Figure 4.
De-GlcNAcylation of TRAK1 does not restore motility to AntA-arrested mitochondria. (A) O-GlcNAc writer/eraser system consists of two engineered enzymes, an OGT and an O-GlcNAcase. Each enzyme is linked to a GFP nanobody that, when transfected into cells, can selectively target a GFP-tagged protein to add or remove O-GlcNAc. (B) O-GlcNAc writer and eraser can add and remove O-GlcNAc from GFP-tagged hTRAK1. GFP-hTRAK1 and the components of the O-GlcNAc writer or/eraser system were co-expressed in HEK293T cells. GFP-hTRAK1 was then immunoprecipitated using an anti-TRAK1 antibody. Immunoprecipitates and lysates were blotted and then probed with anti-RL2 (an antibody that recognizes O-GlcNAcylations), anti-GFP, and anti-tubulin. (C) Quantification of TRAK1 GlcNAcylation from blots as in B. n = 5 independent transfections per condition. (D) O-GlcNAC eraser prevents the mitochondrial arrest caused by 3 h of elevated glucose. Hippocampal neurons expressing GFP-hTRAK1, the O-GlcNAc eraser, mito-dsRED, and mNeonGreen were cultured in 5 mM glucose medium until DIV13. On DIV13, cultures were shifted for 3 h to 25 mM glucose medium or 25 mM sorbitol and then imaged for quantification of mitochondrial motility. n = 7–10 axons per treatment from five independent animals. (E) Quantification of mitochondrial motility after 4 nM antimycin treatment in hippocampal cultures expressing GFP-hTRAK1 and the O-GlcNAc eraser system. Cells were incubated for 2 h with 4 nM AntA, and imaging was done on DIV8–10. O-GlcNAc eraser did not prevent the AntA-induced arrest. n = 6–10 axons per treatment from four independent animals. (F) Example kymographs of mitochondrial movement in axons as in E. Mitochondria are magenta, and axons are green. For all images, scale bars = 20 μm, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. The P value for C was calculated by performing two-tailed, unpaired t tests with Welch’s correction. For D and E, P values were calculated by performing a blocked one-way ANOVA with Tukey’s multiple comparison correction. Only P values ≤0.05 are shown. Source data are available for this figure: SourceData F4.

De-GlcNAcylation of TRAK1 does not restore motility to AntA-arrested mitochondria. (A) O-GlcNAc writer/eraser system consists of two engineered enzymes, an OGT and an O-GlcNAcase. Each enzyme is linked to a GFP nanobody that, when transfected into cells, can selectively target a GFP-tagged protein to add or remove O-GlcNAc. (B) O-GlcNAc writer and eraser can add and remove O-GlcNAc from GFP-tagged hTRAK1. GFP-hTRAK1 and the components of the O-GlcNAc writer or/eraser system were co-expressed in HEK293T cells. GFP-hTRAK1 was then immunoprecipitated using an anti-TRAK1 antibody. Immunoprecipitates and lysates were blotted and then probed with anti-RL2 (an antibody that recognizes O-GlcNAcylations), anti-GFP, and anti-tubulin. (C) Quantification of TRAK1 GlcNAcylation from blots as in B. n = 5 independent transfections per condition. (D) O-GlcNAC eraser prevents the mitochondrial arrest caused by 3 h of elevated glucose. Hippocampal neurons expressing GFP-hTRAK1, the O-GlcNAc eraser, mito-dsRED, and mNeonGreen were cultured in 5 mM glucose medium until DIV13. On DIV13, cultures were shifted for 3 h to 25 mM glucose medium or 25 mM sorbitol and then imaged for quantification of mitochondrial motility. n = 7–10 axons per treatment from five independent animals. (E) Quantification of mitochondrial motility after 4 nM antimycin treatment in hippocampal cultures expressing GFP-hTRAK1 and the O-GlcNAc eraser system. Cells were incubated for 2 h with 4 nM AntA, and imaging was done on DIV8–10. O-GlcNAc eraser did not prevent the AntA-induced arrest. n = 6–10 axons per treatment from four independent animals. (F) Example kymographs of mitochondrial movement in axons as in E. Mitochondria are magenta, and axons are green. For all images, scale bars = 20 μm, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. The P value for C was calculated by performing two-tailed, unpaired t tests with Welch’s correction. For D and E, P values were calculated by performing a blocked one-way ANOVA with Tukey’s multiple comparison correction. Only P values ≤0.05 are shown. Source data are available for this figure: SourceData F4.

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