Intermitochondrial Junctions Research Articles

Dysregulated inter-mitochondrial crosstalk in glioblastoma cells revealed by in situ cryo-electron tomography

Glioblastomas (GBMs) are the most lethal primary brain tumors with limited survival, even under aggressive treatments. The current therapeutics for GBMs are flawed due to the failure to accurately discriminate between normal proliferating cells and distinctive tumor cells. Mitochondria are essential to GBMs and serve as potential therapeutical targets. Here, we utilize cryo-electron tomography to quantitatively investigate nanoscale details of randomly sampled mitochondria in their native cellular context of GBM cells. Our results show that compared with cancer-free brain cells, GBM cells own more inter-mitochondrial junctions of several types for communications. Furthermore, our tomograms unveil microtubule-dependent mitochondrial nanotunnel-like bridges in the GBM cells as another inter-mitochondrial structure. These quantified inter-mitochondrial features, together with other mitochondria-organelle and intra-mitochondrial ones, are sufficient to distinguish GBM cells from cancer-free brain cells under scrutiny with predictive modeling. Our findings decipher high-resolution inter-mitochondrial structural signatures and provide clues for diagnosis and therapeutic interventions for GBM and other mitochondria-related diseases.

Inhibition of mitoNEET induces Pink1-Parkin-mediated mitophagy

MitoNEET, a mitochondrial outer membrane protein containing the Asn-Glu-Glu-Thr (NEET) sequence, controls the formation of intermitochondrial junctions and confers autophagy resistance. Moreover, mitoNEET as a mitochondrial substrate undergoes ubiquitination by activated Parkin during the initiation of mitophagy. Therefore, mitoNEET is linked to the regulation of autophagy and mitophagy. Mitophagy is the selective removal of the damaged or unnecessary mitochondria, which is crucial to sustaining mitochondrial quality control. In numerous human diseases, the accumulation of damaged mitochondria by impaired mitophagy has been observed. However, the therapeutic strategy targeting of mitoNEET as a mitophagy-enhancing mediator requires further research. Herein, we confirmed that mitophagy is indeed activated by mitoNEET inhibition. CCCP (carbonyl cyanide m-chlorophenyl hydrazone), which leads to mitochondrial depolarization, induces mitochondrial dysfunction and superoxide production. This, in turn, contributes to the induction of mitophagy; mitoNEET protein levels were initially increased before an increase in LC3-Ⅱ protein following CCCP treatment. Pharmacological inhibition of mitoNEET using mitoNEET Ligand-1 (NL-1) promoted accumulation of Pink1 and Parkin, which are mitophagy-associated proteins, and activation of mitochondria–lysosome crosstalk, in comparison to CCCP alone. Inhibition of mitoNEET using NL-1, or mitoNEET shRNA transfected into RAW264.7 cells, abrogated CCCP-induced ROS and mitochondrial cell death; additionally, it activated the expression of PGC-1α and SOD2, regulators of oxidative metabolism. In particular, the increase in PGC-1α, which is a major regulator of mitochondrial biogenesis, promotes mitochondrial quality control. These results indicated that mitoNEET is a potential therapeutic target in numerous human diseases to enhance mitophagy and protect cells by maintaining a network of healthy mitochondria.

Renovascular Hypertension Induces Myocardial Mitochondrial Damage, Contributing to Cardiac Injury and Dysfunction in Pigs With Metabolic Syndrome.

Renovascular hypertension (RVH) often manifest with metabolic syndrome (MetS) as well. Coexisting MetS and hypertension increases cardiovascular morbidity and mortality, but the mechanisms underlying cardiac injury remain unknown. We hypothesized that superimposition of MetS induces myocardial mitochondrial damage, leading to cardiac injury and dysfunction in swine RVH. Pigs were studied after 16 weeks of diet-induced MetS with or without RVH (unilateral renal artery stenosis), and Lean controls (n = 6 each). Systolic and diastolic cardiac function were assessed by multidetector CT, and cardiac mitochondrial morphology (electron microscopy) and myocardial function in tissue and isolated mitochondria. Body weight was similarly higher in MetS groups vs. Lean. RVH groups achieved significant stenosis and developed hypertension. Mitochondrial matrix density and adenosine triphosphate production were lower and H2O2 production higher in RVH groups vs. Lean and MetS. Lean + RVH (but not MetS + RVH) activated mitophagy, which was associated with decreased myocardial expression of mitophagy-related microRNAs. MetS groups exhibited higher numbers of intermitochondrial junctions, which could have prevented membrane depolarization/activation of mitophagy in MetS + RVH. Cardiac fibrosis, hypertrophy (increased left ventricular muscle mass), and diastolic function (decreased E/A ratio) were greater in MetS + RVH vs. Lean + RVH. MetS+RVH induces myocardial mitochondrial damage and dysfunction. MetS + RVH failed to activate mitophagy, resulting in greater cardiac remodeling, fibrosis, and diastolic dysfunction. Mitochondrial injury and impaired mitophagy may constitute important mechanisms and therapeutic targets to ameliorate cardiac damage and dysfunction in patients with coexisting MetS and RVH.

Ultraviolet irradiation alters the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions in copepod myocytes

The efficient production of energy via oxidative phosphorylation is essential to the growth, survival, and reproduction of eukaryotes. The behavior (position of, and communication between, mitochondria) and morphology of mitochondria play key roles in efficient energy production and are influenced by oxidative stressors such as ultraviolet (UV) radiation. We tested the hypothesis that mitochondria change their behavior and morphology to meet energetic demands of responding to changes in oxidative stress. Specifically, we predicted that UV irradiation would increase the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions to influence whole-animal metabolic rate. Using transmission electron microscopy, we found that both three and six hours of UV-A/B irradiation (0.5 W/m2) increased the proportion of inter-mitochondrial junctions (with increasing mitochondrial aspect ratio) and the density of inner mitochondrial membrane in myocytes of Tigriopus californicus copepods. Mitochondrial density increased following both irradiation treatments, but mitochondrial size decreased under the six hour treatment. Metabolic rate was maintained under three hours of irradiation but decreased following six hours of exposure. These observations demonstrate that the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions can play formative roles in maintaining whole-animal metabolic rate, and ultimately organismal performance, under exposure to an oxidative stressor.

Coordinated development of the mitochondrial reticulum in skeletal muscle

Skeletal muscle cells form a mitochondrial network, or reticulum, in a fiber‐type specific manner with grid‐like and fiber‐perpendicular mitochondrial network configurations found in oxidative and glycolytic fibers, respectively. However, it is not well characterized how the networks form during postnatal muscle development. Using focused ion beam scanning electron microscopy (FIB‐SEM) and machine learning based 3D image segmentation and analysis, we have sought to understand the 3D mitochondrial development in oxidative (soleus) and glycolytic (gastrocnemius) muscles of mice at different postnatal days 1 (P1), 14 (P14), and 42 (P42). Both oxidative and glycolytic fibers show tortuous, parallel mitochondrial formations at P1. At P14, oxidative fibers have more elongated, fiber‐parallel mitochondria, while glycolytic fibers have a more grid‐like mitochondrial reticulum. To better understand the intra‐ and inter‐organelle interactions of the mitochondrial reticulum during development, we additionally assessed intermitochondrial junctions (IMJ) and mitochondrial interactions with lipid droplets (LD). During early maturation (P1‐P14), many IMJ in oxidative fibers are arranged in both diagonal (~40%) and parallel (~40%) directions, yet matured oxidative fibers have almost half (~50%) of IMJ in the perpendicular direction. In contrast, after having ~50% of IMJ along the parallel axis at P1, glycolytic fibers gradually change IMJ orientations more into both perpendicular and diagonal axis and IMJ are located in all directions at P42. Although mitochondria in glycolytic fibers maintain similar sizes of the IMJ contact sites between P1 and P14 and have smaller areas at P42, oxidative fibers have ~2‐fold larger IMJ areas at P14 as compared to early (P1) and matured (P42) fibers. Meanwhile, both oxidative and glycolytic fibers at P1 have many, big LD that sharply decrease in size during the course of postnatal development. In the matured muscles, oxidative fibers have few, tiny LD and glycolytic fibers have no LD. Both fibers maintain similar LD contract areas per mitochondrion across P1‐P42. Notably, mitochondria directly in contact with LD have volumes ~2–3‐fold larger than non‐LD‐contacted mitochondria at all time points. In addition, the LD‐contacted mitochondria have ~2‐fold bigger IMJ areas as compared to non‐contacted one, with the largest differences observed at P14. In conclusion, development of the muscle mitochondrial reticulum occurs in coordination with altered interactions among mitochondria and with LD. However, future studies are warranted to study possible interactive roles of calcium handling structures such as sarcoplasmic reticulum and t‐tubules.Support or Funding InformationThis study is supported by intramural programs within the National Heart, Lung, and Blood Institute and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (grant 1ZIAHL006221‐02).

Mitochondrial behaviour, morphology, and animal performance.

We have a limited understanding of the proximate mechanisms that are responsible for the development of variation in animal performance and life-history strategies. Provided that components of an organism's successful life history - for example, mate competition, gestation, lactation, etc. - are energetically demanding, increased energy production within mitochondria is likely the foundation from which organisms are able to perform these tasks. Mitochondrial behaviour (positioning within the cell and communication between mitochondria) and morphology affect variation in energy production at the molecular, cellular, and organismal levels. Therefore, adaptations in mitochondrial behaviour and morphology that favour efficient energy production likely influence variation in animal performance. Previous work has linked greater proportions of inter-mitochondrial junctions and density of the inner mitochondrial membrane, among other traits, with increased energetic demand. Future research should focus on how inter-mitochondrial junctions and morphology of the inner mitochondrial membrane, in particular, influence animal performance in accordance with mitochondrial density, fission, and fusion.

Intracardiac administration of ephrinA1-Fc preserves mitochondrial bioenergetics during acute ischemia/reperfusion injury

AimsIntracardiac injection of recombinant EphrinA1-Fc immediately following coronary artery ligation in mice reduces infarct size in both reperfused and non-reperfused myocardium, but the cellular alterations behind this phenomenon remain unknown. Main methodsHerein, 10 wk-old B6129SF2/J male mice were exposed to acute ischemia/reperfusion (30minI/24hrsR) injury immediately followed by intracardiac injection of either EphrinA1-Fc or IgG-Fc. After 24 h of reperfusion, sections of the infarct margin in the left ventricle were imaged via transmission electron microscopy, and mitochondrial function was assessed in both permeabilized fibers and isolated mitochondria, to examine mitochondrial structure, function, and energetics in the early stages of repair. Key findingsAt a structural level, EphrinA1-Fc administration prevented the I/R-induced loss of sarcomere alignment and mitochondrial organization along the Z disks, as well as disorganization of the cristae and loss of inter-mitochondrial junctions. With respect to bioenergetics, loss of respiratory function induced by I/R was prevented by EphrinA1-Fc. Preservation of cardiac bioenergetics was not due to changes in mitochondrial JH2O2 emitting potential, membrane potential, ADP affinity, efficiency of ATP production, or activity of the main dehydrogenase enzymes, suggesting that EphrinA1-Fc indirectly maintains respiratory function via preservation of the mitochondrial network. Moreover, these protective effects were lost in isolated mitochondria, further emphasizing the importance of the intact cardiomyocyte ultrastructure in mitochondrial energetics. SignificanceCollectively, these data suggest that intracardiac injection of EphrinA1-Fc protects cardiac function by preserving cardiomyocyte structure and mitochondrial bioenergetics, thus emerging as a potential therapeutic strategy in I/R injury.

Abstract 556: Intramyocardial EphrinA1-Fc Preserves Mitochondrial Structure and Bioenergetics Post-Myocardial Infarction

EphrinA1, a receptor tyrosine kinase ligand expressed in the plasma membrane of healthy cardiomyocytes, is reduced following acute myocardial ischemia (I)/reperfusion (R) injury in mice. Intramyocardial injection of recombinant EphrinA1-Fc at the time of LAD ligation attenuated infarct injury by 46% and completely preserved cardiac function in I/R (30min I/24hrs R). This was accompanied by reduced inflammation, attenuation of apoptosis, enhanced autophagy, and modulation of metabolic pathways that promote more efficient energy utilization. To examine the mechanism by which this occurs, mitochondrial structure and function was examined in 10 wk-old B6129SF2/J male mice given intramyocardial EphrinA1-Fc or IgG-Fc at LAD occlusion and subsequently reperfused for 24hrs. Permeabilized cardiac muscle fibers were isolated from a portion of the infarct margin of the left ventricle to assess mitochondrial function and energetics. EphrinA1-Fc prevented the 30% loss (p<0.005) in mitochondrial respiratory capacity in the absence of changes in H 2 O 2 emitting potential. Moreover, using a titrated creatine kinase energetic clamp, we found that EphrinA1-Fc improved the mitochondrial thermodynamic force-flow relationship by increasing OXPHOS conductance (+55% vmax, p<0.005), without changes in ADP affinity. Another small portion of the infarct margin was fixed and processed for transmission electron microscopy imaging which revealed that EphrinA1-Fc treatment prevented the MI-induced loss of sarcomere alignment and mitochondrial organization along the Z disks, as well as disorganization of the cristae. There was also significantly less lipid droplet accumulation and preservation of intermitochondrial junctions. Morphometric analyses of mitochondria are in progress as are Western blot assays to identify the proteins involved. Myocardial strain analyses are also underway to explore and pinpoint regional changes in contractile function. Collectively, these data demonstrate that intramyocardial injection of EphrinA1-Fc attenuates I/R injury by preserving the mitochondrial network structure and bioenergetic capacity. The robust cardioprotection afforded by ephrinA1-Fc offers promise as a novel therapeutic for the treatment of acute MI.

Creatine-Kinase Shuttle and Rapid Mitochondrial Membrane Potential Conductivity are Needed Simultaneously to Maintain Uniform Metabolite Distributions in the Cardiac Cell Contraction Cycle

Cardiac muscle cells are reported to contain a cell wide mitochondrial reticulum where mitochondria are interconnected in networks though intermitochondrial junctions. We have also observed that mitochondria form heterogeneous clusters within localised regions of the cell. Recent studies suggest that conduction of membrane potential through the mitochondrial networks can act as one of the dominant mechanisms to distribute energy across the cell. There are several other myofibril based metabolite diffusion systems present in cardiomyocytes which can also contribute to intracellular energy transport. What is the interplay between these different mechanisms and the heterogeneous distribution of mitochondria? We are developing detailed finite element models of cardiac ultrastructure to investigate the role that mitochondrial membrane potential, oxygen and the creatine-kinase shuttle have in homogenizing ATP supply and demand within a heterogeneous cellular architecture. Our results show that diffusion of O2 is insufficient to reach interior of a cardiomyocyte in a high workload - leading to a regional hypoxia at the core of the cell. Our simulations also indicate that rapid diffusion of creatine and phosphocreatine may be still be required to ensure that metabolites like ATP and ADP are uniformly distributed in the midst of a heterogeneous distribution of mitochondria that we observe in the cell. However, our current model simulations suggest that conduction of membrane potential from the mitochondria at the cell periphery can provide sufficient protonomotive force to the mitochondria at the cell core to sustain a uniform distribution of metabolites and force dynamics across the cell cross section.

MitoNEET-dependent formation of intermitochondrial junctions

MitoNEET (mNEET) is a dimeric mitochondrial outer membrane protein implicated in many facets of human pathophysiology, notably diabetes and cancer, but its molecular function remains poorly characterized. In this study, we generated and analyzed mNEET KO cells and found that in these cells the mitochondrial network was disturbed. Analysis of 3D-EM reconstructions and of thin sections revealed that genetic inactivation of mNEET did not affect the size of mitochondria but that the frequency of intermitochondrial junctions was reduced. Loss of mNEET decreased cellular respiration, because of a reduction in the total cellular mitochondrial volume, suggesting that intermitochondrial contacts stabilize individual mitochondria. Reexpression of mNEET in mNEET KO cells restored the WT morphology of the mitochondrial network, and reexpression of a mutant mNEET resistant to oxidative stress increased in addition the resistance of the mitochondrial network to H2O2-induced fragmentation. Finally, overexpression of mNEET increased strongly intermitochondrial contacts and resulted in the clustering of mitochondria. Our results suggest that mNEET plays a specific role in the formation of intermitochondrial junctions and thus participates in the adaptation of cells to physiological changes and to the control of mitochondrial homeostasis.

Power Grid Protection of the Muscle Mitochondrial Reticulum

Mitochondrial network connectivity enables rapid communication and distribution of potential energy throughout the cell. However, this connectivity putsthe energy conversion system at risk, because damaged elements could jeopardize the entire network. Here, we demonstrate the mechanisms for mitochondrial network protection in heart and skeletal muscle (SKM). We find that the cardiac mitochondrial reticulum is segmented into subnetworks comprising many mitochondria linked through abundant contact sites at highly specific intermitochondrial junctions (IMJs). In both cardiac and SKM subnetworks, a rapid electrical and physical separation of malfunctioning mitochondriaoccurs, consistent with detachment of IMJs andretraction of elongated mitochondria into condensed structures. Regional mitochondrial subnetworks limit the cellular impact of local dysfunction while the dynamic disconnection of damaged mitochondria allows the remaining mitochondria toresume normal function within seconds. Thus, mitochondrial network security is comprised of both proactive and reactive mechanisms in striated muscle cells.

Trans-mitochondrial coordination of cristae at regulated membrane junctions.

Reminiscent of bacterial quorum sensing, mammalian mitochondria participate in inter-organelle communication. However, physical structures that enhance or enable interactions between mitochondria have not been defined. Here we report that adjacent mitochondria exhibit coordination of inner mitochondrial membrane cristae at inter-mitochondrial junctions (IMJs). These electron-dense structures are conserved across species, resistant to genetic disruption of cristae organization, dynamically modulated by mitochondrial bioenergetics, independent of known inter-mitochondrial tethering proteins mitofusins and rapidly induced by the stable rapprochement of organelles via inducible synthetic linker technology. At the associated junctions, the cristae of adjacent mitochondria form parallel arrays perpendicular to the IMJ, consistent with a role in electrochemical coupling. These IMJs and associated cristae arrays may provide the structural basis to enhance the propagation of intracellular bioenergetic and apoptotic waves through mitochondrial networks within cells.

Electron microscope analysis of cardiomyocytes in the rat left ventricle under simulation of weightlessness effects and artificial gravitation

Electron microscopic study of left ventricle cardiomyocytes and quantitative analysis of their mitochondriom was performed in rats exposed to tail-suspension, as a model of weightlessness effects, to artificial gravity produced by intermittent 2G centrifugation and a combination of these effects. It was found that the cardiomyocytes ultrastructure changed slightly after tail-suspension and after intermittent 2G influence, as well as under a combination of these effects. However, the number of intermitochondrial junctions increased significantly in the interfibrillar zone of cardiomyocytes under a combination of tail-suspension and intermittent 2G influence, which agrees with the cell hypertrophy described earlier.

Changes in mitochondria and inter-mitochondrial junctions of the rat cardiomyocytes upon adrenergic stimulation by isoproterenol

Changes in rat cardiomyocytes and their mitochondria and intermitochondrial junctions (IMJs) upon beta-adrenoreceptor stimulation with isoproterenol were studied by the methods of light and electron microscopy and computer-aided morphometry. It was found that isoproterenol injections (0.3 mg/kg for eight days) resulted in myocardial hypertrophy, which was more pronounced in the right than in the left ventricle. In the hypertrophied cardiomyocytes of both ventricles, an adaptive response of mitochondria was observed: their ultrastructure, size, and number changed, and the number and average length of IMJs increased. A positive correlation between the degree of cell hypertrophy and the number of IMJs was revealed. The reactive properties of mitochondria, including IMJ formation, differed depending on their location in the cell (i.e., in the paranuclear, intermyofibrillar, or subsarcolemmal regions). These results suggest that the rates and intensities of adaptive compensatory processes developing in the mitochondria of cardiomyocytes exposed to beta-adrenoreceptor stimulation differ in the left and right ventricles.

Morphometric and Ultrastructural Characteristics of the Mitochondrial System of Cardiomyocytes in the Rat Left Ventricle under Conditions of Hypergravity

For the first time, electron-microscopic morphometric analysis of the mitochondrial system in cardiomyocytes of the left ventricle was performed in rats exposed to hypergravity (2 g). After five days of exposure, the number of long mitochondria sharply increased in the interfibrillar zone of cardiomyocytes. The numbers of intermitochondrial junctions (IMJs) were increased in all zones of mitochondria localization. The ultrastructure and numerical density of mitochondria remained within the normal limits. The same picture was revealed on day 19 (the end of exposure), but the numbers of IMJs in the perinuclear and subsarcolemmal pericapillary zones were lower than on day 5. One months after the end of 19-day exposure at 2 g, the test parameters of the mitochondrial system did not return to the norm. Apparently, this is why repeated exposure to hypergravity (2 g for five days after 30-day rest) failed to evoke a similar response from the mitochondrial system of cardiomyocytes.

Ultrastructure of intermitochondrial junctions in glomus cells : Identification of proteic material

Ultrastructure of intermitochondrial junctions in glomus cells : Identification of proteic material

Intermitochondrial junctions in chemoreceptor cells and lymphocytes: Ultrastructural and experimental investigations

Intermitochondrial junctions in chemoreceptor cells and lymphocytes: Ultrastructural and experimental investigations

Ultrastructural studies of spermatogenesis in Antalis entalis (Scaphopoda, Mollusca)

Mature spermatozoa and spermatogenesis of Antalis entalis were studied by light, scanning and transmission electron microscopy. During spermatogenesis the spherical proacrosome arises from a single Golgi complex, migrates toward the anterior pole of the spermatid, and there differentiates to form an electron-dense collar. This is unique among examples of the simple acrosome of primitive spermatozoa. Nuclear condensation does not occur until the proacrosome and mitochondria migrate towards the future anterior and posterior poles of the spermatid. Chromatin condensation occurs by the formation of heterochromatin granules. No manchette of microtubules is observed in the course of nuclear condensation and elongation. Mitochondria achieve their mature spherical shape by decreasing in number and increasing in diameter at an early stage of spermiogenesis. No inter-mitochondrial or mitochondrianuclear (or centriolar) junctions occur. The centriolar satellite is formed in association with the distal centriole late in spermiogenesis. Earlier, the typical ‘9 + 2 ’ microtubular axoneme is coiled within the spermatid cytoplasm and the flagellum emerges only late in spermiogenesis. Beta glycogen granules appear late in the development of the spermatid, and are distributed in the centriolar and periacrosomal regions.

Intermitochondrial junctions in a subpopulation of peripheral blood lymphocytes from healthy subjects*

This paper reports the observation of intermitochondrial junctions (IMJ) with a periodicity of 15.4 +/- 0.65 nm in peripheral blood lymphocytes of healthy subjects. The percentage of IMJ-containing cells appears constant (about 10% of examined lymphocytes) and is independent of the delay of fixation. Immunogold staining reveals that lymphocytes with IMJ exhibit a T-phenotype. IMJ have been reported in other types of tissues but, to our knowledge, have not been previously described in the blood cell. The signification of these structures is discussed.

Intermitochondrial junctions in the heart of the frog, Rana esculenta. A thin-section and freeze-fracture study.

In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4 +/- 0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3 +/- 0.29 nm. There are 65.3 +/- 2 periodic structures per micron of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11 +/- 0.3 nm thick and are separated from each other by 16.5 +/- 0.46 nm, their density being 65 +/- 2.28 per micron of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.