AMPK contributes to autophagosome maturation and lysosomal fusion

Abstract

AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK α1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-independent autophagy inducer were used to examine the role of AMPK in autophagosome maturation and lysosomal fusion. While the treatment of control cells with either compound C or trehalose induces activation of autophagosomes as well as autolysosomes, the treatment of AMPK α1 knockout cells with compound C or trehalose induces mainly activation of autophagosomes, but not autolysosomes. We demonstrate that this effect is due to interference with the fusion of autophagosomes with lysosomes in AMPK α1 knockout cells. The transient expression of AMPK α1 can rescue autophagosome maturation. These results indicate that AMPK α1 is required for efficient autophagosome maturation and lysosomal fusion.

Figure 1

Generation of AMPK α1 knockout (KO) HEK293T cells. (A) Validation of AMPK α1 KO by T7 endonuclease 1 (T7E1) assay. HEK293T cells were transfected with either pX459/AMPK α1 gRNA #1 or pX459/AMPK α1 gRNA #2, and single colonies were isolated. The genomic PCR products were analyzed by T7E1 assay. Two different clones (KO #1 and KO #2) were used for the further experiments. (B) DNA sequencing analysis revealed the presence of AMPK α1 mutation in KO #1 and KO #2 (Top). The CRISPR-Cas9 system introduced an indel mutation in the target sites of the AMPK α1 gene (bottom). (C) Validation of AMPK α1 KO by Western blotting. Equal amounts of HEK293T wild-type (WT) and AMPK α1 KO cell lysates were subjected to Western blotting with an anti-AMPK α1 antibody. Experiments were repeated three times with similar results. (D) Proliferation of AMPK α1 KO cell lines was analyzed by MTT assay. The MTT assay was performed in triplicate, and the graph shows the average and standard deviation (SD). Control vs. knockout cells. *P < 0.05, **P < 0.01. (E) Phase contrast microscope images (400x) of control cells and AMPK α1 knockout cells.

Figure 2

AMPK α1 knockout inhibits autophagy initiation upon starvation. (A) Control cells and AMPK α1 knockout cells were transfected with a plasmid encoding mRFP-GFP-LC3, and cells were either mock-treated or starved in HBSS medium for the indicated time (1 h or 2 h). Bars: 10 μm. (B) The numbers of LC3 puncta in control cells and AMPK α1 knockout cells were counted (n = 20). Control cells vs. AMPK knockout cells, *P < 0.05, **P < 0.0005. (C) AMPK α1 knockout represses starvation-induced autophagy. Control cells and AMPK α1 knockout cells were incubated in HBSS medium, and the cell lysates were probed with anti-LC3 and anti-p62 antibodies. (D) The LC3-II, LC3-I and p62 bands were quantified, and the relative expression levels of LC3-II/LC3-I and p62 are shown in the graph. 0 h vs. 1 h or 2 h, *P < 0.05.

Figure 3

AMPK α1 knockout represses autophagic flux. (A) Control cells and AMPK α1 knockout cells were transfected with a plasmid encoding mRFP-GFP-LC3, and cells were either mock-treated (vehicle alone) or treated with compound C (cC, 10 μM) for 18 h. Bars: 10 μm. (B) Ratio quantification of autophagosomal LC3 puncta to autolysosomal LC3 puncta (n = 10). Control cells vs. knockout cells, *P < 0.0001. (C,D) HEK293T cells and AMPK α1 knockout cells were treated with compound C (10 μM) for 18 h, and the cell lysates were probed with anti-LC3 and anti-p62 antibodies. The p62 bands were quantified, and the relative expression levels are shown in the graph. Control cells vs. AMPK α1 knockout cells, *P < 0.05, **P < 0.005.

Figure 4

AMPK α1 knockout interferes with the formation of autolysosomes. (A) Control cells and AMPK α1 knockout cells were transfected with the plasmid encoding mRFP-LC3 and treated with compound C for 18 h. Cells were fixed and stained with an anti-LAMP1 antibody. Bars: 10 μm. (B) Ratio quantification of autophagosomal LC3 puncta (RFP-LC3 only) to autolysosomal LC3 puncta (RFP-LC3 and LAMP-1) Each data represents the mean and standard deviation of two independent experiments (n = 30), control cells vs AMPK Knockout cells, *P < 0.05. (C) Representative electron micrograph of HEK293T cells and AMPK α1 knockout cells after compound C treatment. Yellow arrows denote the autophagosome; blue arrows, autolysosomes; and red arrows, lysosomes. Bars: 1 μm. (D) Ratio quantification of autophagosomal puncta to autolysosomal puncta. We counted autophagosomes and autolysosomes in 54 individual cells (n = 54) of each group, and calculated the relative abundance. Graph shows the average and standard deviations. Control cells vs. knockout cells, *P < 0.0001.

Figure 5

AMPK α1 knockout deregulates AMPK-related signaling. (A) AMPK α1 knockout decreases the phosphorylation level of acetyl-CoA carboxylase (ACC). Control cells and AMPK α1 knockout cells were either mock-treated (vehicle alone) or treated with compound C (cC, 10 μM) for 18 h, and the equal amount of cell lysates were probed with the indicated antibodies. (B) Proliferation of AMPK α1 KO cell lines after compound C treatment. HEK293T control and knockout clones were seeded into 24-well plates and incubated with the indicated concentrations of compound C. MTT assays were performed in triplicate, and the graph shows the average and standard deviation (SD). (C) Cell viability of AMPK α1 KO cell lines were measured with Trypan blue assay after compound C treatment. Control cells vs. knockout cells, NS: not significant.

Figure 6

Expression of AMPK α1 recues the AMPK α1 knockout phenotype. (A) AMPK α1 knockout cells were transfected with mRFP-GRP-LC3 in the presence or absence of the AMPK plasmid. Twenty-four h after transfection, cells were treated with compound C for 18 h. Bars: 10 μm. (B) Ratio quantification of autophagosomal LC3 puncta to autolysosomal LC3 puncta (n = 10). Vector vs AMPK α1 plasmid, *P < 0.0001. (C) AMPK α1 knockout cells were transfected with the plasmid encoding AMPK α1. Twenty-four hour after transfection, cells were treated with compound C for 18 h. Equal amounts of cell lysates were probed with the indicated antibodies.

Figure 7

AMPK α1 knockout represses trehalose induced autophagic flux. (A) Control cells and AMPK α1 knockout cells were transfected with mRFP-GRP-LC3. Twenty-four h after transfection, cells were either mock-treated (vehicle only) or treated with trehalose (50 mM) for 4 h. (B,C) HEK293T cells and AMPK α1 knockout cells were treated with trehalose (50 mM) for 6 h, and the cell lysates were probed with anti-LC3 and anti-p62 antibodies. The p62 bands were quantified, and the relative expression levels are shown in the graph. Control cells vs. AMPK α1 knockout cells, *P < 0.05. (D,E) Cells were transfected with mRFP-LC3 plasmid. 24 h after, cells were treated with Trehalose for 6 h. Cells were fixed and stained with anti-LAMP1 antibody. Ratio quantification of autophagosomal LC3 puncta (RFP-LC3 only) to autolysosomal LC3 puncta (RFP-LC3 and LAMP-1). Each data represents the mean and standard deviation of two independent experiments (n = 30), control cells vs AMPK Knockout cells, *P < 0.05.

Figure 8

Expression of AMPK α1 recovers trehalose induced autophagic flux. (A,B) AMPK α1 knockout cells were transfected with mRFP-GFP-LC3 in the presence or absence of the AMPK plasmid. Twenty-four hour after, cells were treated with trehalose for 6 h. Ratio quantification of autophagosomal LC3 (GFP+/RFP+) puncta to autolysosomal LC3 puncta (GFP−/RFP+) Bars: 10 μm. (n = 15, vector vs AMPK plasmid, *P < 0.0001). (C) AMPK α1 knockout cells were transfected with AMPK plasmid. Twenty-four hour after transfection, cells were treated with trehalose for 6 h. Cell lysates were probed with the indicated antibody.