RESUMEN
Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. Here, we report a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. We establish the mitochondrial motor-adaptor protein Milton as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. We find that the GlcNAcylation state of Milton is altered by extracellular glucose and that OGT alters mitochondrial motility in vivo. Our findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Glucosa/metabolismo , Mitocondrias/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Axones/metabolismo , Proteínas Portadoras , Drosophila melanogaster , Técnicas de Silenciamiento del Gen , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , N-Acetilglucosaminiltransferasas/genética , Ratas , Alineación de SecuenciaRESUMEN
Point-scanning imaging systems are among the most widely used tools for high-resolution cellular and tissue imaging, benefiting from arbitrarily defined pixel sizes. The resolution, speed, sample preservation and signal-to-noise ratio (SNR) of point-scanning systems are difficult to optimize simultaneously. We show these limitations can be mitigated via the use of deep learning-based supersampling of undersampled images acquired on a point-scanning system, which we term point-scanning super-resolution (PSSR) imaging. We designed a 'crappifier' that computationally degrades high SNR, high-pixel resolution ground truth images to simulate low SNR, low-resolution counterparts for training PSSR models that can restore real-world undersampled images. For high spatiotemporal resolution fluorescence time-lapse data, we developed a 'multi-frame' PSSR approach that uses information in adjacent frames to improve model predictions. PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed and sensitivity. All the training data, models and code for PSSR are publicly available at 3DEM.org.
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Aprendizaje Profundo , Algoritmos , Microscopía Electrónica/métodos , Relación Señal-RuidoRESUMEN
Remodelling neuronal connections by synaptic activity requires membrane trafficking. We present evidence for a signalling pathway by which synaptic activity and its consequent Ca(2+) influx activate the small GTPase Ral and thereby recruit exocyst proteins to postsynaptic zones. In accord with the ability of the exocyst to direct delivery of post-Golgi vesicles, constitutively active Ral expressed in Drosophila muscle causes the exocyst to be concentrated in the region surrounding synaptic boutons and consequently enlarges the membrane folds of the postsynaptic plasma membrane (the subsynaptic reticulum, SSR). SSR growth requires Ral and the exocyst component Sec5 and Ral-induced enlargement of these membrane folds does not occur in sec5(-/-) muscles. Chronic changes in synaptic activity influence the plastic growth of this membrane in a manner consistent with activity-dependent activation of Ral. Thus, Ral regulation of the exocyst represents a control point for postsynaptic plasticity. This pathway may also function in mammals as expression of activated RalA in hippocampal neurons increases dendritic spine density in an exocyst-dependent manner and increases Sec5 in spines.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Animales , Animales Modificados Genéticamente , Señalización del Calcio , Espinas Dendríticas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Exocitosis , Genes de Insecto , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/metabolismo , Neuronas/metabolismo , Transporte de Proteínas , Ratas , Transducción de Señal , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.
Asunto(s)
Acetilglucosamina , Metabolismo Energético , Mitocondrias , N-Acetilglucosaminiltransferasas , Neuronas , Neuronas/metabolismo , Animales , Mitocondrias/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Acetilglucosamina/metabolismo , Ratones , Hipocampo/metabolismo , Hipocampo/citología , Glucosa/metabolismo , Ratones Endogámicos C57BL , Plasticidad Neuronal/fisiologíaRESUMEN
The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.
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Línea Celular , Proteínas de la Membrana/metabolismo , Distrofia Muscular de Cinturas/genética , Mioblastos/metabolismo , Animales , Proliferación Celular , Disferlina , Rayos Láser/efectos adversos , Proteínas de la Membrana/genética , Ratones , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Compuestos de Piridinio/efectos adversos , Compuestos de Amonio Cuaternario/efectos adversos , Heridas y Lesiones/genéticaRESUMEN
Plate-based quantitative metabolic flux analysis has emerged as the central technology to examine cellular metabolism and mitochondrial bioenergetics. However, accurate interpretation of metabolic activity between different experimental conditions in multi-well microplates requires data normalization based on in situ cell counts. Here, we describe FluxNorm, a platform-independent semi-automated computational workflow, validated for three different cell types, to normalize cell density for accurate assessment of cellular bioenergetics.
RESUMEN
Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-GlcNAc transferase regulates neuronal activity-driven mitochondrial bioenergetics. We show that neuronal activity upregulates O-GlcNAcylation mainly in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven fuel consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.
RESUMEN
Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1's regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1's mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1's O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.
RESUMEN
Mitochondria are vital organelles with distinct morphological features and functional properties. The dynamic network of mitochondria undergoes structural and functional adaptations in response to cell-type-specific metabolic demands. Even within the same cell, mitochondria can display wide diversity and separate into functionally distinct subpopulations. Mitochondrial heterogeneity supports unique subcellular functions and is crucial to polarized cells, such as neurons. The spatiotemporal metabolic burden within the complex shape of a neuron requires precisely localized mitochondria. By travelling great lengths throughout neurons and experiencing bouts of immobility, mitochondria meet distant local fuel demands. Understanding mitochondrial heterogeneity and homeostasis mechanisms in neurons provides a framework to probe their significance to many other cell types. Here, we put forth an outline of the multifaceted role of mitochondria in regulating neuronal physiology and cellular functions more broadly.
Asunto(s)
Mitocondrias , Neuronas , Homeostasis , Mitocondrias/metabolismo , Neuronas/metabolismo , Orgánulos/metabolismoRESUMEN
Mitochondrial movement and distribution are fundamental to their function. Here we report a mechanism that regulates mitochondrial movement by anchoring mitochondria to the F-actin cytoskeleton. This mechanism is activated by an increase in glucose influx and the consequent O-GlcNAcylation of TRAK (Milton), a component of the mitochondrial motor-adaptor complex. The protein four and a half LIM domains protein 2 (FHL2) serves as the anchor. FHL2 associates with O-GlcNAcylated TRAK and is both necessary and sufficient to drive the accumulation of F-actin around mitochondria and to arrest mitochondrial movement by anchoring to F-actin. Disruption of F-actin restores mitochondrial movement that had been arrested by either TRAK O-GlcNAcylation or forced direction of FHL2 to mitochondria. This pathway for mitochondrial immobilization is present in both neurons and non-neuronal cells and can thereby adapt mitochondrial dynamics to changes in glucose availability.
Asunto(s)
Actinas/metabolismo , Glucosa/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Mitocondrias/metabolismo , Proteínas Musculares/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Humanos , Dinámicas Mitocondriales , RatasRESUMEN
The movement of intracellular cargo, such as transcripts, proteins, and organelles, is fundamental to cellular function. Neurons, due to their long axons and dendrites, are particularly dependent on proper intracellular trafficking and vulnerable to defects in the movement of intracellular cargo that are noted in neurodegenerative and neurodevelopmental disorders. Accurate quantification of intracellular transport is therefore needed for studying the mechanisms of cargo trafficking, the influence of mutations, and the effects of potentially therapeutic pharmaceuticals. In this article, we introduce an algorithm called "Kymolyzer." The algorithm can quantify intracellular trafficking along a defined path, such as that formed by the aligned microtubules of axons and dendrites. Kymolyzer works as a semi-autonomous kymography software application. It constructs and analyzes kymographs to measure the movement and distribution of fluorescently tagged objects along a user-defined path. The algorithm can be used under a wide variety of experimental conditions and can extract a diverse array of motility parameters describing intracellular movement, including time spent in motion, percentage of objects in motion, percentage of objects that are stationary, and velocities of motile objects. This article serves as a user manual describing the design of Kymolyzer, providing a stepwise protocol for its use and illustrating its functions with common examples. © 2020 Wiley Periodicals LLC Basic Protocol: Kymolyzer, a semi-autonomous kymography tool to analyze intracellular motility.
Asunto(s)
Transporte Biológico/fisiología , Quimografía , Microtúbulos/metabolismo , Orgánulos/metabolismo , Algoritmos , Animales , Axones/metabolismo , Movimiento Celular/fisiología , Quimografía/métodos , Transporte de Proteínas/fisiología , Programas InformáticosRESUMEN
Neurons are highly polarized cells with extraordinary energy demands, which are mainly fulfilled by mitochondria. In response to altered neuronal energy state, mitochondria adapt to enable energy homeostasis and nervous system function. This adaptation, also called mitochondrial plasticity, can be observed as alterations in the form, function and position. The primary site of energy consumption in neurons is localized at the synapse, where mitochondria are critical for both pre- and postsynaptic functions. In this review, we will discuss molecular mechanisms regulating mitochondrial plasticity at the synapse and how they contribute to information processing within neurons.
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Mitocondrias , Sinapsis , Homeostasis , Plasticidad Neuronal , NeuronasRESUMEN
Eukaryotic cells modulate their metabolism by organizing metabolic components in response to varying nutrient availability and energy demands. In rat axons, mitochondria respond to glucose levels by halting active transport in high glucose regions. We employ quantitative modeling to explore physical limits on spatial organization of mitochondria and localized metabolic enhancement through regulated stopping of processive motion. We delineate the role of key parameters, including cellular glucose uptake and consumption rates, that are expected to modulate mitochondrial distribution and metabolic response in spatially varying glucose conditions. Our estimates indicate that physiological brain glucose levels fall within the limited range necessary for metabolic enhancement. Hence mitochondrial localization is shown to be a plausible regulatory mechanism for neuronal metabolic flexibility in the presence of spatially heterogeneous glucose, as may occur in long processes of projection neurons. These findings provide a framework for the control of cellular bioenergetics through organelle trafficking.
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Axones/metabolismo , Glucosa/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Algoritmos , Animales , Transporte Biológico , Metabolismo Energético , Humanos , Modelos Biológicos , RatasRESUMEN
To maintain homeostasis, every cell must constantly monitor its energy level and appropriately adjust energy, in the form of ATP, production rates based on metabolic demand. Continuous fulfillment of this energy demand depends on the ability of cells to sense, metabolize, and convert nutrients into chemical energy. Mitochondria are the main energy conversion sites for many cell types. Cellular metabolic states dictate the mitochondrial size, shape, function, and positioning. Mitochondrial shape varies from singular discrete organelles to interconnected reticular networks within cells. The morphological adaptations of mitochondria to metabolic cues are facilitated by the dynamic events categorized as transport, fusion, fission, and quality control. By changing their dynamics and strategic positioning within the cytoplasm, mitochondria carry out critical functions and also participate actively in inter-organelle cross-talk, assisting metabolite transfer, degradation, and biogenesis. Mitochondrial dynamics has become an active area of research because of its particular importance in cancer, metabolic diseases, and neurological disorders. In this review, we will highlight the molecular pathways involved in the regulation of mitochondrial dynamics and their roles in maintaining energy homeostasis.
Asunto(s)
Metabolismo Energético , Homeostasis , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Animales , Susceptibilidad a Enfermedades , Humanos , Orgánulos/metabolismoRESUMEN
Improving axonal transport in the injured and diseased central nervous system has been proposed as a promising strategy to improve neuronal repair. However, the contribution of each cargo to the repair mechanism is unknown. DRG neurons globally increase axonal transport during regeneration. Because the transport of specific cargos after axonal insult has not been examined systematically in a model of enhanced regenerative capacity, it is unknown whether the transport of all cargos would be modulated equally in injured central nervous system neurons. Here, using a microfluidic culture system we compared neurons co-deleted for PTEN and SOCS3, an established model of high axonal regeneration capacity, to control neurons. We measured the axonal transport of three cargos (mitochondria, synaptic vesicles and late endosomes) in regenerating axons and found that the transport of mitochondria, but not the other cargos, was increased in PTEN/SOCS3 co-deleted axons relative to controls. The results reported here suggest a pivotal role for this organelle during axonal regeneration.
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Axones/fisiología , Mitocondrias/metabolismo , Regeneración Nerviosa/fisiología , Animales , Transporte Biológico , Células Cultivadas , Corteza Cerebral/citología , Femenino , Inmunohistoquímica , Ratones Transgénicos , Microscopía Confocal , Neuronas/citología , Neuronas/metabolismo , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Ratas , Proteína 3 Supresora de la Señalización de Citocinas/deficiencia , Proteína 3 Supresora de la Señalización de Citocinas/genética , Imagen de Lapso de Tiempo , Tubulina (Proteína)/metabolismoRESUMEN
Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.
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Biología/métodos , Decapodiformes/ultraestructura , Animales , Centrifugación por Gradiente de Densidad/métodos , Microscopía Electrónica/métodos , Lóbulo Óptico de Animales no Mamíferos/ultraestructura , Vesículas Sinápticas/ultraestructuraRESUMEN
Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(-/-) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues.