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The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin. Endoplasmic reticulum (ER)-interorganelle membrane contact sites have emerged as key regulators of organelle dynamics. Here, the authors report that the ER-resident protein SNX19 mediates ER-endolysosome membrane contacts to maintain the perinuclear distribution of endolysosomes and restrict their motility.

The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in β-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease. The internal organization of the cell has been enriched by the discovery that organelles establish membrane contact sites, however the entire repertoire of these contacts is still being explored. Here the authors systematically identify the landscape of cellular contact sites in yeast, discovering four potential novel contact sites and two tether proteins for the peroxisome-mitochondria contact site.

Key Points The endoplasmic reticulum (ER) forms tight membrane contact sites (MCSs) with several organelles in animal cells and yeast. The function of MCSs between the ER and mitochondria and endosomes are summarized in this Review. Electron microscopy studies reveal that although MCSs are less than 30 nm apart, the membranes do not fuse and each organelle maintains its identity. Ribosomes are excluded from the ER membrane at MCSs, and the distance between the ER and other membranes is close enough to suggest that the two organelles are tethered together by other proteins located on apposing membranes. Live-cell fluorescence microscopy reveals that ER-organelle MCSs can remain stable while both organelles traffic through the cell on the cytoskeleton. Recently identified factors have been shown to regulate organelle trafficking through MCS formation. ER–organelle MCSs regulate the lipid environment of the organelle membrane apposed to the ER. Lipid-synthesis proteins on the ER can modify lipids on the membrane of another organelle or on protein complexes. ER MCS may also transfer lipids between membranes. ER–organelle MCSs are sites of dynamic Ca 2+ crosstalk. Organelles can sequester Ca 2+ released from the ER, which can regulate processes in these organelles. Additionally, ER Ca 2+ release may regulate protein complexes at ER MCS. Mitochondria and endosomes undergo fission and fusion to, respectively, maintain their integrity and properly sort signalling receptors in the cell. ER–organelle MCSs define the position of fission for both mitochondria and endosomes, and the ER could have a variety of roles at those specific MCSs that are destined for fission. Endoplasmic reticulum (ER) is typically associated with protein biogenesis. However, recent studies suggest that it additionally synchronizes and regulates a plethora of intracellular events owing to its ability to form tight membrane associations, so-called membrane contact sites (MCSs), with other organelles. The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

Lipid mobilization through fatty acid β-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where β-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in β-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although β-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1’s role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites. Enkler et al. show that a pool of Arf1 at lipid droplets is implicated in mitochondrial ATP production control through regulation of fatty acid metabolism and acetyl-CoA transfer to mitochondria.

Eukaryotic cells contain several membrane-separated organelles to compartmentalize distinct metabolic reactions. However, it has remained unclear how these organelle systems are coordinated when cells adapt metabolic pathways to support their development, survival or effector functions. Here we present OrgaPlexing, a multi-spectral organelle imaging approach for the comprehensive mapping of six key metabolic organelles and their interactions. We use this analysis on macrophages, immune cells that undergo rapid metabolic switches upon sensing bacterial and inflammatory stimuli. Our results identify lipid droplets (LDs) as primary inflammatory responder organelle, which forms three- and four-way interactions with other organelles. While clusters with endoplasmic reticulum (ER) and mitochondria (mitochondria–ER–LD unit) help supply fatty acids for LD growth, the additional recruitment of peroxisomes (mitochondria–ER–peroxisome–LD unit) supports fatty acid efflux from LDs. Interference with individual components of these units has direct functional consequences for inflammatory lipid mediator synthesis. Together, we show that macrophages form functional multi-organellar units to support metabolic adaptation and provide an experimental strategy to identify organelle-metabolic signalling hubs. Zimmermann et al. present OrgaPlexing, an imaging pipeline mapping metabolic organelles and their interactions. They find changes in mitochondria, ER, peroxisome and lipid droplet dynamics that impact macrophage inflammatory lipid mediator synthesis.

Tissue ischemia adversely affects the function of mitochondria, which results in impairment of oxidative phosphorylation and compromised recovery of the affected organ. The impact of ischemia on mitochondrial function has been extensively studied in the heart because of the morbidity and mortality associated with injury to this organ. As conventional methods to preserve cardiac cell viability and contractile function following ischemia are limited in their efficacy, we developed a unique approach to protect the heart by transplanting respiration-competent mitochondria to the injured region. Our previous animal experiments showed that transplantation of isolated mitochondria to ischemic heart tissue leads to decreases in cell death, increases in energy production, and improvements in contractile function. We also discovered that exogenously-derived mitochondria injected or perfused into ischemic hearts were rapidly internalised by cardiac cells. Here, we used three-dimensional super-resolution microscopy and transmission electron microscopy to determine the intracellular fate of endocytosed exogenous mitochondria in human iPS-derived cardiomyocytes and primary cardiac fibroblasts. We found isolated mitochondria are incorporated into cardiac cells within minutes and then transported to endosomes and lysosomes. The majority of exogenous mitochondria escape from these compartments and fuse with the endogenous mitochondrial network, while some of these organelles are degraded through hydrolysis.

Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.

Formation of inter-organelle contacts between mitochondria and lysosomes, regulated by lysosomal RAB7 GTP hydrolysis, allows for bidirectional regulation of mitochondrial and lysosomal dynamics. Contacts inside cell walls Cellular organelles such as mitochondria and lysosomes are dynamic entities that communicate with each other not just through vesicular trafficking but also by direct, albeit transient, contact between different organelles. Dimitri Krainc and colleagues report the existence of inter-organelle contacts between mitochondria and lysosomes—a phenomenon that seems to be independent of the association between damaged mitochondria and lysosomes in the context of the degradative process of mitophagy. The authors also identify organelle-specific molecules that mediate the tethering between these two organelles and demonstrate that lysosome–mitochondrion contacts allow bidirectional regulation of the dynamics of these organelles. Both mitochondria and lysosomes are essential for maintaining cellular homeostasis, and dysfunction of both organelles has been observed in multiple diseases 1 , 2 , 3 , 4 . Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network, which drives cellular metabolism 5 . Lysosomes similarly undergo constant dynamic regulation by the RAB7 GTPase 1 , which cycles from an active GTP-bound state into an inactive GDP-bound state upon GTP hydrolysis. Here we have identified the formation and regulation of mitochondria–lysosome membrane contact sites using electron microscopy, structured illumination microscopy and high spatial and temporal resolution confocal live cell imaging. Mitochondria–lysosome contacts formed dynamically in healthy untreated cells and were distinct from damaged mitochondria that were targeted into lysosomes for degradation 6 , 7 . Contact formation was promoted by active GTP-bound lysosomal RAB7, and contact untethering was mediated by recruitment of the RAB7 GTPase-activating protein TBC1D15 to mitochondria by FIS1 to drive RAB7 GTP hydrolysis and thereby release contacts. Functionally, lysosomal contacts mark sites of mitochondrial fission, allowing regulation of mitochondrial networks by lysosomes, whereas conversely, mitochondrial contacts regulate lysosomal RAB7 hydrolysis via TBC1D15. Mitochondria–lysosome contacts thus allow bidirectional regulation of mitochondrial and lysosomal dynamics, and may explain the dysfunction observed in both organelles in various human diseases.

Mitochondria are the key organelles for sensing oxygen, which is consumed by oxidative phosphorylation to generate ATP. Lysosomes contain hydrolytic enzymes that degrade misfolded proteins and damaged organelles to maintain cellular homeostasis. Mitochondria physically and functionally interact with lysosomes to regulate cellular metabolism. However, the mode and biological functions of mitochondria-lysosome communication remain largely unknown. Here, we show that hypoxia remodels normal tubular mitochondria into megamitochondria by inducing broad inter-mitochondria contacts and subsequent fusion. Importantly, under hypoxia, mitochondria-lysosome contacts are promoted, and certain lysosomes are engulfed by megamitochondria, in a process we term megamitochondria engulfing lysosome (MMEL). Both megamitochondria and mature lysosomes are required for MMEL. Moreover, the STX17-SNAP29-VAMP7 complex contributes to mitochondria-lysosome contacts and MMEL under hypoxia. Intriguingly, MMEL mediates a mode of mitochondrial degradation, which we termed mitochondrial self-digestion (MSD). Moreover, MSD increases mitochondrial ROS production. Our results reveal a mode of crosstalk between mitochondria and lysosomes and uncover an additional pathway for mitochondrial degradation. Several organelle membranes make contact in the cell, with many contacts being spatially segregated sites dedicated to specific functions. Here, Hao et al. show that hypoxia increases mitochondria-lysosome contacts, leading to engulfment and degradation of the mitochondria.

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites 1 – 3 , but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum–mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture 4 – 6 . Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells. Integrative structural biology combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling enables in situ determination of the structure of the endoplasmic reticulum–mitochondria encounter complex in yeast.