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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

Mitf controls transcription of V-ATPase genes. (A) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes. Arrowheads point to areas with distinctive pattern of expression. (B) Schematics of the structure and function of V-ATPase with positioning of the subunits analyzed. (C) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes upon overexpression of Mitf. Note that V-ATPase subunit expression but not Lamp1 expression is controlled by Mitf. (D) Transcript levels of the indicated putative Mitf targets in wing discs by Q-PCR. RpL32 has been used as housekeeping control. The values represents means ± s.d of 2 independent experiments.
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f0003: Mitf controls transcription of V-ATPase genes. (A) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes. Arrowheads point to areas with distinctive pattern of expression. (B) Schematics of the structure and function of V-ATPase with positioning of the subunits analyzed. (C) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes upon overexpression of Mitf. Note that V-ATPase subunit expression but not Lamp1 expression is controlled by Mitf. (D) Transcript levels of the indicated putative Mitf targets in wing discs by Q-PCR. RpL32 has been used as housekeeping control. The values represents means ± s.d of 2 independent experiments.

Mentions: To assess whether Mitf acts as a master regulator of lysosomal gene expression in Drosophila, we took advantage of available GFP and YFP knock-in lines in the Drosophila orthologs of a subset of TFEB target genes (Fig. 3A). We used 3 lines with insertions in genes encoding components of the cytoplasmic V1 sector of V-ATPase: YFP::Vha55/V1B, GFP::VhaSFD/V1H, and GFP::Vha13/V1G. Two lines with insertions in genes encoding components of the membrane-embedded V0 sector: GFP::Vha16-1/V0c and GFP::VhaAC45/AC45 (see Fig. 3B for a schematic of the V-ATPase). Finally, we used YFP::Lamp1, tagging the single Drosophila gene whose product is the ortholog of mammalian Lysosomal-associated membrane protein 1 (LAMP1).25,28,29 Complementation analysis with existing mutants and deficiencies reveals that most knock-in lines in V-ATPase genes behaved as loss-of-function mutants (Table S1), but that all were viable and fertile in heterozygosis. The absence of dominant effects indicates that the exon traps can be used in heterozygosity to study regulation of expression in vivo.Figure 3.


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)

Mitf controls transcription of V-ATPase genes. (A) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes. Arrowheads point to areas with distinctive pattern of expression. (B) Schematics of the structure and function of V-ATPase with positioning of the subunits analyzed. (C) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes upon overexpression of Mitf. Note that V-ATPase subunit expression but not Lamp1 expression is controlled by Mitf. (D) Transcript levels of the indicated putative Mitf targets in wing discs by Q-PCR. RpL32 has been used as housekeeping control. The values represents means ± s.d of 2 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0003: Mitf controls transcription of V-ATPase genes. (A) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes. Arrowheads point to areas with distinctive pattern of expression. (B) Schematics of the structure and function of V-ATPase with positioning of the subunits analyzed. (C) Pattern of expression in wing discs of the tagged lines for the indicated subset of Drosophila orthologs of TFEB target genes upon overexpression of Mitf. Note that V-ATPase subunit expression but not Lamp1 expression is controlled by Mitf. (D) Transcript levels of the indicated putative Mitf targets in wing discs by Q-PCR. RpL32 has been used as housekeeping control. The values represents means ± s.d of 2 independent experiments.
Mentions: To assess whether Mitf acts as a master regulator of lysosomal gene expression in Drosophila, we took advantage of available GFP and YFP knock-in lines in the Drosophila orthologs of a subset of TFEB target genes (Fig. 3A). We used 3 lines with insertions in genes encoding components of the cytoplasmic V1 sector of V-ATPase: YFP::Vha55/V1B, GFP::VhaSFD/V1H, and GFP::Vha13/V1G. Two lines with insertions in genes encoding components of the membrane-embedded V0 sector: GFP::Vha16-1/V0c and GFP::VhaAC45/AC45 (see Fig. 3B for a schematic of the V-ATPase). Finally, we used YFP::Lamp1, tagging the single Drosophila gene whose product is the ortholog of mammalian Lysosomal-associated membrane protein 1 (LAMP1).25,28,29 Complementation analysis with existing mutants and deficiencies reveals that most knock-in lines in V-ATPase genes behaved as loss-of-function mutants (Table S1), but that all were viable and fertile in heterozygosis. The absence of dominant effects indicates that the exon traps can be used in heterozygosity to study regulation of expression in vivo.Figure 3.

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