Inter-organelle Communication Research Articles

Imaging interorganelle contacts at a glance.

Eukaryotic cells are compartmentalized into membrane-bound organelles that must coordinate their responses to stimuli. One way that organelles communicate is via membrane contact sites (MCSs), sites of close apposition between organelles used for the exchange of ions, lipids and information. In this Cell Science at a Glance article and the accompanying poster, we describe an explosion of new methods that have led to exciting progress in this area and discuss key examples of how these methods have advanced our understanding of MCSs. We discuss how diffraction-limited and super-resolution fluorescence imaging approaches have provided important insight into the biology of interorganelle communication. We also describe how the development of multiple proximity-based methods has enabled the detection of MCSs with high accuracy and precision. Finally, we assess how recent advances in electron microscopy (EM), considered the gold standard for detecting MCSs, have allowed the visualization of MCSs and associated proteins in 3D at ever greater resolution.

The lysosomal lipid transporter LIMP-2/SCARB2 is part of lysosome-endoplasmic reticulum STARD3-VAPB-dependent contact sites.

SCARB2/LIMP-2 is an abundant lysosomal membrane protein. Previous studies have shown LIMP-2 functions as a virus receptor, a chaperone for lysosomal enzyme targeting, and a lipid transporter. The large luminal domain of LIMP-2 contains a hydrophobic tunnel that enables transport of phospholipids, sphingosine and cholesterol from the lysosomal lumen to the membrane. The question about the fate of the lipids after LIMP-2-mediated transport is largely unexplored. To elucidate whether LIMP-2 is part of contact sites between lysosomes and the endoplasmic reticulum (ER), we performed a proximity-based interaction screen. This revealed that LIMP-2 interacts with the endosomal protein STARD3 and the ER-resident protein VAPB. Using imaging and co-immunoprecipitation, we demonstrated colocalization and physical interaction between LIMP-2 and these proteins. Moreover, we found that interaction of LIMP-2 with VAPB required the presence of STARD3. Our findings suggest that LIMP-2 is part of ER-lysosome contact sites, possibly facilitating cholesterol transport from the lysosomal to the ER membrane. This suggests a novel mechanism for inter-organelle communication and lipid trafficking mediated by LIMP-2.

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.

Imaging and proteomics toolkits for studying organelle contact sites.

Organelle contact sites are regions where two heterologous membranes are juxtaposed by molecular tethering complexes. These contact sites are important in inter-organelle communication and cellular functional integration. However, visualizing these minute foci and identifying contact site proteomes have been challenging. In recent years, fluorescence-based methods have been developed to visualize the dynamic physical interaction of organelles while proximity labeling approaches facilitate the profiling of proteomes at contact sites. In this review, we explain the design principle for these contact site reporters: a dual-organelle interaction mechanism based on how endogenous tethers and/or tethering complexes localize to contact sites. We classify the contact site reporters into three categories: (i) single-protein systems, (ii) two-component systems with activated reporter signal upon organelle proximity, and (iii) reporters for contact site proteomes. We also highlight advanced imaging analysis with high temporal-spatial resolution and the use of machine-learning algorithms for detecting contact sites.

Altered Endoplasmic Reticulum Integrity and Organelle Interactions in Living Cells Expressing INF2 Variants.

The cytoskeleton mediates fundamental cellular processes by organizing inter-organelle interactions. Pathogenic variants of inverted formin 2 (INF2) CAAX isoform, an actin assembly factor that is predominantly expressed in the endoplasmic reticulum (ER), are linked to focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth (CMT) neuropathy. To investigate how pathogenic INF2 variants alter ER integrity, we used high-resolution live imaging of HeLa cells. Cells expressing wild-type (WT) INF2 showed a predominant tubular ER with perinuclear clustering. Cells expressing INF2 FSGS variants that cause mild and intermediate disease induced more sheet-like ER, a pattern similar to that seen for cells expressing WT-INF2 that were treated with actin and microtubule (MT) inhibitors. Dual CMT-FSGS INF2 variants led to more severe ER dysmorphism, with a diffuse, fragmented ER and coarse INF2 aggregates. Proper organization of both F-actin and MT was needed to modulate the tubule vs. sheet conformation balance, while MT arrays regulated spatial expansion of tubular ER in the cell periphery. Pathogenic INF2 variants also induced mitochondria fragmentation and dysregulated mitochondria distribution. Such mitochondrial abnormalities were more prominent for cells expressing CMT-FSGS compared to those with FSGS variants, indicating that the severity of the dysfunction is linked to the degree of cytoskeletal disorganization. Our observations suggest that pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton that in turn impairs inter-organelle communication, especially at ER-mitochondria contact sites. ER continuity defects may be a common disease mechanism involved in both peripheral neuropathy and glomerulopathy.

Lysosomes drive the piecemeal removal of mitochondrial inner membrane.

Mitochondrial membranes define distinct structural and functional compartments. Cristae of the inner mitochondrial membrane (IMM) function as independent bioenergetic units that undergo rapid and transient remodelling, but the significance of this compartmentalized organization is unknown1. Using super-resolution microscopy, here we show that cytosolic IMM vesicles, devoid of outer mitochondrial membrane or mitochondrial matrix, are formed during resting state. These vesicles derived from the IMM (VDIMs) are formed by IMM herniation through pores formed by voltage-dependent anion channel 1 in the outer mitochondrial membrane. Live-cell imaging showed that lysosomes in proximity to mitochondria engulfed the herniating IMM and, aided by the endosomal sorting complex required for transport machinery, led to the formation of VDIMs in a microautophagy-like process, sparing the remainder of the organelle. VDIM formation was enhanced in mitochondria undergoing oxidative stress, suggesting their potential role in maintenance of mitochondrial function. Furthermore, the formation of VDIMs required calcium release by the reactive oxygen species-activated, lysosomal calcium channel, transient receptor potential mucolipin 1, showing an interorganelle communication pathway for maintenance of mitochondrial homeostasis. Thus, IMM compartmentalization could allow for the selective removal of damaged IMM sections via VDIMs, which should protect mitochondria from localized injury. Our findings show a new pathway of intramitochondrial quality control.

Analysis of Brain, Blood, and Testis Phenotypes Lacking the Vps13a Gene in C57BL/6N Mice.

The Vps13a gene encodes a lipid transfer protein called VPS13A, or chorein, associated with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), mitochondria-endosomes, and lipid droplets. This protein plays a crucial role in inter-organelle communication and lipid transport. Mutations in the VPS13A gene are implicated in the pathogenesis of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder characterized by chorea, orofacial dyskinesias, hyperkinetic movements, seizures, cognitive impairment, and acanthocytosis. Previous mouse models of ChAc have shown variable disease phenotypes depending on the genetic background. In this study, we report the generation of a Vps13a flox allele in a pure C57BL/6N mouse background and the subsequent creation of Vps13a knockout (KO) mice via Cre-recombination. Our Vps13a KO mice exhibited increased reticulocytes but not acanthocytes in peripheral blood smears. Additionally, there were no significant differences in the GFAP- and Iba1-positive cells in the striatum, the basal ganglia of the central nervous system. Interestingly, we observed abnormal spermatogenesis leading to male infertility. These findings indicate that Vps13a KO mice are valuable models for studying male infertility and some hematological aspects of ChAc.

Volume microscopic analysis of membrane contact sites in mouse kidney renal proximal tubule epithelial cells

Subcellular organelles communicate with one another and with the plasma membrane (PM). A wide variety of membrane contact sites (MCS) tether subcellular structures to one another and mediate inter-organelle communication and exchange. The nature, distribution and function of membrane contact sites within the segment-specific architecture of renal epithelial cells has only just begun to be elucidated. We have used advanced imaging techniques, including Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and machine learning-based segmentation, to create a comprehensive, high resolution, 3D and quantifiable volume microscopic atlas of the ultrastructure of proximal tubule epithelial cells of the mouse kidney. We applied this data set to identify and quantify MCS between the endoplasmic reticulum and mitochondria and the PM in these cells. FIB-SEM was performed on a 50 μm by 50 μm by 44 μm block of tissue prepared from the renal cortex of a perfusion fixed 12-week-old male mouse. OsO4-stained tissue was embedded in Durcupan and a 3D X-ray tomogram of the block was used to select a volume of interest that included extended lengths of multiple proximal tubule (PT). The volume was imaged by a customized enhanced FIB-SEM system. The X-Y scanning resolution was set to 4-nm, and 4-nm FIB milling was used to expose surfaces for successive imaging. Generation of the complete image stack required ~11,000 cycles of scanning/FIB ablation over ~4 weeks of uninterrupted imaging. Segmentation of the resultant image set was performed with Zeiss Arivis Pro technology. The boundary box for reconstruction had the dimensions of 29.2 μm x 24.8 μm x 8 μm with a volume of 5793.280 μm3 comprising of 2000 consecutive images at an isotropic voxel size of 4 nm. Deep learning segmentation teased apart 10 cells with distinct boundaries in the PT structure. The volume of the cells in our clipped volume ranged from 8.79 μm3 to 1438.53 μm3 with a median of 45.20 μm3. In one representative proximal tubule cell the volume of mitochondria makes up 84.91 μm3 (31.62%) of the cell volume and the volume of Endoplasmic Reticulum (ER) makes up 23.92 μm3 (8.91%) of the cell volume. The 3D reconstruction and quantitative analysis revealed that there is one dominant ER object accounting for most of the ER volume in the whole cell: i.e., the largest ER object takes up 22.37 μm3 or 93.5% of the ER volume in the cell. A post deep-learning processing algorithm used 3D object modifications to identify 11,109 3D MCS between mitochondria and ER in the 10 cells. The Mito-ER MCS ranged from 512 nm3 to 354,304 nm3 with a median volume of 1792 nm3. Visually we can see Mito-ER and Mito-PM MCS extending across a region of up to ~45 nm between the two organelles. Taken together, these data show that the basal lateral surfaces of PT cell PMs are generously invested with fenestrated smooth ER, as are mitochondria and peroxisomes. Extensive regions of direct contact between mitochondria and PM are also observed. The biochemical identities of these contacts and their physiological functions are being actively pursued. NIDDK DK072612 NIDDK DK120534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Emerging functions of the mitochondria-ER-lipid droplet three-way junction in coordinating lipid transfer, metabolism, and storage in cells.

Over the past two decades, we have witnessed a growing appreciation for the importance of membrane contact sites (CS) in facilitating direct communication between organelles. CS are tiny regions where the membranes of two organelles meet but do not fuse and allow the transfer of metabolites between organelles, playing crucial roles in the coordination of cellular metabolic activities. The significant advancements in imaging techniques and molecular and cell biology research have revealed that CS are more complex than what originally thought, and as they are extremely dynamic, they can remodel their shape, composition, and functions in accordance with metabolic and environmental changes and can occur between more than two organelles. Here, we describe how recent studies led to the identification of a three-way mitochondria-ER-lipid droplet CS and discuss the emerging functions of these contacts in maintaining lipid storage, homeostasis, and balance. We also summarize the properties and functions of key protein components localized at the mitochondria-ER-lipid droplet interface, with a special focus on lipid transfer proteins. Understanding tripartite CS is essential for unraveling the complexities of inter-organelle communication and cooperation within cells.

MCSdb, a database of proteins residing in membrane contact sites

Organelles do not act as autonomous discrete units but rather as interconnected hubs that engage in extensive communication by forming close contacts called “membrane contact sites (MCSs)”. And many proteins have been identified as residing in MCS and playing important roles in maintaining and fulfilling specific functions within these microdomains. However, a comprehensive compilation of these MCS proteins is still lacking. Therefore, we developed MCSdb, a manually curated resource of MCS proteins and complexes from publications. MCSdb documents 7010 MCS protein entries and 263 complexes, involving 24 organelles and 44 MCSs across 11 species. Additionally, MCSdb orchestrates all data into different categories with multitudinous information for presenting MCS proteins. In summary, MCSdb provides a valuable resource for accelerating MCS functional interpretation and interorganelle communication deciphering.

Viral regulation of organelle membrane contact sites.

At the core of organelle functions lies their ability and need to form dynamic organelle-organelle networks that drive intracellular communication and coordination of cellular pathways. These networks are facilitated by membrane contact sites (MCSs) that promote both intra-organelle and inter-organelle communication. Given their multiple functions, MCSs and the proteins that form them are commonly co-opted by viruses during infection to promote viral replication. This Essay discusses mechanisms acquired by diverse human viruses to regulate MCS functions in either proviral processes or host defense. It also examines techniques used for examining MCSs in the context of viral infections.

Combinatorial selective ER-phagy remodels the ER during neurogenesis

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells.

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.

Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Mitochondria-derived vesicles: potential nano-batteries to recharge the cellular powerhouse.

Mitochondria dysfunction is increasingly recognized as a critical factor in various pathogenic processes. The mechanism governing mitochondrial quality control serves as an adaptive response, ensuring the preservation of mitochondrial morphology, quantity, and overall function, crucial for cell survival. The generation of mitochondria-derived vesicles (MDVs) is one of the processes of mitochondrial quality control. Recent literature has suggested MDV heterogeneity; however, the detailed characteristics of various MDV subtypes still need to be studied better. Recent studies have shown that MDVs also play a role in inter-organelle communication for mitochondria besides quality control. For instance, Hazan et al. demonstrated that functional mitochondria from Saccharomyces cerevisiae release vesicles independent of the fission machinery. These vesicles, falling within the typical size range of MDVs, were selectively loaded with mitochondrial proteins, especially with functional ATP synthase subunits. Intriguingly, these MDVs maintained membrane potential and could generate ATP. Moreover, MDVs could fuse with naïve mitochondria, transferring their ATP generation machinery. Lastly, this study revealed a potential delivery mechanism of ATP-producing vesicles, presenting a promising avenue to rejuvenate ATP-deficient mitochondria. Overall, this study unveils a novel mechanism for inter-organelle communication by vesicles, which is crucial for maintaining cellular homeostasis and could also be important in pathological conditions.

C. elegans Presenilin Mediates Inter-Organelle Contacts and Communication that Is Required for Lysosome Activity.

Compromised lysosome function is implicated in the pathology of many neurodegenerative diseases, including Alzheimer's disease (AD). Familial Alzheimer's disease (fAD) is caused primarily by mutations in the presenilin encoding genes, but the underlying mechanism remains obscure. Loss of the conserved C. elegans presenilin orthologue SEL-12 results in increased mitochondrial calcium, which promotes neurodegeneration. Here, we find that sel-12 mutant lysosomes, independent of SEL-12 proteolytic activity, are significantly enlarged and more alkaline due to increased ER-to-mitochondrial calcium signaling and concomitant mitochondrial oxidative stress. These defects and their dependence on mitochondrial calcium are recapitulated in human fAD fibroblasts, demonstrating a conserved role for mitochondrial calcium in presenilin-mediated lysosome dysfunction. sel-12 mutants also have increased contact surface area between the ER, mitochondria, and lysosomes, suggesting sel-12 has an additional role in modulating organelle contact and communication. Overall, we demonstrate that SEL-12 maintains lysosome acidity and lysosome health by controlling ER-to-mitochondrial calcium signaling.

ER CALCIUM SIGNALING COORDINATES MITOCHONDRIAL DYNAMICS AND METABOLIC FUNCTION TO PROMOTE LONGEVITY

Abstract Inter-organelle communication is a critical determinant of cellular metabolic homeostasis, and defects in the interactions between organelles are increasingly correlated with age-dependent diseases. Despite these promising links, exploration of how inter-organelle signaling pathways can be targeted to promote healthy organismal aging is a nascent area of research. We have established a foundation in C. elegans to investigate how the communication between endoplasmic reticulum (ER) and mitochondria, two of the most central metabolic hubs in cells, controls the aging process. Combining electron microscopy, live-imaging and biochemical approaches, we provide evidence that C. elegans forms ER-mitochondrial contacts and that through molecular mechanisms conserved to mammals, ER calcium flux potently stimulates mitochondrial bioenergetics. Furthermore, genetic manipulation of the ER inositol triphosphate receptor (InsP3R) reveals that ER calcium signaling promotes mitochondrial homeostasis at multiple other levels, including mitochondrial gene expression and dynamics. Building on this foundation, we now reveal that ER calcium signaling is essential for longevity in contexts of mitochondrial reprogramming. Surprisingly, InsP3R-stimulated mitochondrial bioenergetics do not appear to be the key mechanism of longevity in these contexts, but rather the underappreciated roles of the ER in tuning mitochondrial gene expression and morphology. Our results thus highlight the InsP3R as a potent and central regulator of diverse mitochondrial properties and reveal that the connections between ER and mitochondria are deeper and more complex than current models suggest. Overall, we reveal that inter-organelle communication between ER and mitochondria is an essential mechanism of longevity in paradigms involving mitochondrial reprogramming.

DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress

Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages. Solution structure studies affirm the interaction between the Diaphanous Inhibitory Domain and the cytosolic GTPase domain of MFN2. In male rodent and human cardiomyocytes, DIAPH1-MFN2 interaction regulates mitochondrial turnover, mitophagy, and oxidative stress. Introduction of synthetic linker construct, which shorten the mitochondria-SR/ER distance, mitigated the molecular and functional benefits of DIAPH1 silencing in ischemia. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of mitochondria-SR/ER contact networks. We propose that targeting pathways that regulate DIAPH1-MFN2 interactions may facilitate recovery from tissue ischemia.