Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: MTMR4, a phosphoinositide-specific 3'-phosphatase, regulates TFEB activity and the endocytic and autophagic pathways.

doi: 10.1111/gtc.12609

Figure Lengend Snippet: FIGURE 6 MTMR4 knockdown inhibits starvation-induced increase in dephosphorylated TFEB/total TFEB ratio. (a) Phosphorylation of TFEB and other proteins and total amounts of MTMR4 and MTMR3 in control and MTMR4-depleted cells. A549 cells transfected with control- or MTMR4-siRNAs were placed in growth medium (fed) with or without 100 μM chloroquine or in HBSS for 4 hr, and subjected to western blot analyses. S6K, S6 kinase. (b) Quantified data of total TFEB protein level (sum of the dephosphorylated and phosphorylated forms of TFEB) (left), phospho-TFEB level (normalized to GAPDH) (middle) and ratio of dephosphorylated TFEB/total TFEB (right) in control and MTMR4–depleted cells. (c) Time course of starvation-induced change in phosphorylation of TFEB and S6K. A549 cells were starved by culture in HBSS for the indicated time periods and subjected to western blot analyses. Stv, starved. (d) Phosphorylation of TFEB in control and MTMR4-depleted cells under both fed and starved conditions with or without cyclosporine A treatment. Control and MTMR4-depleted A549 cells were incubated in growth medium (fed) or HBSS (starved) with or without cyclosporine-A (10 μM) for 4 hr

Article Snippet: GFP- TFEB expression vector (# 38119) was provided by Dr. Shawn Ferguson through Addgene (Roczniak- Ferguson et al., 2012).

Techniques: Knockdown, Phospho-proteomics, Control, Transfection, Western Blot, Incubation

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: MTMR4, a phosphoinositide-specific 3'-phosphatase, regulates TFEB activity and the endocytic and autophagic pathways.

doi: 10.1111/gtc.12609

Figure Lengend Snippet: FIGURE 8 PI(3)P-phosphatase MTMR4 regulates endocytic and autophagic pathways. MTMR4 is localized mainly in LEs and APs to regulate PI(3)P levels. MTMR4 regulates the fusion, fission, motility and maturation of endosomes, the autophagic pathway, and lysosomal biogenesis. MTMR4 is also required for activation of TFEB. This action of MTMR4 contributes to MTMR4 regulation of the endocytic and autophagic pathways

Article Snippet: GFP- TFEB expression vector (# 38119) was provided by Dr. Shawn Ferguson through Addgene (Roczniak- Ferguson et al., 2012).

Techniques: Activation Assay

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: TFEB translocates to the nucleus during mitophagy in a Parkin- and PINK1-dependent manner. (A) YFP-Parkin HeLa cells were treated with O/A for up to 10 h, fractionated, and immunoblotted. (B) Quantification of data in A. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (C) mCherry-Parkin HeLa cells were left untreated (Control), starved (2 h), or treated with torin 1 (2 h), O/A (6 h), or valinomycin (Val; 6 h). CIP treatment of cell lysates was performed before immunoblotting. (D) WT and mCherry-Parkin HeLa cells were treated with DMSO or O/A (6 h), lysed, and immunoblotted. A CIP-treated control was included as a reference for total TFEB dephosphorylation. (E) WT and mCherry-Parkin HeLa cells were treated with DMSO, torin 1, or O/A for 18 h and analyzed by quantitative PCR for TFEB target gene expression. Data are means ± SD ( n = 3). (F) WT and PINK1 KO HeLa cells stably expressing TFEB-GFP with or without mCherry-Parkin were treated as in C. Fixed cells were stained with DAPI and analyzed by immunofluorescence. Bars, 10 µm. See Fig. S1 F for quantification. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Expressing, Western Blot, De-Phosphorylation Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Staining, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Analysis of Parkin-dependent effects on mTORC1 activity and TFEB association with 14-3-3 proteins. (A and C) WT and YFP-Parkin HeLa cells were treated with DMSO, O/A, or torin 1 as indicated, lysed, and immunoblotted. (B and D) Quantification of data in A and C, respectively. Protein levels were normalized to actin and the ratio of phosphorylated to total protein in treated samples is expressed relative to DMSO controls. Data are means ± SD ( n = 3); no differences observed were statistically significant. (E) HeLa cells stably expressing TFEB-GFP were transfected with control or untagged Parkin DNA and treated the next day with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Cells were lysed and TFEB-GFP was immunoprecipitated with anti-GFP beads. Cell lysates (Input) and immunoprecipitated proteins were immunoblotted. Images are representative of n = 2 experiments.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Parkin acts upstream of Rag GTPases to regulate TFEB subcellular localization. (A and B) HeLa cells stably expressing TFEB-GFP with (B) or without (A) mCherry-Parkin were transfected with empty vector or active or inactive RagB/D heterodimer DNA (detected with αHA antibody). The indicated cells were starved (2 h) or treated with DMSO or O/A (6 h) before fixation and immunofluorescence analysis. Images are representative of n = 2 experiments. 75–100% of cells observed (mean of 175 cells per condition) exhibited the given phenotype. Asterisks indicate αHA-negative cells. Bars, 10 µm.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Atg5 and Atg9A are required for Parkin-mediated TFEB translocation. (A) WT and Atg5 KO cells stably expressing mCherry-Parkin were treated with DMSO or O/A (6 h), fixed, immunostained for TFEB, and analyzed by immunofluorescence. Bars, 10 µm. (B) Quantification of endogenous TFEB nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) Untreated WT and Atg5 KO cells stably expressing mCherry-Parkin and GFP-Atg5 as indicated were lysed and immunoblotted. (D) Cells from C were treated with DMSO or O/A (6 h), lysed, fractionated, and immunoblotted. (E) Quantification of data in D. Endogenous TFEB expression was normalized to GAPDH (cytosol) or histone H3 (nuclear) and nuclear TFEB was expressed as a percentage of total TFEB. Data are means ± SD ( n = 3). (F) WT and Atg5 KO HeLa cells stably expressing mCherry-Parkin as indicated were treated with DMSO (6 h), torin 1 (2 h), CIP (1 h), and O/A (6 h) as indicated, lysed, and immunoblotted. Images are representative of n = 3 experiments. (G) WT and Atg5 KO cells expressing mCherry-Parkin as indicated were starved (2 h) or left untreated, lysed, fractionated, and immunoblotted. Images are representative of n = 3 experiments. (H) WT and Atg9A KO HeLa cells expressing mCherry-Parkin as indicated were starved (2 h), or treated with DMSO (Ctrl) or O/A for 6 h. Cell lysates were processed as in D. (I) Quantification of endogenous TFEB nuclear localization in H, performed as in E. Data are means ± SD ( n = 3). C, cytosol; N, nuclear. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Translocation Assay, Stable Transfection, Expressing, Immunofluorescence, Fluorescence

Journal: The Journal of Cell Biology

Article Title: MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

doi: 10.1083/jcb.201501002

Figure Lengend Snippet: Parkin- and Atg5-dependent regulation of TFEB homologue subcellular localization. (A) WT HeLa cells stably expressing mCherry-Parkin, TFE3-GFP, MITF1-GFP, and TFEC-YFP as indicated were treated with DMSO (6 h), torin 1 (2 h), or O/A (6 h). Fixed cells were analyzed by immunofluorescence. (B) Quantification of ectopic TFE3, MITF1, and TFEC nuclear localization in A. The nuclear/cytosol ratio for each condition was calculated from mean fluorescence intensity/volume measurements made for each compartment across a field (four to seven) of cells (40–60 cells/field). Data are means ± SD ( n = 3). (C) WT and Atg5 KO cells stably expressing mCherry-Parkin treated with DMSO or O/A (6 h) were fixed, immunostained for TFE3 or MITF, and analyzed by immunofluorescence. (D) Quantification of endogenous TFE3 and MITF nuclear localization in C. Analysis was performed as in B (40–60 cells/field, 4 fields/condition, n = 3 experiments). Data are means ± SD. For all graphs: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.

Article Snippet: The viral TFEB-GFP expression vector was generated by PCR amplification of the full-length encoding sequence from pEGFP-N1-TFEB followed by in-frame cloning into HindIII–SalI sites of the retrovirus pBMN-Z vector (Orbigen).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Fluorescence

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 1. Peptides derived from transcription factor EB bind to CN in mouse brain lysates. (A) The TFEB-YLENP peptide binds CNA in mouse brain lysates in pull-down assays. Bound CNA was visualized by western blot with monoclonal anti-CNA antibody, shown in the upper blot; the input is shown in the middle panel. The bottom blot confirms that equal amounts of GST fusion proteins were used in the reactions. (B) Alterations of the TFEB-YLENP peptide decrease the interaction between peptides and CNA in mouse brain lysates assayed by GST pull-down. (C) The TFEB-YLENP derivatives TFEB-YLENPN78V and TFEB-YLENPE77A, N78V interact more strongly with CNA. (D) Densitometric quantification of CNA bound by TFEB-YLENPE77A and TFEB-YLENPE77A, N78V and histograms showing the relative intensity units of bound CN. Data were presented as mean ± SEM (n ¼ 3), *p < 0.05; **p < 0.01 compared with the TFEB-YLENP group.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Derivative Assay, Western Blot

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 2. Peptides derived from transcription factor EB bind to purified recombinant CN. (A) The affinity of FAM-labeled TFEB-YLENP peptide for CN. (B) The affinity of FAM- labeled TFEB-YLAVP peptide for CN. CN was titrated against fixed concentrations of labeled TFEB-YLENP (40 nM) and TFEB-YLAVP (20 nM). The top panel shows the isotherm derived from the raw data, fitted to a sigmoidal dose-response curve. The bottom panel displays the raw data for thermophoresis recorded at 20 C using 20% LED power and 80% MST power. (C) Comparison of Kd values between TFEB-YLENP/CN and TFEB-YLAVP/CN from three individual experiments. The individual experimental values were showed. (D) The interaction between CN and 300 nM FAM-labeled TFEB-YLAVP measured by fluorescence polarization. (E) Complex formation between CN and 300 nM FAM-labeled TFEB-YLAVP in the presence of unlabeled NFATc1-YLAVP peptide by fluorescence polarization.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Derivative Assay, Recombinant, Labeling, Comparison

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 3. A similar binding pattern for both TFEB-YLENP and LxVP-type peptides to CN. (A and B) The distance (upper) between C-alpha atoms of the peptide and CN, and their interaction energy (lower) during the simulations. (C) The YLENP (shiny) and YLAVP (brushed) peptides bind to CN in a similar manner, as shown in the last snapshots of the simulation. Both CNA (ice blue) and CNB (orange) are shown as transparent surfaces and cartoons, whereas the LxxP peptides are drawn as sticks. (D) Binding between CN and the TFEB-YLENP peptide is weaker than that between CN and the TFEB-YLAVP peptide, as shown by the average interaction energies based on the complete trajectories.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Binding Assay

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 4. CsA bound to cyclophilin competes with activated CN for the mutant TFEB. (A) Competition for binding of CN to TFEB-YLAVP by CsA alone and by the CsA-CyP complex (20 mM and 200 nM, respectively). (B) Binding of GST-CNA and its deletion mutants to GFP-TFEB in HeLa cells. (C) TFEB bound by CNA and its mutants was measured densito- metrically, and the histograms show the relative intensity units of bound TFEB. TFEB bound by CNA is set at 100%. Data were presented as mean ± SEM (n ¼ 3), *p < 0.05 compared with CNA group. (D) Quercetin (50 mM and 100 mM) competes with binding of GFP-TFEB to GST-CNA. (E) The bar graph depicts the ratios of bound TFEB and GST-CN in the presence of quercetin as percentages of the control binding (100%). Data were presented as mean ± SEM (n ¼ 3), **p < 0.01; ***p < 0.001 compared with the control group.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Mutagenesis, Binding Assay, Control

Journal: Biochimie

Article Title: Peptides derived from transcription factor EB bind to calcineurin at a similar region as the NFAT-type motif.

doi: 10.1016/j.biochi.2017.09.002

Figure Lengend Snippet: Fig. 5. Expression of NFATc1-YLAVP peptide blocks TFEB activation in starved HeLa cells. (A) Starvation induces nuclear translocation of GFP-TFEB. (B) NFATc1-YLAVP peptide inhibits TFEB nuclear translocation. The graph shows the percentages of TFEB translocation in starved and peptide-treated cells. Data were presented as mean ± SEM (n ¼ 3), *p < 0.05; **p < 0.01 compared with the starved group. (C) Transfection of NFATc1-YLAVP cDNA induces TFEB phosphorylation in starved HeLa cells. Empty vector cDNA and NFATc1- YLAVP cDNA were transfected into HeLa cells, and the cells were starved. The blot shows the expression of GFP-TFEB in total lysates and indicates the positions of phosphorylated and dephosphorylated proteins.

Article Snippet: GFP-tagged TFEB plasmid was purchased from OriGene Technologies (Beijing, China).

Techniques: Expressing, Activation Assay, Translocation Assay, Transfection, Phospho-proteomics, Plasmid Preparation

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis.

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: Figure 2. Conditional Deletion of TFEB in OL Lineage Cells Causes Ectopic Myelination in the Cerebellar Molecular Layer (A and B) Genetic labeling reveals OPCs in the P14 mouse cerebellar molecular layer (red in A and B, indicated by white arrowheads). ML, molecular layer; GL, granule layer.

Article Snippet: GFP-TFEB expression construct was generated by subcloning mouse TFEB cDNA into a pEGFP-N1-Flag vector, which was a gift from Patrick Calsou (Addgene plasmid, #60360) (Britton et al., 2014).

Techniques: Labeling

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis.

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: Figure 4. TFEB cKO Mice Harbor Thicker Myelin Sheaths and Are Precociously Myelinated in White Matter (A and A’) Representative TEM micrographs from P21 Ctl (TFEBF/F) and TFEB cKO (Olig2-Cre; TFEBF/F) CC showing that myelinated axon density is increased in the absence of TFEB. (B) Quantification of myelinated axon number (left), total axon number (middle), and myelinated axon percentage (right) in the CC from TFEBF/F (Flox), Olig2-Cre; TFEBF/+ (Het) and Olig2-Cre; TFEBF/F (cKO) mice. (C and C’) Representative high-magnification TEM micrographs from P21 Ctl and TFEB cKO CC. (D and E) Quantification of average g-ratios of myelinated axons (E), and as a function of axon diameter (D), in P21 TFEB cKO mice (red) compared with littermate Ctls (TFEBF/F in black and Olig2-Cre; TFEBF/+ in blue). n R 180 axons from 3 animals per genotype. (F and F’) Representative high-magnification TEM micrographs from 6-month-old Ctl and TFEB cKO CC. (G and H) Quantification of average g-ratios of myelinated axons (H), and as a function of axon diameter (G), in 6-month-old TFEB cKO mice (red) as compared with littermate Ctls n R 180 axons from 3 animals per genotype. Error bars represent SEM. Scale bars: 5 mm in (A’) for (A) and (A’); and 0.5 mm in (F’) for (C)–(F’). *p < 0.05.

Article Snippet: GFP-TFEB expression construct was generated by subcloning mouse TFEB cDNA into a pEGFP-N1-Flag vector, which was a gift from Patrick Calsou (Addgene plasmid, #60360) (Britton et al., 2014).

Techniques:

Journal: Cell

Article Title: Spatiotemporal Control of CNS Myelination by Oligodendrocyte Programmed Cell Death through the TFEB-PUMA Axis.

doi: 10.1016/j.cell.2018.10.044

Figure Lengend Snippet: Figure 6. TFEB Promotes the Expression of Subsets of ER Stress Genes and Induces the Pro-apoptotic Factor PUMA in Pre-OLs (A) Volcano plot showing differentially expressed genes in pre-OLs acutely purified from P12 TFEB cKO (Olig2-Cre; TFEBF/F) compared with those purified from littermate Ctl mice (Olig2-Cre; TFEBF/+). Each dot represents a gene. Blue color dots are genes up- or downregulated more than 2-fold. Four selected genes are denoted and highlighted in red. (B) Heatmap showing expression levels (in z scores) of genes up- or downregulated more than 2-fold in TFEB cKO pre-OLs by RNA sequencing (RNA-seq). Replicates of TFEB heterozygous Ctl cells (Het) and KO cells from distinct clusters are shown by the dendrogram. Each row corresponds to a gene, and rows are ordered by fold change values from the highest to the lowest. See Table S1 for the full gene list. (C) Gene ontology (GO) term analysis (Protein Analysis through Evolutionary Relationships [PANTHER] Overrepresentation Test) by using genes downregulated more than 2-fold as input. The top 10 most enriched biological process (BP) terms are shown. Only the narrowest term from each hierarchical group in the analysis is plotted. The full list of enriched terms can be found in Table S2. (D) qRT-PCR of PUMA(Bbc3) mRNA expression in differentiating OLs from TFEB cKO mice and littermate Ctls. n = 3 biological replicates for both genotypes and for each time points. Error bars represent SEM. *p < 0.05.

Article Snippet: GFP-TFEB expression construct was generated by subcloning mouse TFEB cDNA into a pEGFP-N1-Flag vector, which was a gift from Patrick Calsou (Addgene plasmid, #60360) (Britton et al., 2014).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR