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Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster.

Tognon E, Kobia F, Busi I, Fumagalli A, De Masi F, Vaccari T - Autophagy (2016)

Bottom Line: In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development.Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity.Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

View Article: PubMed Central - PubMed

Affiliation: a IFOM - FIRC Institute of Molecular Oncology , Milan , Italy.

ABSTRACT
In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H(+)-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

No MeSH data available.


Related in: MedlinePlus

The TFEB-V-ATPase axis regulates lysosomal function and basal N signaling in human cells. (A) LTR assay indicates that acute treatment with both BafA1 and FCCP, which dissipates lysosomal pH independently of V-ATPase, impairs acidification in MCF10A cells. (B and C) Chronic inhibition of V-ATPase or KD of ATP6V0C in contrast causes an expansion of the acidified (B) and LAMP1-positive lysosomal organelles (C). (D) Relative to the control, western blot analysis of MTOR signaling of lysate from MCF10A cells treated as indicated reveals a reduction in the level of phosphorylated RPS6KB (pS6K), a measure of active MTOR signaling. (E) Western blot analysis of MCF10A lysates treated as indicated shows that chronic inhibition of V-ATPase leads to accumulation of the 52 and 44 KDa immature CTSD forms (iCTSD), a sign of impaired lysosomal function. (F) Chronic V-ATPase inhibition results in high levels of nuclear TFEB compared to control. (G) siRNA against TFEB reduce the level of TFEB, ATP6V0C and HES1 mRNA expression compared to control. A similar reduction in the level of N signaling is observed after knocking down the expression of the γ-secretase component, PSENEN or of the NOTCH1 receptor. In contrast, efficient knockdown of ADAM10, which is dispensable for ligand-independent signaling, does not reduce HES1 levels. (H) Chronic treatment with both BafA1 and FCCP compromises γ-secretase substrate cleavage. (I) Chronic inhibition of the V-ATPase reduces the levels of expression of N receptors among other genes.
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f0008: The TFEB-V-ATPase axis regulates lysosomal function and basal N signaling in human cells. (A) LTR assay indicates that acute treatment with both BafA1 and FCCP, which dissipates lysosomal pH independently of V-ATPase, impairs acidification in MCF10A cells. (B and C) Chronic inhibition of V-ATPase or KD of ATP6V0C in contrast causes an expansion of the acidified (B) and LAMP1-positive lysosomal organelles (C). (D) Relative to the control, western blot analysis of MTOR signaling of lysate from MCF10A cells treated as indicated reveals a reduction in the level of phosphorylated RPS6KB (pS6K), a measure of active MTOR signaling. (E) Western blot analysis of MCF10A lysates treated as indicated shows that chronic inhibition of V-ATPase leads to accumulation of the 52 and 44 KDa immature CTSD forms (iCTSD), a sign of impaired lysosomal function. (F) Chronic V-ATPase inhibition results in high levels of nuclear TFEB compared to control. (G) siRNA against TFEB reduce the level of TFEB, ATP6V0C and HES1 mRNA expression compared to control. A similar reduction in the level of N signaling is observed after knocking down the expression of the γ-secretase component, PSENEN or of the NOTCH1 receptor. In contrast, efficient knockdown of ADAM10, which is dispensable for ligand-independent signaling, does not reduce HES1 levels. (H) Chronic treatment with both BafA1 and FCCP compromises γ-secretase substrate cleavage. (I) Chronic inhibition of the V-ATPase reduces the levels of expression of N receptors among other genes.

Mentions: We and others previously reported that V-ATPase sustains N signaling activity in Drosophila epithelial tissue.9,10 Recently, we have shown that mild reduction of V-ATPase activity affects N signaling also in human MCF10A breast epithelial cells in culture, which endogenously express N receptors,7 suggesting that MCF10A cells could be a relevant model to study the impact of the TFEB-V-ATPase axis on functionality of the endolysosomal system and on N signaling activation. Acute treatment of MCF10A cells with the V-ATPase inhibitor bafilomycin A1 (BafA1) or with FCCP, which dissipates lysosomal acidification without impairing V-ATPase activity,44 led to strong reduction in LTR incorporation when compared to mock-treated controls or cells treated with the γ-secretase inhibitor (GSI) N-[2S-(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester (DAPT), that blocks N signaling activation (Fig. 8A). In sheer contrast, chronic reduction of V-ATPase activity by treatment with a very low dose of BafA1 (3 nM) over 7 d, a regimen that did not prevent MCF10A growth (Fig. S2J),7 caused expansion of the lysosomal compartment, as revealed by LTR incorporation or LAMP1 staining. Similar effects were found 7 d after siRNA-mediated downregulation of ATP6V0C/V-ATPase subunit c expression (Fig. 8B and C). In these conditions, phosporylation of RPS6KB/S6K and maturation of CTSD (cathepsin D), which are measures of MTOR signaling and lysosomal activity, respectively,13,45 were impaired (Fig. 8D and E). Consistent with the described compensatory feedback loop between TFEB and V-ATPase activity,27 the effects described above correlated with increased localization of TFEB in the nucleus (Fig. 8F). These data indicate that in MCF10A cells lysosomal compartment size and function are inversely correlated by the activity of the TFEB-V-ATPase axis.Figure 8.


Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster.

Tognon E, Kobia F, Busi I, Fumagalli A, De Masi F, Vaccari T - Autophagy (2016)

The TFEB-V-ATPase axis regulates lysosomal function and basal N signaling in human cells. (A) LTR assay indicates that acute treatment with both BafA1 and FCCP, which dissipates lysosomal pH independently of V-ATPase, impairs acidification in MCF10A cells. (B and C) Chronic inhibition of V-ATPase or KD of ATP6V0C in contrast causes an expansion of the acidified (B) and LAMP1-positive lysosomal organelles (C). (D) Relative to the control, western blot analysis of MTOR signaling of lysate from MCF10A cells treated as indicated reveals a reduction in the level of phosphorylated RPS6KB (pS6K), a measure of active MTOR signaling. (E) Western blot analysis of MCF10A lysates treated as indicated shows that chronic inhibition of V-ATPase leads to accumulation of the 52 and 44 KDa immature CTSD forms (iCTSD), a sign of impaired lysosomal function. (F) Chronic V-ATPase inhibition results in high levels of nuclear TFEB compared to control. (G) siRNA against TFEB reduce the level of TFEB, ATP6V0C and HES1 mRNA expression compared to control. A similar reduction in the level of N signaling is observed after knocking down the expression of the γ-secretase component, PSENEN or of the NOTCH1 receptor. In contrast, efficient knockdown of ADAM10, which is dispensable for ligand-independent signaling, does not reduce HES1 levels. (H) Chronic treatment with both BafA1 and FCCP compromises γ-secretase substrate cleavage. (I) Chronic inhibition of the V-ATPase reduces the levels of expression of N receptors among other genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836007&req=5

f0008: The TFEB-V-ATPase axis regulates lysosomal function and basal N signaling in human cells. (A) LTR assay indicates that acute treatment with both BafA1 and FCCP, which dissipates lysosomal pH independently of V-ATPase, impairs acidification in MCF10A cells. (B and C) Chronic inhibition of V-ATPase or KD of ATP6V0C in contrast causes an expansion of the acidified (B) and LAMP1-positive lysosomal organelles (C). (D) Relative to the control, western blot analysis of MTOR signaling of lysate from MCF10A cells treated as indicated reveals a reduction in the level of phosphorylated RPS6KB (pS6K), a measure of active MTOR signaling. (E) Western blot analysis of MCF10A lysates treated as indicated shows that chronic inhibition of V-ATPase leads to accumulation of the 52 and 44 KDa immature CTSD forms (iCTSD), a sign of impaired lysosomal function. (F) Chronic V-ATPase inhibition results in high levels of nuclear TFEB compared to control. (G) siRNA against TFEB reduce the level of TFEB, ATP6V0C and HES1 mRNA expression compared to control. A similar reduction in the level of N signaling is observed after knocking down the expression of the γ-secretase component, PSENEN or of the NOTCH1 receptor. In contrast, efficient knockdown of ADAM10, which is dispensable for ligand-independent signaling, does not reduce HES1 levels. (H) Chronic treatment with both BafA1 and FCCP compromises γ-secretase substrate cleavage. (I) Chronic inhibition of the V-ATPase reduces the levels of expression of N receptors among other genes.
Mentions: We and others previously reported that V-ATPase sustains N signaling activity in Drosophila epithelial tissue.9,10 Recently, we have shown that mild reduction of V-ATPase activity affects N signaling also in human MCF10A breast epithelial cells in culture, which endogenously express N receptors,7 suggesting that MCF10A cells could be a relevant model to study the impact of the TFEB-V-ATPase axis on functionality of the endolysosomal system and on N signaling activation. Acute treatment of MCF10A cells with the V-ATPase inhibitor bafilomycin A1 (BafA1) or with FCCP, which dissipates lysosomal acidification without impairing V-ATPase activity,44 led to strong reduction in LTR incorporation when compared to mock-treated controls or cells treated with the γ-secretase inhibitor (GSI) N-[2S-(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester (DAPT), that blocks N signaling activation (Fig. 8A). In sheer contrast, chronic reduction of V-ATPase activity by treatment with a very low dose of BafA1 (3 nM) over 7 d, a regimen that did not prevent MCF10A growth (Fig. S2J),7 caused expansion of the lysosomal compartment, as revealed by LTR incorporation or LAMP1 staining. Similar effects were found 7 d after siRNA-mediated downregulation of ATP6V0C/V-ATPase subunit c expression (Fig. 8B and C). In these conditions, phosporylation of RPS6KB/S6K and maturation of CTSD (cathepsin D), which are measures of MTOR signaling and lysosomal activity, respectively,13,45 were impaired (Fig. 8D and E). Consistent with the described compensatory feedback loop between TFEB and V-ATPase activity,27 the effects described above correlated with increased localization of TFEB in the nucleus (Fig. 8F). These data indicate that in MCF10A cells lysosomal compartment size and function are inversely correlated by the activity of the TFEB-V-ATPase axis.Figure 8.

Bottom Line: In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development.Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity.Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

View Article: PubMed Central - PubMed

Affiliation: a IFOM - FIRC Institute of Molecular Oncology , Milan , Italy.

ABSTRACT
In vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H(+)-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

No MeSH data available.


Related in: MedlinePlus