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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 localization in wing imaginal discs. (A) In situ hybridization using labeled sense and antisense RNA probe for Mitf transcripts in wing discs from yellow white (control) animals and from animals overexpressing Mitf in wing disc (ms1096 Mitf). The sense probe has been used as a negative control. Dorsal is up, anterior to the left. All wing discs shown in figures are oriented as such. (B) Control wing disc and wing disc overexpressing Mitf or Mitf DN stained with anti- Drosophila Mitf antibody. (C and D) High magnifications of wing pouch tissue of the indicated genotype stained as indicated. Note that Mitf is present in the nucleus when overexpressed and in some lysosomes (close-up in D). (E) Quantification of colocalization of YFP:Lamp1 and Mitf. Colocalization across 80 regions of interest (ROI) per discs is shown as overlap. Proximity denotes nonoverlapping signal that falls within the ROI. Averages of 3 discs per genotype are graphed. -ve controls are obtained by rotating one of the 2 channels by 90 degrees.
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f0001: Mitf localization in wing imaginal discs. (A) In situ hybridization using labeled sense and antisense RNA probe for Mitf transcripts in wing discs from yellow white (control) animals and from animals overexpressing Mitf in wing disc (ms1096 Mitf). The sense probe has been used as a negative control. Dorsal is up, anterior to the left. All wing discs shown in figures are oriented as such. (B) Control wing disc and wing disc overexpressing Mitf or Mitf DN stained with anti- Drosophila Mitf antibody. (C and D) High magnifications of wing pouch tissue of the indicated genotype stained as indicated. Note that Mitf is present in the nucleus when overexpressed and in some lysosomes (close-up in D). (E) Quantification of colocalization of YFP:Lamp1 and Mitf. Colocalization across 80 regions of interest (ROI) per discs is shown as overlap. Proximity denotes nonoverlapping signal that falls within the ROI. Averages of 3 discs per genotype are graphed. -ve controls are obtained by rotating one of the 2 channels by 90 degrees.

Mentions: To explore whether Drosophila Mitf possesses functions of mammalian TFEB in vivo, we first characterized expression and function of endogenous and overexpressed Mitf in the wing imaginal disc of Drosophila melanogaster, an epithelial organ that will give rise to the adult thorax and wing (Fig. 1). By in situ hybridization, we observed that endogenous Mitf mRNA is expressed at low uniform level in wing disc tissue (Fig. 1A). This finding was consistent with expression of endogenous Mitf protein (Fig. 1B), using a specific antibody that we have generated (Fig. S1A; Material and Methods). Upon overexpression of both a functional Mitf and a dominant negative form that cannot bind DNA (Mitf DN)24 in the wing pouch with Bxms1096-Gal4 (ms1096; Fig. 1B, Fig. S1B and C), large cytoplasmic puncta and nuclear localization in a subset of cells were detected (Fig. 1C and D). Approximately half of the puncta were positive for YFP-Lamp1, which localizes to lysosomes,25 with a slight increase when Mitf is overexpressed (Fig. 1D, quantification in E). These data are consistent with the reported TFEB localization in mammalian cells.26,27Figure 1.


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 localization in wing imaginal discs. (A) In situ hybridization using labeled sense and antisense RNA probe for Mitf transcripts in wing discs from yellow white (control) animals and from animals overexpressing Mitf in wing disc (ms1096 Mitf). The sense probe has been used as a negative control. Dorsal is up, anterior to the left. All wing discs shown in figures are oriented as such. (B) Control wing disc and wing disc overexpressing Mitf or Mitf DN stained with anti- Drosophila Mitf antibody. (C and D) High magnifications of wing pouch tissue of the indicated genotype stained as indicated. Note that Mitf is present in the nucleus when overexpressed and in some lysosomes (close-up in D). (E) Quantification of colocalization of YFP:Lamp1 and Mitf. Colocalization across 80 regions of interest (ROI) per discs is shown as overlap. Proximity denotes nonoverlapping signal that falls within the ROI. Averages of 3 discs per genotype are graphed. -ve controls are obtained by rotating one of the 2 channels by 90 degrees.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0001: Mitf localization in wing imaginal discs. (A) In situ hybridization using labeled sense and antisense RNA probe for Mitf transcripts in wing discs from yellow white (control) animals and from animals overexpressing Mitf in wing disc (ms1096 Mitf). The sense probe has been used as a negative control. Dorsal is up, anterior to the left. All wing discs shown in figures are oriented as such. (B) Control wing disc and wing disc overexpressing Mitf or Mitf DN stained with anti- Drosophila Mitf antibody. (C and D) High magnifications of wing pouch tissue of the indicated genotype stained as indicated. Note that Mitf is present in the nucleus when overexpressed and in some lysosomes (close-up in D). (E) Quantification of colocalization of YFP:Lamp1 and Mitf. Colocalization across 80 regions of interest (ROI) per discs is shown as overlap. Proximity denotes nonoverlapping signal that falls within the ROI. Averages of 3 discs per genotype are graphed. -ve controls are obtained by rotating one of the 2 channels by 90 degrees.
Mentions: To explore whether Drosophila Mitf possesses functions of mammalian TFEB in vivo, we first characterized expression and function of endogenous and overexpressed Mitf in the wing imaginal disc of Drosophila melanogaster, an epithelial organ that will give rise to the adult thorax and wing (Fig. 1). By in situ hybridization, we observed that endogenous Mitf mRNA is expressed at low uniform level in wing disc tissue (Fig. 1A). This finding was consistent with expression of endogenous Mitf protein (Fig. 1B), using a specific antibody that we have generated (Fig. S1A; Material and Methods). Upon overexpression of both a functional Mitf and a dominant negative form that cannot bind DNA (Mitf DN)24 in the wing pouch with Bxms1096-Gal4 (ms1096; Fig. 1B, Fig. S1B and C), large cytoplasmic puncta and nuclear localization in a subset of cells were detected (Fig. 1C and D). Approximately half of the puncta were positive for YFP-Lamp1, which localizes to lysosomes,25 with a slight increase when Mitf is overexpressed (Fig. 1D, quantification in E). These data are consistent with the reported TFEB localization in mammalian cells.26,27Figure 1.

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