Figure S5.
Controls supporting a model in which AMPK regulates mitochondrial movement by phosphorylating TRAK1. (A) Representative images and kymographs of data quantified in Fig. 5 C. Briefly, time-lapse images were acquired to measure mitochondrial motility in hippocampal cultures expressing constitutive active AMPK (CA-AMPK) or empty vector control, as well as mito-dsRED and meGFP. Mitochondria are magenta, and axons are green. (B) Quantification of mitochondrial motility in hippocampal cultures expressing constitutive active AMPK (CA-AMPK) or empty vector control. The cultures were treated for 4 h with 5 µM LatA or vehicle. n = 7–10 axons per treatment from 2 independent animals. The bars on each boxplot represent the min to max values. (C) Representative images and kymographs of data quantified in Fig. 5 D. Briefly, time-lapse images were acquired to measure mitochondrial motility in hippocampal cultures expressing dominant negative AMPK (DN-AMPK) or empty vector control, as well as mito-dsRED and meGFP. Neuron cultures were treated with 4 nM AntA or vehicle for 1 h and then imaged. Mitochondria are magenta, and axons are green. (D) Representative western blot of endogenous AMPKα1 in either untransduced fibroblasts or cells transduced with lentivirus expressing a shRNA targeting AMPKα1 or a nontargeting shRNA control. The blot was probed for AMPKα1 and GAPDH. (E) Quantification of AMPKα1 levels from blots as in D. n = 4 lysates from four independent animals. (F) Quantification of Rab5 motility in cultured hippocampal neurons expressing mEmerald-Rab5a, mCherry2, and either CA-AMPK or an empty vector control. Imaging was performed after 2 days of expression in Hibernate E media to reduce background fluorescence. n = 10 neurites per treatment from three independent animals. (G) HEK293T cells were cotransfected with CA-AMPK and MYC-tagged hTRAK1, hTRAK2, or an empty vector. MYC-hTRAK1 or MYC-hTRAK2 was then immunoprecipitated using an anti-MYC antibody. Immunoprecipitates and lysates were blotted and then probed for anti-phosphorylated AMPK substrate, anti-MYC, and anti-GAPDH immunoreactivity. (H) Comparison of the AMPK consensus sequence with the region surrounding S200 and S201 in human and rat TRAK1 and TRAK2. Amino acid properties of the consensus are as follows: Φ = hydrophobic; X = any amino acid; B = basic; S/T = phosphosite. Multiple sequence alignment was done with Clustal Omega and visualized in Jalview version 2 (Madeira et al., 2024; Waterhouse et al., 2009). UniProt and NCBI protein sequence identifiers are as follows: human TRAK1 = Q9UPV9, rat TRAK1 = NP_001128037.1, human TRAK2 = O60296, rat TRAK2 = Q8R2H7. A blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction was performed for E. The P value for F was calculated by a two-tailed, unpaired t test with Welch’s correction. Source data are available for this figure: SourceData FS5.

Controls supporting a model in which AMPK regulates mitochondrial movement by phosphorylating TRAK1. (A) Representative images and kymographs of data quantified in Fig. 5 C. Briefly, time-lapse images were acquired to measure mitochondrial motility in hippocampal cultures expressing constitutive active AMPK (CA-AMPK) or empty vector control, as well as mito-dsRED and meGFP. Mitochondria are magenta, and axons are green. (B) Quantification of mitochondrial motility in hippocampal cultures expressing constitutive active AMPK (CA-AMPK) or empty vector control. The cultures were treated for 4 h with 5 µM LatA or vehicle. n = 7–10 axons per treatment from 2 independent animals. The bars on each boxplot represent the min to max values. (C) Representative images and kymographs of data quantified in Fig. 5 D. Briefly, time-lapse images were acquired to measure mitochondrial motility in hippocampal cultures expressing dominant negative AMPK (DN-AMPK) or empty vector control, as well as mito-dsRED and meGFP. Neuron cultures were treated with 4 nM AntA or vehicle for 1 h and then imaged. Mitochondria are magenta, and axons are green. (D) Representative western blot of endogenous AMPKα1 in either untransduced fibroblasts or cells transduced with lentivirus expressing a shRNA targeting AMPKα1 or a nontargeting shRNA control. The blot was probed for AMPKα1 and GAPDH. (E) Quantification of AMPKα1 levels from blots as in D. n = 4 lysates from four independent animals. (F) Quantification of Rab5 motility in cultured hippocampal neurons expressing mEmerald-Rab5a, mCherry2, and either CA-AMPK or an empty vector control. Imaging was performed after 2 days of expression in Hibernate E media to reduce background fluorescence. n = 10 neurites per treatment from three independent animals. (G) HEK293T cells were cotransfected with CA-AMPK and MYC-tagged hTRAK1, hTRAK2, or an empty vector. MYC-hTRAK1 or MYC-hTRAK2 was then immunoprecipitated using an anti-MYC antibody. Immunoprecipitates and lysates were blotted and then probed for anti-phosphorylated AMPK substrate, anti-MYC, and anti-GAPDH immunoreactivity. (H) Comparison of the AMPK consensus sequence with the region surrounding S200 and S201 in human and rat TRAK1 and TRAK2. Amino acid properties of the consensus are as follows: Φ = hydrophobic; X = any amino acid; B = basic; S/T = phosphosite. Multiple sequence alignment was done with Clustal Omega and visualized in Jalview version 2 (Madeira et al., 2024; Waterhouse et al., 2009). UniProt and NCBI protein sequence identifiers are as follows: human TRAK1 = Q9UPV9, rat TRAK1 = NP_001128037.1, human TRAK2 = O60296, rat TRAK2 = Q8R2H7. A blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction was performed for E. The P value for F was calculated by a two-tailed, unpaired t test with Welch’s correction. Source data are available for this figure: SourceData FS5.

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