Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) – comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI – is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions, including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Oscillations in insulin secretion, driven by islet Ca²⁺ waves, are crucial for glycemic control. Prior studies, performed with single-plane imaging, suggest that subpopulations of electrically coupled β-cells have privileged roles in leading and coordinating the propagation of Ca²⁺ waves. Here, we used three-dimensional (3D) light-sheet imaging to analyze the location and Ca²⁺ activity of single β-cells within the entire islet at >2 Hz. In contrast with single-plane studies, 3D network analysis indicates that the most highly synchronized β-cells are located at the islet center, and remain regionally but not cellularly stable between oscillations. This subpopulation, which includes ‘hub cells’, is insensitive to changes in fuel metabolism induced by glucokinase and pyruvate kinase activation. β-Cells that initiate the Ca²⁺ wave (leaders) are located at the islet periphery, and strikingly, change their identity over time via rotations in the wave axis. Glucokinase activation, which increased oscillation period, reinforced leader cells and stabilized the wave axis. Pyruvate kinase activation, despite increasing oscillation frequency, had no effect on leader cells, indicating the wave origin is patterned by fuel input. These findings emphasize the stochastic nature of the β-cell subpopulations that control Ca²⁺ oscillations and identify a role for glucokinase in spatially patterning ‘leader’ β-cells.