16 research outputs found
<i>als<sup>RNAi</sup></i> causes notched wing margins.
<p><i>c765-Gal4</i> drives the expression of different <i>UAS-als<sup>RNAi</sup></i> transgenes in the entire wing primordium at 29°C, which results in nicked wing margins, accompanied by loss of sensory bristles. (A) <i>Gal4</i> driver only, (B) VDRC line 39000, (C) VDRC line 105104, (D) oligo3, (E) oligo2.</p
<i>als</i> acts in a cell-autonomous manner.
<p>Wg target gene expression (Sens) is affected cell-autonomously upon impaired <i>als</i> function as judged from wing disc analysis at the late L3 stage (A–A″). GFP expression indicates cell clones expressing <i>als<sup>RNAi</sup></i> line <i>oligo3<sup>10UAS</sup></i> (A and A″). Adult wing phenotypes coincide with cell clones that experienced <i>als</i> depletion, as marked by <i>forked</i> wing hairs (B–E). This is reminiscent of adult wing phenotypes obtained when Wg signaling is impaired (Lgs<sup>17E</sup> expression) (F–I). Red asterisks (B and F) indicate notches at the wing margin; red arrows (C and G) indicate wing margin notches accompanied by loss of mechano- and sensory bristles; red arrows (D and H) and brackets (E and I) indicate broadened veins.</p
Human UBXN6 is the functional ortholog of Als.
<p>(A–D) Human UBXN6 expression could largely or completely rescue <i>als<sup>RNAi</sup></i> wing and eye phenotypes. (E and E′) When expressed in wing imaginal discs <sup>HA</sup>UBXN6 localization resembles that of <sup>HA</sup>Als (cf. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001988#pbio.1001988.s008" target="_blank">Figure S8A–S8E</a>). (F) UBXN6 and Armless belong to the same protein subfamily of UBX domain proteins (blue). (G–G″) Expression of the WNT target genes <i>SP5</i>, <i>AXIN2</i>, and <i>FZD1</i> was upregulated upon stimulation of the pathway with mWnt3a (green bar), and was reduced upon siRNA against human <i>UBXN6</i> in HEK-293 cells. Depletion of β-Catenin served as a control. (H) β-Catenin levels were reduced upon siRNA against human <i>UBXN6</i> in HEK-293 cells (lane 6, cf. lane 1; and lane 3, cf. 2), whereas depletion of <i>UBXN6</i> had no effect on β-Catenin levels upon inhibition of the proteasome (MG132; lane 5, cf. lane 4). (I) WNT target genes were upregulated upon GSK3β inhibition (CHIR; green bars), and were reduced upon depletion of <i>UBXN6</i> (blue bars).</p
Protection of Armadillo/β-Catenin by Armless, a Novel Positive Regulator of Wingless Signaling
<div><p>The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. We identified a novel Wg/Wnt pathway component, Armless (Als), which is required for Wg target gene expression in a cell-autonomous manner. We found by genetic and biochemical analyses that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. We show by biochemical and in vivo studies that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. We suggest that Als antagonizes Ter94's positive effect on E3 ligase function and propose that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated.</p></div
<i>als</i> is required for Wg-dependent target expression.
<p>L3 wing imaginal discs; anterior is to the left, posterior is to the right. <i>als<sup>RNAi</sup></i> expression in the P-compartment of wing imaginal discs (visualized by the co-expression of <i>UAS-GFP</i>, green fluorescence) (A–C′) or in the entire wing pouch (D–G) causes loss of expression of Wg-signaling-dependent target genes—Senseless (A and A′) (line 39000), Distalless (B and B′) (line oligo3), <i>frizzled3</i> (C and C′) (line oligo3), or <i>wingful</i> (D and E; white arrows indicate the Wg-dependent expression domain of <i>wf</i> at the D/V border which is lost in E.) (line 39000)—and ectopic expression of <i>arrow</i>, which is a negative target (G c.f. F; white arrows indicate the extent of the domain that is free from <i>arrow-lacZ</i> expression) (line oligo3).</p
<i>als</i> functions downstream of the Arm/β-Catenin destruction complex and upstream of activated Armadillo.
<p>Overexpression of the positive components Arrow (A), Dishevelled (L), Arm<sup>S10</sup> (F and Q), or Arm (G and R) and depletion of negative components such as <i>wingful</i> (B), <i>shaggy</i> (C, H, and I), <i>apc</i> (D and J), <i>slimb</i> (E, O, and P), and <i>axin</i> (K) cause phenotypes that reflect ectopic Wg signaling: ectopic sensory bristles (A–G), ectopic veins (A–C), and tissue overgrowth (A, H–K, O, and P), but also reduced organ size upon strong pathway activation (B–F, L, M, and Q) and caused ectopic head cuticle (H–J, L, and M). Co-expression of <i>als<sup>RNAi</sup></i> could suppress these phenotypes in the wing (A′–D′) and the eye (H′–M′). Phenotypes based on <i>slimb<sup>RNAi</sup></i> and Arm<sup>S10</sup> expression could not be suppressed upon <i>als</i> depletion (E′, O′, P′, F′, and Q′). Arm overexpession could not rescue <i>als<sup>RNAi</sup></i> phenotypes (G′ and R′). Heads were photographed from dorsal views (left pictures of H–K′) or lateral views, anterior to the left (right pictures of H–K′, L–R′). <i>als<sup>RNAi</sup></i> lines: <i>oligo3<sup>10UAS</sup></i> (A′–E′) and for the eye analysis; <i>oligo2_5′utr</i> (G′).</p
Reduction of Armadillo levels upon <i>als</i> depletion.
<p>(A–A″) <i>als</i> depletion (line <i>oligo3<sup>10UAS</sup></i>) in the P-compartment of wing imaginal discs caused reduced Arm protein levels compared to the A-compartment (A′ and A″). CD8-GFP expression marks the cell membranes of the P-compartment, and DAPI staining marks all cellular nuclei. (B and B′) The expression of Arm driven from a transgene by <i>nub</i>-<i>Gal4</i> is similar to endogenous Arm (cf. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001988#pbio.1001988.s008" target="_blank">Figure S8D</a>′). (C and C′) Upon <i>als</i> depletion (line 39000), Arm levels were strongly reduced. (D–G′) DAPI staining (D′–G′) visualizes the large nuclei of the peripodial membrane (blue, out of the focal plane), which overlies the apical part of wing disc cells including Arm (in the focal plane of D–E′). The expression of Arm<sup>S10-Myc</sup> (F–G′) was not altered upon depletion of <i>als</i> (line <i>oligo3<sup>10UAS</sup></i>). (D–E′): apical confocal sections; (F–G′): deeper confocal <i>Z</i>-sections, which show enhanced cytoplasmic Arm<sup>S10</sup> levels.</p
Als co-localizes and interacts with Ter94.
<p>(A) Western blot analysis after immunoprecipitation (IP) of <sup>FLAG</sup>Als or Ter94<sup>HA</sup>. Condition 1: negative control (empty <i>pUAST-attB</i> vector), condition 2: <sup>FLAG</sup>Als (60 kDa), condition 3: negative control (<sup>FLAG</sup>Gal4, 115 kDa), condition 4: <sup>FLAG</sup>ΔN-Als (40 kDa); Ter94<sup>HA</sup> (105 kDa) was co-expressed in conditions 2–4. Ter94 binds to full-length Als (condition 2) and ΔN-Als (condition 4), and vice versa. The anti-human-UBXN6 peptide antibody recognizes only the full-length Als, but not ΔN-Als. (B–B″) Transgene expression by <i>nubbin</i>-<i>Gal4</i> in wing imaginal disc cells shows that <sup>FLAG</sup>Als co-localizes with Ter94<sup>HA</sup>. (C and C′) Co-expression of Als-VC and Ter94-VN with <i>GMR-Gal4</i> in eye imaginal discs results in a Venus YFP fluorescence signal, which demonstrates a physical interaction between Als and Ter94.</p
<i>als</i> acts downstream of Wg.
<p>Control eye with <i>ey-Gal4, GMR-Gal4/+</i> (A). The small eye phenotype based on <i>sev-wg</i> expression (B) can be suppressed by the expression of different <i>UAS</i>-<i>als<sup>RNAi</sup></i> transgenes (C–E), by expression of <i>UAS</i>-<i>als<sup>HA(DN)</sup></i> (F), and by overexpression of Lgs<sup>17E</sup>, which suppresses Wg signaling (G). The small eye phenotype based on enhanced apoptosis (H) is not altered upon <i>als</i> depletion (I and J).</p
Characterization of the larval hemolymph proteome.
<p>(A) Workflow of the analyses. Hemolymph samples from fed and starved larvae were digested in solution. Tryptic peptides were separated by isoelectric focusing for complexity reduction. Peptides were analyzed using microcapillary liquid chromatography–electrospray ionization–tandem MS (µLC-ESI-MS/MS). SEQUEST spectral search was performed for peptide spectrum matching. (B) Venn diagram illustrating the number of gene models detected in hemolymph from fed and starved larvae, respectively.</p