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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

Vha16-1 expression is elevated in SOPs. (A) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in ac-expressing tissue. (B) In situ hybridization using labeled sense and antisense RNA probes for Vha16-1 transcripts in control wing discs. Note the high expression in 2 stripes of tissue abutting the anterior D/V boundary (arrow) when using the antisense probe. (C) Schematic representation of the patterning of a larval wing disc. The patterning features relevant for this study are indicated. Dl, N ligand Delta. (D and E) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in neur-LacZ-positive cell (D, arrowheads) and low in E(spl)m4 -positive cells (E, arrowheads). Arrowheads in E indicate examples of SOP cells that are not E(spl)m4-positive and have high GFP::Vha16-1 expression. (F and G) In situ hybridization using an antisense RNA probe for Vha16-1 transcripts of discs overexpressing NICD or Mitf. Note the very different transcriptional modulation of Vha16-1 expression upon overexpression. (H) Discs of the indicated genotype stained to detect the N target cut (ct). Note that GFP::Vha16-1 expression is very low and not patterned in ct -positive tissue. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (I and J) GFP::Vha16-1 wing disc overexpressing the N target E(Spl) genes m8, and m4 under ms1096-Gal4. Note that overexpression of E(Spl)m8 results in loss of sensory organs and of GFP::Vha16-1 expression at the anterior margin, while overexpression of E(Spl)m4 leads to formation of ectopic SOPs expressing GFP::Vha16-1. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (K) Pupal nota of the indicated genotype dissected 20 h after puparium formation. Note that elevated GFP::Vha16-1 expression is maintained along the SOP lineage. (L) High magnifications of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. The image is a maximal projection of several sections. Note that overexpression of Mitf DN disrupts the pattern of GFP::Vha16-1 expression in SOPs and leads to missing and ectopic SOPs (arrowheads).
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f0004: Vha16-1 expression is elevated in SOPs. (A) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in ac-expressing tissue. (B) In situ hybridization using labeled sense and antisense RNA probes for Vha16-1 transcripts in control wing discs. Note the high expression in 2 stripes of tissue abutting the anterior D/V boundary (arrow) when using the antisense probe. (C) Schematic representation of the patterning of a larval wing disc. The patterning features relevant for this study are indicated. Dl, N ligand Delta. (D and E) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in neur-LacZ-positive cell (D, arrowheads) and low in E(spl)m4 -positive cells (E, arrowheads). Arrowheads in E indicate examples of SOP cells that are not E(spl)m4-positive and have high GFP::Vha16-1 expression. (F and G) In situ hybridization using an antisense RNA probe for Vha16-1 transcripts of discs overexpressing NICD or Mitf. Note the very different transcriptional modulation of Vha16-1 expression upon overexpression. (H) Discs of the indicated genotype stained to detect the N target cut (ct). Note that GFP::Vha16-1 expression is very low and not patterned in ct -positive tissue. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (I and J) GFP::Vha16-1 wing disc overexpressing the N target E(Spl) genes m8, and m4 under ms1096-Gal4. Note that overexpression of E(Spl)m8 results in loss of sensory organs and of GFP::Vha16-1 expression at the anterior margin, while overexpression of E(Spl)m4 leads to formation of ectopic SOPs expressing GFP::Vha16-1. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (K) Pupal nota of the indicated genotype dissected 20 h after puparium formation. Note that elevated GFP::Vha16-1 expression is maintained along the SOP lineage. (L) High magnifications of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. The image is a maximal projection of several sections. Note that overexpression of Mitf DN disrupts the pattern of GFP::Vha16-1 expression in SOPs and leads to missing and ectopic SOPs (arrowheads).

Mentions: Surprisingly, while expression of most knock-in lines appeared uniform in wing imaginal discs, expression of GFP::Vha16-1, GFP::Vha13 and YFP::Lamp1 appeared patterned (Fig. 3A), suggesting that function of the lysosome-associated proteins might be developmentally regulated in epithelial tissue. One exclusive feature of GFP::Vha16-1 expression, that is not displayed by GFP::Vha13, is elevated expression in proneural clusters (PNCs), as revealed by colocalization of GFP::Vha16-1 with the PNC marker ac (achaete) (Fig. 4A; Fig. S1E).30 The GFP::Vha16-1 signal is specific to Vha16-1 as the GFP-exon insertion tags Vha16-1 protein and did not alter significantly mRNA expression (Fig. S1F to H). Also, the GFP::Vha16-1 signal in PNCs is unlikely due to morphology differences between SOPs and surrounding cells or to the transmembrane nature of the protein. In fact, no pattern was observed in wing discs of GFP::CG8668 larvae (Fig. S1I), in which GFP is tagging the gene coding for the plasma membrane transmembrane glycosyl transferase CG8668/Resille.31 Finally, elevated expression of endogenous Vha16-1 mRNA could be observed by in situ hybridization (Fig. 4B, arrow).Figure 4.


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)

Vha16-1 expression is elevated in SOPs. (A) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in ac-expressing tissue. (B) In situ hybridization using labeled sense and antisense RNA probes for Vha16-1 transcripts in control wing discs. Note the high expression in 2 stripes of tissue abutting the anterior D/V boundary (arrow) when using the antisense probe. (C) Schematic representation of the patterning of a larval wing disc. The patterning features relevant for this study are indicated. Dl, N ligand Delta. (D and E) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in neur-LacZ-positive cell (D, arrowheads) and low in E(spl)m4 -positive cells (E, arrowheads). Arrowheads in E indicate examples of SOP cells that are not E(spl)m4-positive and have high GFP::Vha16-1 expression. (F and G) In situ hybridization using an antisense RNA probe for Vha16-1 transcripts of discs overexpressing NICD or Mitf. Note the very different transcriptional modulation of Vha16-1 expression upon overexpression. (H) Discs of the indicated genotype stained to detect the N target cut (ct). Note that GFP::Vha16-1 expression is very low and not patterned in ct -positive tissue. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (I and J) GFP::Vha16-1 wing disc overexpressing the N target E(Spl) genes m8, and m4 under ms1096-Gal4. Note that overexpression of E(Spl)m8 results in loss of sensory organs and of GFP::Vha16-1 expression at the anterior margin, while overexpression of E(Spl)m4 leads to formation of ectopic SOPs expressing GFP::Vha16-1. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (K) Pupal nota of the indicated genotype dissected 20 h after puparium formation. Note that elevated GFP::Vha16-1 expression is maintained along the SOP lineage. (L) High magnifications of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. The image is a maximal projection of several sections. Note that overexpression of Mitf DN disrupts the pattern of GFP::Vha16-1 expression in SOPs and leads to missing and ectopic SOPs (arrowheads).
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Related In: Results  -  Collection

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f0004: Vha16-1 expression is elevated in SOPs. (A) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in ac-expressing tissue. (B) In situ hybridization using labeled sense and antisense RNA probes for Vha16-1 transcripts in control wing discs. Note the high expression in 2 stripes of tissue abutting the anterior D/V boundary (arrow) when using the antisense probe. (C) Schematic representation of the patterning of a larval wing disc. The patterning features relevant for this study are indicated. Dl, N ligand Delta. (D and E) A high magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. Note that GFP::Vha16-1 expression is elevated in neur-LacZ-positive cell (D, arrowheads) and low in E(spl)m4 -positive cells (E, arrowheads). Arrowheads in E indicate examples of SOP cells that are not E(spl)m4-positive and have high GFP::Vha16-1 expression. (F and G) In situ hybridization using an antisense RNA probe for Vha16-1 transcripts of discs overexpressing NICD or Mitf. Note the very different transcriptional modulation of Vha16-1 expression upon overexpression. (H) Discs of the indicated genotype stained to detect the N target cut (ct). Note that GFP::Vha16-1 expression is very low and not patterned in ct -positive tissue. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (I and J) GFP::Vha16-1 wing disc overexpressing the N target E(Spl) genes m8, and m4 under ms1096-Gal4. Note that overexpression of E(Spl)m8 results in loss of sensory organs and of GFP::Vha16-1 expression at the anterior margin, while overexpression of E(Spl)m4 leads to formation of ectopic SOPs expressing GFP::Vha16-1. Insets enlarging corresponding areas of the pouch are show on the sides of each disc. (K) Pupal nota of the indicated genotype dissected 20 h after puparium formation. Note that elevated GFP::Vha16-1 expression is maintained along the SOP lineage. (L) High magnifications of the anterior part of the wing pouch of wing discs of the indicated genotypes stained as indicated. The image is a maximal projection of several sections. Note that overexpression of Mitf DN disrupts the pattern of GFP::Vha16-1 expression in SOPs and leads to missing and ectopic SOPs (arrowheads).
Mentions: Surprisingly, while expression of most knock-in lines appeared uniform in wing imaginal discs, expression of GFP::Vha16-1, GFP::Vha13 and YFP::Lamp1 appeared patterned (Fig. 3A), suggesting that function of the lysosome-associated proteins might be developmentally regulated in epithelial tissue. One exclusive feature of GFP::Vha16-1 expression, that is not displayed by GFP::Vha13, is elevated expression in proneural clusters (PNCs), as revealed by colocalization of GFP::Vha16-1 with the PNC marker ac (achaete) (Fig. 4A; Fig. S1E).30 The GFP::Vha16-1 signal is specific to Vha16-1 as the GFP-exon insertion tags Vha16-1 protein and did not alter significantly mRNA expression (Fig. S1F to H). Also, the GFP::Vha16-1 signal in PNCs is unlikely due to morphology differences between SOPs and surrounding cells or to the transmembrane nature of the protein. In fact, no pattern was observed in wing discs of GFP::CG8668 larvae (Fig. S1I), in which GFP is tagging the gene coding for the plasma membrane transmembrane glycosyl transferase CG8668/Resille.31 Finally, elevated expression of endogenous Vha16-1 mRNA could be observed by in situ hybridization (Fig. 4B, arrow).Figure 4.

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