TFEB links autophagy to lysosomal biogenesis

Abstract

Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal-regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase-dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles.

Fig. 1

TFEB induces autophagy. (A) Inverted color micrograph of control and stably overexpressing TFEB HeLa cells transfected with a GFP-LC3 plasmid and treated as follows: normal medium (normal), 2 hours of bafilomycin 400 nM (bafilomycin) and 2 hours in medium without nutrients (starvation); ~100 cells were analyzed for each treatment. Graph shows means of GFP-positive vesicles per cell. (B) Immunoblot analysis of LC3 in stable TFEB-overexpressing cells starved (Starv) for the indicated time (h, hours) represented as quantification of LC3-II intensity (relative to actin). (C) Cellular lysates isolated from TFEB-RNAi (+) and from scrambled RNAi-treated cells (–) cultured in normal medium, starved medium, or starved mediumsupplemented with bafilomycin (baf, 400 nM for 4 hours) as the quantification of LC3-II intensity (relative to actin). (D) TFEBmRNA levels from cells transfected with siRNA oligomers targeting TFEB or a scrambled sequence (ctr). (E) Representative confocal images of fixed HeLa cells stably expressing GFP-mRFP-LC3 transfected with empty (control) or TFEB vector shown as the average of vesicles per cell relative to the control (%). Aminimum of 2000 cells was counted. AL (autolysosomes) = (mRFP-positive vesicles)/(GFP-negative vesicles); total, mRFP-positive vesicles. All values are means TSEM of at least three independent experiments. Student’s t test (unpaired); *P < 0.05, **P < 0.01.

Fig. 2

Starvation regulates TFEB nuclear translocation and activity. (A) The expression levels of 51 autophagy-related genes were compared in control and TFEB-overexpressing HeLa cells ultured under different conditions. The results were represented as scatter-plot graphs where circles represent genes with increased (red) or decreased (green) fold change (logarithmic value); x axis, control group; y axis, treated group. (B) Representative images of HeLa cells stably overexpressing TFEB cultured in normal or starved medium for 4 hours. Five fields containing 50 to 100 cells each from five independent experiments were analyzed for TFEB nuclear localization: nucleus, 4′,6′-diamidino-2-phenylindole (DAPI); TFEB, Flag. Values are means ± SEM; Student’s t test (unpaired) **P<0.01. (C) Cells were subjected to nuclear and/or cytosolic fractionation and blotted with antibody against Flag. H3 and tubulin were used as nuclear and cytosolic markers, respectively. (D) Starved cells were treated as indicated (EGF, epidermal growth factor; FGF, fibroblast growth factor; PMA, phorbol 12-myristate 13-acetate). Nuclear fractions were blotted with antibodies against Flag and H3 (loading control). (E) Immunoblot analysis of Flag, tubulin, and H3 in nuclear extracts prepared from normal, starved, and starved then stimulated cells in normal medium for 1 hour (normal) or pretreated with AKT inhibitor, rapamycin mTOR inhibitor, and MAPK inhibitors 1 hour before media stimulation. Total extracts were used to verify the efficiency of the inhibitors (p-ERK, phosphorylated ERK kinase; Rap, rapamycin).

Fig. 3

Serine phosporylation regulates TFEB activation. (A) Flag immunostaining of TFEB subcellular localization in HeLa cells expressing mutated versions of TFEB-3xFlag. Five fields from three independent experiments, containing 50 to 100 cells each were analyzed. (B) Quantitative polymerase chain reaction (QPCR) analysis of TFEB target gene expression 24 hours after transfection with empty, normal, and mutant TFEB plasmids. (C) Immunoblot analysis of LC3-II in protein extracts from HeLa cells transfected with equal amounts of empty (pcDNA), WT-TFEB, or TFEBS142A-3xFlag vectors. Bafilomycin was added where indicated (400 nM for 4 hours). Quantification of LC3-II level was normalized to actin levels. (D) Analysis of autophagolysosomes (AL = RFP positive/GFP negative) in HeLa cells stably expressing GFP-mRFP-LC3 and transfected with pcDNA, WT-TFEB or TFEBS142A-3xFlag for 24 hours before harvesting. (E) In vitro kinase assay. Recombinant kinases were incubated in the presence of [γ-³²P]adenosine triphosphate and of a peptide spanning amino acids 120 to 170 of TFEB protein (TFEB-S-142) or with a similar peptide in which serine 142 was replaced with alanine (TFEB-A-142). Phosphorylation efficiency was measured as the amount of radioactivity incorporated by the peptides. (F) HeLa stable clones overexpressing TFEB were transfected with siRNA oligomers specific for ERK1/2 or with control siRNA. After 48 hours, cells were left untreated, serum-starved, or serum- and amino acid (a.a.)– starved for 4 hours; harvested; and subjected to nuclear isolation and Flag and H3 immunoblotting. Total lysates were probed with ERK-specific antibody. Values are means ± SEM of at least three independent experiments. Student’s t test (unpaired) *P < 0.05, **P < 0.001.

Fig. 4

In vivo analysis of TFEB-mediated induction of autophagy. (A and B) Analysis of TFEB subcellular localization in 2-month-old WT mice infected with AAV2/9 Tcfeb-HA and fasted 16 hours before being killed. (A) Quantification of nuclear HA signal. HA-immunofluorescence analysis (red) and DAPI staining (blue); 100 transduced cells were counted for each liver. (B) Immunoblot analysis of HA, tubulin, and H3 in liver specimens subjected to nuclear fractionation. Total liver lysates were probed with an HA-specific antibody to verify comparable transgene expression between fed and fasted animals. (C) Immunofluorescence of GFP and DNA (DAPI) staining in cryopreserved liver slices from 2-month-old GFP-LC3 transgenic mice injected with either AAV-Tcfeb-HA or saline solution (control group) and fed ad libitum or fasted for 24 hours before being killed. Quantification of GFP-positive vesicles is shown in the graph. (D) Immunoblot analysis of LC3 and actin in liver protein extracts from Alb-CRE, Tcfeb-3xFlag, and Tcfeb-3xFlag; Alb-CRE mice. (E) QPCR analysis of both autophagic and lysosomal TFEB target gene expression in liver samples isolated from Alb-CRE, Tcfeb-3xFlag, and Tcfeb-3xFlag;Alb-CRE mice. (F) Model of phosphodependent TFEB regulation of the autophagic-lysosomal network during nutrient starvation. Values are means ± SEM; at least five mice per group were analyzed; *P < 0.05, **P < 0.001.