Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in <i>Drosophila melanogaster</i>

<p>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 <i>Drosophila</i>, controls expression of vacuolar-type H⁺-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of <i>Vha16-1</i> and <i>Vha13</i>, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, <i>Vha16-1</i> 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 <i>Drosophila</i>, in human breast epithelial cells we observe that impairment of the <i>Vha16-1</i> human ortholog <i>ATP6V0C</i> 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.</p

ESCRT-0 is not required for ectopic Notch activation and tumor suppression in Drosophila.

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis

<i>Hrs</i>, <i>Stam</i> or <i>Hrs</i>, <i>Stam</i> double mutant tissue do not display altered tissue architecture.

<p>(A–H) Epithelial morphology of mosaic FE cells (A–D) and eye discs (E–H) revealed by phalloidin staining to detect F-actin. Follicle cells of 5–7 stage egg chambers homozygous for the mutations (GFP-negative) show normal epithelial architecture compared to WT (GFP-positive). Eye disc cells homozygous for the mutations (GFP-negative) do not show any disruption of tissue architecture. (I–L) High magnification of a region of mosaic eye imaginal discs. Homozygous cells are marked by the absence of GFP. Apoptotic Caspase-3 (magenta) is activated cell autonomously in a subset of <i>Hrs</i> and <i>Stam</i> as well as <i>Hrs</i>, <i>Stam</i> mutant cells, compared to WT. (M–P) WT and predominantly mutant eye-antennal discs for the indicated gene stained with phalloidin revealed that <i>Hrs</i>, <i>Stam</i> mutant discs form morphologically normal eye-antennal discs. (Q–T) Adult eyes deriving from mosaic discs of the indicated genotype. Clones or WT (Q) or mutant cells (R–T) are marked by the absence of red pigment in bright field images indicating that mutant tissue can form photoreceptors.</p

ESCRT-0 mutations lead to accumulation of ubiquitylated cargoes, as well as of Notch and Dome in endosomes.

<p>(A–F) High magnification of a region of mosaic eye imaginal discs (A–D), or of FE (E–F) shows accumulation of ubiquitylated cargoes in mutant cells (GFP-negative), as revealed by an antibody against mono- and poly- ubiquitin chains (Ubi). High magnification of the boxed areas is shown in insets. (G–H) Mutant FE cells (GFP-negative) show accumulation of the Notch receptor. Notch receptor has been revealed using anti-NICD specific to the intracellular domain of Notch. Apical as well as intracellular accumulations of Notch ICD epitope is seen in <i>Hrs</i> and <i>Stam</i> FE mutant cells. High magnification of the boxed areas is shown in insets. (I–K) Co-localization with anti Notch ECD (NECD) or Notch ICD (NICD) and Avl, marking early endosomes, in mosaic eye imaginal discs. Notch ECD is mainly accumulated in early endosomes in GFP-negative mutant tissue. (L–L’) Mosaic eye imaginal discs were stained with Ubi and anti-Domeless (Dome). <i>Hrs</i>, <i>Stam</i> mutant cells (GFP-negative) accumulate ubiquitylated cargoes and moderate levels of Dome, compared to WT. (M–O) Endocytic trafficking assay with anti-Notch ECD to label Notch at the surface of living imaginal discs. In WT tissue, after labeling (0 hrs), Notch is present mostly at the apical surface of the cell. After a 5-hour chase (5 hrs) Notch is completely degraded in WT but still present in endosomes in <i>Stam</i> mutant discs, indicating that Notch is internalized but it is not degraded.</p

Under H₂O₂-induced ox-stress elevated ceramide co-localizes with active EGFR and c-Src.

<p>A549 cells were seeded on cover-glasses, serum starved for 1 h and treated (or not) with 1 U/ml GO as indicated. After treatments, ceramide (<b>A</b> and <b>B</b>), EGFR phosphorylated on Y1173 (<b>A</b>) and Y416 phosphorylated c-Src (<b>B</b>) were localized <i>in situ</i> by IF as indicated in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023240#s4" target="_blank">Material and Methods</a>”; nuclei were stained by DAPI. White arrows indicate regions where p-Y1173 EGFR and ceramide (<b>A</b>) or p-Y416 c-Src (p-Src) and ceramide (<b>B</b>) co-localized under ox-stress (GO). Z-stack sections of cells have been discriminated by confocal microscopy: the panels show the merge of all of the Z-stack sections.</p

H₂O₂ activation of EGFR is temperature-dependent.

<p>Serum-starved A549 cells were left intact or incubated for 30 min. with 1 U/ml GO or 15 min. 100 ng/ml EGF at the indicated temperatures (T). EGFR was IPed from cell lysates and IBed for total and Tyr-phosphorylated EGFR, as indicated.</p

Cholesterol levels modulate EGFR activation by H₂O₂.

<p><b>A</b>. Serum-starved A549 cells were treated, or not, with 100 ng/ml EGF for 15 min. or 2% (w/v) MβCD for 1 h and cell lysates were IBed for total and Tyr-phosphorylated EGFR. <b>B</b> and <b>C</b>. Cells were treated with EGF as in <b>A</b> or with 1 U/ml GO for 30 min. in the absence or presence of 2 mM MβCD-cholesterol complex (CC), prepared as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023240#s4" target="_blank">Material and Methods</a>”. Cell lysates were separated by SDS-PAGE and IBed for total and Tyr-phosphorylated EGFR (<b>B</b>) or total and Y416 phosphorylated Src (<b>C</b>).</p

Proposed model of EGFR activation under ox-stress.

<p><b>A.</b> Conventional activation/dimerization of EGFR upon stimulation by the ligand EGF. <b>B.</b> Ox-stress could cause a change in membrane structure/fluidity by increasing cellular ceramide levels, which could affect cholesterol distribution. This, together with possible direct effect of ox-stress on EGFR, induces, or stabilizes, a novel acquired active conformation of EGFR that is bound by active c-Src and caveolin-1 (Cav-1). Such aberrantly active EGFR does not dimerize “conventionally” and becomes resistant to TKI drug, while it traffics via caveolae to an unidentified peri-nuclear region, where it remains active. Please note: Cav-1 binding to EGFR under ox-stress was demonstrated by our group before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023240#pone.0023240-Khan2" target="_blank">[26]</a>.</p