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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 endolysosomal system at sites of PNC development is distinctive. (A and E) High magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes, stained as indicated. Arrowheads point to the approximate location of PNC. Note that compared to surrounding epithelial cells, PNC cells show a slightly higher amount of YFP::Lamp1-positive lysosomes (A), a higher number of acidified organelles (B) and of GFP-hLAMP1 puncta (C), overall less N (D) and more endolysosomal N (E).
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f0007: The endolysosomal system at sites of PNC development is distinctive. (A and E) High magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes, stained as indicated. Arrowheads point to the approximate location of PNC. Note that compared to surrounding epithelial cells, PNC cells show a slightly higher amount of YFP::Lamp1-positive lysosomes (A), a higher number of acidified organelles (B) and of GFP-hLAMP1 puncta (C), overall less N (D) and more endolysosomal N (E).

Mentions: Is the function of V-ATPase and Mitf in proneural development linked to regulation of the endolysosomal system? To assess this, we tested whether PNCs possess an endolysosomal compartment that is different to that of surrounding cells. Consistent with observations in Figs. 1 and 2, expression of YFP::Lamp1 was mildly upregulated in PNC regions abutting the D/V margin, whereas we did not detect significant differences in endogenous expression or localization of Mitf across the wing pouch (Fig. 7A). To further assess lysosome abundance, we labeled acidified compartments in the disc with LTR. We found that LTR incorporation was high in PNCs compared to other epithelial cells of the disc, suggesting that PNCs might possess more lysosomes than surrounding cells (Fig. 7B). Upon ubiquitous expression of GFP-hLamp1 in the disc with actin-GAL4, we found that PNCs were more GFP-positive than surrounding cells (Fig. 7C). GFP-hLamp1 is a lysosome-anchored GFP form that has been developed as a sensor for lysosomal degradation, because the GFP is exposed to the lysosomal lumen, where it is degraded by resident hydrolases. In conditions of impaired lysosome function, GFP-hLamp1 is less degraded leading to a high GFP signal.9 Thus, PNC cells possess lysosomes with less degradative capacity than surrounding cells. In contrast, localization of Syx7 (Syntaxin7), a marker of early endosomes,40 was uniform across the disc tissue (Fig. 7D). Consistent with previous evidence indicating transcriptional down-regulation of N in PNCs,41,42 we found that overall N protein levels in the endolysosomal system of PNCs were lower than in the rest of the disc (Fig. 7D). We next determined N stability in the endolysosomal system of PNCs and pro-vein cells. To this end, we analyzed expression of Ni-GFP4-Cherry5, a functional N form tagged with fast-maturing, pH-sensitive GFP and a slow-maturing, pH-insensitive mCherry. It has been recently reported that the GFP signal of such N form indicates the newly synthetized N found at the plasma membrane, while the mCherry signal highlights old N molecules that reach the endolysosomal compartment on their way to degradation.43 Using this sensor, we found that the amount of mCherry-positive N in the endolysosomal compartment was higher in the PNCs than in surrounding cells (Fig. 7E). Overall, these data indicate that PNCs might possess an expanded, less degradative and more N -rich lysosomal system than surrounding cells.Figure 7.


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 endolysosomal system at sites of PNC development is distinctive. (A and E) High magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes, stained as indicated. Arrowheads point to the approximate location of PNC. Note that compared to surrounding epithelial cells, PNC cells show a slightly higher amount of YFP::Lamp1-positive lysosomes (A), a higher number of acidified organelles (B) and of GFP-hLAMP1 puncta (C), overall less N (D) and more endolysosomal N (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0007: The endolysosomal system at sites of PNC development is distinctive. (A and E) High magnification of the anterior part of the wing pouch of wing discs of the indicated genotypes, stained as indicated. Arrowheads point to the approximate location of PNC. Note that compared to surrounding epithelial cells, PNC cells show a slightly higher amount of YFP::Lamp1-positive lysosomes (A), a higher number of acidified organelles (B) and of GFP-hLAMP1 puncta (C), overall less N (D) and more endolysosomal N (E).
Mentions: Is the function of V-ATPase and Mitf in proneural development linked to regulation of the endolysosomal system? To assess this, we tested whether PNCs possess an endolysosomal compartment that is different to that of surrounding cells. Consistent with observations in Figs. 1 and 2, expression of YFP::Lamp1 was mildly upregulated in PNC regions abutting the D/V margin, whereas we did not detect significant differences in endogenous expression or localization of Mitf across the wing pouch (Fig. 7A). To further assess lysosome abundance, we labeled acidified compartments in the disc with LTR. We found that LTR incorporation was high in PNCs compared to other epithelial cells of the disc, suggesting that PNCs might possess more lysosomes than surrounding cells (Fig. 7B). Upon ubiquitous expression of GFP-hLamp1 in the disc with actin-GAL4, we found that PNCs were more GFP-positive than surrounding cells (Fig. 7C). GFP-hLamp1 is a lysosome-anchored GFP form that has been developed as a sensor for lysosomal degradation, because the GFP is exposed to the lysosomal lumen, where it is degraded by resident hydrolases. In conditions of impaired lysosome function, GFP-hLamp1 is less degraded leading to a high GFP signal.9 Thus, PNC cells possess lysosomes with less degradative capacity than surrounding cells. In contrast, localization of Syx7 (Syntaxin7), a marker of early endosomes,40 was uniform across the disc tissue (Fig. 7D). Consistent with previous evidence indicating transcriptional down-regulation of N in PNCs,41,42 we found that overall N protein levels in the endolysosomal system of PNCs were lower than in the rest of the disc (Fig. 7D). We next determined N stability in the endolysosomal system of PNCs and pro-vein cells. To this end, we analyzed expression of Ni-GFP4-Cherry5, a functional N form tagged with fast-maturing, pH-sensitive GFP and a slow-maturing, pH-insensitive mCherry. It has been recently reported that the GFP signal of such N form indicates the newly synthetized N found at the plasma membrane, while the mCherry signal highlights old N molecules that reach the endolysosomal compartment on their way to degradation.43 Using this sensor, we found that the amount of mCherry-positive N in the endolysosomal compartment was higher in the PNCs than in surrounding cells (Fig. 7E). Overall, these data indicate that PNCs might possess an expanded, less degradative and more N -rich lysosomal system than surrounding cells.Figure 7.

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