SUMMARY Cells allocate substantial resources towards 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.

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.

Point-scanning imaging systems are among the most widely used tools for high-resolution cellular and tissue imaging, benefitting 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 timelapse data, we developed a “multi-frame” PSSR approach that utilizes information in adjacent frames to improve model predictions. In conclusion, 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 . Editor’s summary Point-scanning super-resolution imaging uses deep learning to supersample undersampled images and enable time-lapse imaging of subcellular events. An accompanying “crappifier” rapidly generates quality training data for robust performance.

Remodelling neuronal connections by synaptic activity requires membrane trafficking. We present evidence for a signalling pathway by which synaptic activity and its consequent Ca2+ 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.

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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.

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