Fig. 2: Lysosomal AMPK activity is rapidly induced in an LKB1-dependent manner.
From: Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter
a (Top) Domain layout and representative image of lyso-ExRai AMPKAR in MEFs stained with the lysosomal marker LysoTracker Red. (Bottom) Domain layout and representative image of mito-ExRai AMPKAR in MEFs stained with the mitochondrial marker MitoTracker Red. b Average response of lyso-ExRai AMPKAR in WT (black, n = 5 cells from two experiments) and AMPKα KO MEFs (pink, n = 6 cells from two experiments) to 2-DG (40 mM) stimulation, along with maximum ratio change (p = 9.66 × 10−6, unpaired t-test, two-tailed). c Average response of mito-ExRai AMPKAR in WT (black, n = 10 cells from five experiments and AMPKα KO MEFs (pink, n = 7 cells from three experiments) treated with 2-DG (40 mM) along with maximum ratio change (**p = 0.0012, unpaired t-test, two-tailed). d Time to half-maximal response (t1/2) of lyso-ExRai AMPKAR (teal), cytoplasmic ExRai AMPKAR (pink), and mito-ExRai AMPKAR (black) following treatment with 2-DG (**p = 0.0033; ****p = 4.43 × 10−10, one-way ANOVA with Dunnett’s multiple comparisons test). e Average response to 2-DG (40 mM) stimulation of cytoplasmic ExRai AMPKAR in WT (black, reproduced from Fig. 1e) and LKB1 KO MEFs (pink, n = 7 cells from four experiments), along with maximum ratio changes (****p = 1.57 × 10−6, unpaired t-test, two-tailed). f Average response to 2-DG (40 mM) stimulation of lyso-ExRai AMPKAR in WT (black, reproduced from b) and LKB1 KO MEFs (pink, n = 5 cells from four experiments), along with maximum ratio changes (ns p = 0.084, unpaired t-test, two-tailed). g Average response to 2-DG (40 mM) stimulation of mito-ExRai AMPKAR in WT (black, reproduced from c) and LKB1 KO MEFs (pink, n = 13 cells from five experiments), along with maximum ratio changes (ns p = 0.09, unpaired t-test, two-tailed). h t1/2 of lyso-ExRai AMPKAR (teal, n = 6 cells from four experiments), cytoplasmic ExRai AMPKAR (pink, n = 5 cells from four experiments), and mito-ExRai AMPKAR (black, n = 10 cells from five experiments) following treatment with 2-DG in LKB1 KO MEFs (ns p ≥ 0.55, one-way ANOVA with Dunnett’s multiple comparisons test). For all figures, time courses show the mean ± SD, dot plots show the mean ± SEM. Scale bars, 20 µm.
