select LD proteins for clearance by the 26S proteasome as they transit between the ER and LDs. These findings implicate the ER as a site of LD protein degradation and underscore the high degree of crosstalk between ER and LDs.

Morphological Pathways of Mitochondrial Division.

Mitochondrial fission is essential for distributing cellular energy throughout cells and for isolating damaged regions of the organelle that are targeted for degradation. Excessive fission is associated with the progression of cell death as well. Therefore, this multistep process is tightly regulated and several physiologic cues directly impact mitochondrial division. The double membrane structure of mitochondria complicates this process, and protein factors that drive membrane scission need to coordinate the separation of both the outer and inner mitochondrial membranes. In this review, we discuss studies that characterize distinct morphological changes associated with mitochondrial division. Specifically, coordinated partitioning and pinching of mitochondria have been identified as alternative mechanisms associated with fission. Additionally, we highlight the major protein constituents that drive mitochondrial fission and the role of connections with the endoplasmic reticulum in establishing sites of membrane division. Collectively, we review decades of research that worked to define the molecular framework of mitochondrial fission. Ongoing studies will continue to sort through the complex network of interactions that drive this critical event.

The endoplasmic reticulum-mitochondria coupling in health and disease: Molecules, functions and significance.

The close apposition between endoplasmic reticulum (ER) and mitochondria represents a key platform, capable to regulate different fundamental cellular pathways. Among these, Ca2+ signaling and lipid homeostasis have been demonstrated over the last years to be deeply modulated by ER-mitochondria cross-talk. Given its importance in cell life/death decisions, increasing evidence suggests that alterations of the ER-mitochondria axis could be responsible for the onset and progression of several diseases, including neurodegeneration, cancer and obesity. However, the molecular identity of the proteins controlling this inter-organelle apposition is still debated. In this review, we summarize the main cellular pathways controlled by ER-mitochondria appositions, focusing on the principal molecules reported to be involved in this interplay and on those diseases for which alterations in organelles communication have been reported.