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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 regulates lysosomal biogenesis. (A) Wing discs of the indicated genotypes subjected to incorporation of LTR. High magnifications of portions of the overexpressing tissue are shown below the discs. (B) Quantification of LTR puncta density, size and normalized intensity are graphed for the indicated samples. Overexpression of Mitf or Mitf DN increases LTR incorporation in wing disc cells, while overexpression of activated NICD slightly reduces it. (C and D) Immunolocalization of ref(2)P and Atg8a in discs of the indicated genotype. Side panels are higher magnifications of the overexpressing tissue with insets shown below them.
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f0002: Mitf regulates lysosomal biogenesis. (A) Wing discs of the indicated genotypes subjected to incorporation of LTR. High magnifications of portions of the overexpressing tissue are shown below the discs. (B) Quantification of LTR puncta density, size and normalized intensity are graphed for the indicated samples. Overexpression of Mitf or Mitf DN increases LTR incorporation in wing disc cells, while overexpression of activated NICD slightly reduces it. (C and D) Immunolocalization of ref(2)P and Atg8a in discs of the indicated genotype. Side panels are higher magnifications of the overexpressing tissue with insets shown below them.

Mentions: To test whether Drosophila Mitf promotes activation of catabolic pathways, we labeled acidified lysosomes in wild-type and Mitf-overexpressing discs with the acidophilic dye LysoTracker Red (LTR). Compared to the control, Mitf overexpression increased the size of LTR-positive puncta, indicating that Mitf might control lysosomal biogenesis (Fig. 2A, quantification in B). To determine whether Mitf regulates autophagy, we labeled discs to detect ref(2)P (human SQSTM1/p62), and Atg8a (human MAP1LC3/LC3). Overexpression of Mitf led to a mild increase in the ref(2)P and Atg8a signal (Fig. 2C and D), relative to the basal low levels observed in control discs, suggesting that Mitf might affect autophagy. Finally, we find that overexpression of Mitf in the wing discs led to formation of a low number of apoptotic cells, as shown by expression of activated product of the gene Decay/caspase 3, which are normally not present in control discs (Fig. S1D).Figure 2.


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 regulates lysosomal biogenesis. (A) Wing discs of the indicated genotypes subjected to incorporation of LTR. High magnifications of portions of the overexpressing tissue are shown below the discs. (B) Quantification of LTR puncta density, size and normalized intensity are graphed for the indicated samples. Overexpression of Mitf or Mitf DN increases LTR incorporation in wing disc cells, while overexpression of activated NICD slightly reduces it. (C and D) Immunolocalization of ref(2)P and Atg8a in discs of the indicated genotype. Side panels are higher magnifications of the overexpressing tissue with insets shown below them.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0002: Mitf regulates lysosomal biogenesis. (A) Wing discs of the indicated genotypes subjected to incorporation of LTR. High magnifications of portions of the overexpressing tissue are shown below the discs. (B) Quantification of LTR puncta density, size and normalized intensity are graphed for the indicated samples. Overexpression of Mitf or Mitf DN increases LTR incorporation in wing disc cells, while overexpression of activated NICD slightly reduces it. (C and D) Immunolocalization of ref(2)P and Atg8a in discs of the indicated genotype. Side panels are higher magnifications of the overexpressing tissue with insets shown below them.
Mentions: To test whether Drosophila Mitf promotes activation of catabolic pathways, we labeled acidified lysosomes in wild-type and Mitf-overexpressing discs with the acidophilic dye LysoTracker Red (LTR). Compared to the control, Mitf overexpression increased the size of LTR-positive puncta, indicating that Mitf might control lysosomal biogenesis (Fig. 2A, quantification in B). To determine whether Mitf regulates autophagy, we labeled discs to detect ref(2)P (human SQSTM1/p62), and Atg8a (human MAP1LC3/LC3). Overexpression of Mitf led to a mild increase in the ref(2)P and Atg8a signal (Fig. 2C and D), relative to the basal low levels observed in control discs, suggesting that Mitf might affect autophagy. Finally, we find that overexpression of Mitf in the wing discs led to formation of a low number of apoptotic cells, as shown by expression of activated product of the gene Decay/caspase 3, which are normally not present in control discs (Fig. S1D).Figure 2.

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