Extended Data Fig. 7: Hexokinase 1 O-GlcNAcylation and neuronal functional measurements.
From: Organization of a functional glycolytic metabolon on mitochondria for metabolic efficiency
a, Representative images of neuronal soma expressing shRNA-resistant BFP tagged WT or T259A-HK1 (blue) with HK1-shRNA, mCherry cell filler (magenta), and vGLUT1-pH (green). b-c, Retrospective quantification of WT-HK1 and T259A-HK1-BFP, and vGLUT1-pH (c) in rat hippocampal neurons used for imaging experiments depicted in Fig. 7. Scale bar represents 5 µm. All values are presented as mean ± SEM. n = 12–13 neurons from three biological replica (unpaired two-tailed t-test). d-g, The experimental design outlines the timeline for the plating, transfection, Tetrodotoxin (TTX) treatment, and imaging of cultured rat hippocampal neurons. (e) Hippocampal neurons were transfected with shRNA-resistant BFP-tagged WT or T259A-HK1-BFP, HK1-shRNA, and vGLUT1-pH. Following transfection, 1 µM TTX was added to neuronal culture. Two hours before imaging, TTX was washed-off as shown in (d). Neurons were electrically stimulated with 100 APs 10 Hz. Images showing vGLUT1-pH (pseudo-color, fire) and the cell filler mCherry (gray) before and after stimulation with WT-HK1 or T259A-HK1-BFP expressing neurons. Neutralization of vGLUT1-pH vesicles with NH4Cl reveals total axonal vesicle pool. (f) Average traces of vGLUT1-pH with 100 APs 10 Hz stimulation in WT-HK1 (black) or T259A-HK1 (orange) expressing neurons, previously TTX treated. ∆F values were normalized to maximal ∆F obtained from NH4Cl treatment. Error bars represent SEM. n = 8-9 neurons and 20–55 presynaptic boutons from four biological replicas. (g) Baseline and maximal (after electrical stimulation) vGLUT1-pH ∆F/F values. All values are shown as mean ± SEM (unpaired two-tailed t-test).
