RNA-Seq of early follicle cells – EGFRact Rep2 Read2

Paired-end RNA-Sequencing data from early follicle cells Genotype 109-30-Gal4, UAS-mCD8::GFP, UAS-EGFR[lambda]top Replicate #2, Read

<i>Drosophila</i> MAPK <i>rolled</i> is directing HOW phosphorylation in muscles and regulation of Sallimus levels.

<p>A. HOW was immunoprecipitated from lysates of 3^rd instar larvae either expressing <i>rolled</i> RNAi in muscles under <i>mef2</i>-GAL4 (left) or of wild-type <i>mef2</i>-GAL4 heterozygotes (w−, right) and reacted with a pTP antibody to observe its phosphorylation level and with anti-HOW to compare total HOW levels. The crude extract was reacted with anti-HOW and anti-Tubulin as a loading control, showing that the total quantity of HOW is not significantly altered by MAPK/Rolled levels. Note the reduction in HOW phosphorylation following expression of <i>rolled</i> RNAi. B. Reduction in MAPK/Rolled levels elevates Sls protein levels. Same genotypes as in A were analyzed by Western with the following antibodies (from top to bottom): anti-Sls, anti-MHC, anti-MSP300, anti-pERK (to verify the reduction in activated MAPK in these larvae) and anti-α-Actinin as a loading control. C–H. Wild-type (C–E) and <i>mef2</i>-GAL4><i>rolled</i> RNAi (F–H) larvae were stained with anti-Sls antibody (red, C,F) and anti MSP-300 (blue, D,G). Merge is shown in E,H. Note the significant elevation in Sls levels following down regulation of MAPK/Rolled levels.</p

HOW regulates the levels of Sls RNA and protein in a phosphorylation-dependent manner.

<p>A. Sls and MSP-300 are directly or indirectly regulated by HOW. Extracts from larvae in which HOW was down-regulated by expressing <i>how</i> RNAi (aimed against all HOW isoforms) under <i>mef2</i>-GAL4, on the background of <i>how^stru</i> heterozygotes, were reacted with anti-Sls, MSP-300, and HOW antibodies and compared to larvae heterozygotes for <i>mef2</i>-GAL4 in Western blot analysis. Down-regulation of HOW (lower panel) led to upregulation of Sls (upper panel) and down-regulation of MSP-300 (middle panel). B–D. Overexpression of HOW leads to reduction of Sls protein levels, in a phosphorylation-dependent manner. Body wall muscles from 3rd instar larvae expressing <i>mef2</i>-GAL4 alone (B,B′,B″), UAS-HOW(L)^WT-HA, or UAS-HOW(L)^TTAA-HA driven by the <i>mef2</i>-GAL4 driver (C,C′,C″ and D,D′,D″, respectively) were stained with anti-Sls (B,C,D - red), anti-MSP-300 (B′,C′,D′- blue) and anti-HA (B″,C″,D″ - green, shown within merge). Quantification of at least three larvae from each genotype showed that HOW(L)^WT over-expression reduced Sls intensity to 0.71±0.05 of the intensity of the <i>mef2</i> -GAL4 heterozygotes, while the HOW(L)^TTAA protein only reduced it to 0.83±0.03 of control (P = 0.049, ANOVA test). E. Overexpression of HOW(L) in larval muscles alters the levels of multiple muscle proteins, of which only Sls is affected in a HOW phosphorylation-dependent manner. Protein extracts from 2^nd instar larvae expressing <i>mef2</i>-GAL4 alone (middle), or either HOW(L)^WT (left) or HOW(L)^TTAA (right) driven by <i>mef2</i>-GAL4 were analyzed by Western blotting with the following antibodies (from top to bottom): anti-Sls, anti-MHC, anti-MSP300 (reacts with three different isoforms), anti-MLP84B, anti-Actin, and anti-HOW. Note the comparable levels of HOW(L)^WT and HOW(L)^TTAA expression, and the relatively mild reduction in Sls when HOW(L)^TTAA is overexpressed. F–F′. HOW regulates Sls at the RNA level. RNA was extracted from single 3^rd instar larvae, and real-time PCR was performed with <i>sls</i> primers (F) and <i>how</i> primers (F′), both normalized to <i>rp49</i> as a control. From left to right: <i>mef2</i>-GAL4 heterozygotes (w−), <i>how^stru+/−</i>, <i>mef2</i>><i>how</i> RNAi (RNAi), and <i>mef2</i>-GAL4 driving either HOW(L)^WT or HOW(L)^TTAA. Error bars indicate SEM. While reduction of HOW levels elevated <i>sls</i> (1.38±0.11, P = 0.039), overexpression of HOW(L)^WT reduced it more efficiently (0.54±0.20, P = 0.08) than HOW(L)^TTAA (0.92±0.18, P = 0.78) (Student's t-test, n = 3 or 4).</p

HOW(L) is phosphorylated <i>in vitro</i> and in S2R+ cells by MAPK/ERK on one or more TP sites.

<p>A. ERK2 phosphorylates HOW <i>in vitro</i>. HOW(L)^WT and HOW(L)^TTAA (phospho mutant) constructs were <i>in vitro</i> translated (using S³⁵ labeling) and incubated with activated ERK2. The reaction was resolved on an SDS gel. The left part of the panel (lanes 1–4) shows phosphorylation of the Yan (Aop) protein as a control. HOW(L)^WT protein, but not the HOW(L)^TTAA mutant (lanes 7–8) exhibits a molecular weight shift of a fraction of the protein when incubated with the kinase (lane 5) relative to HOW without the kinase (lane 6). B. HOW(L), but not HOW(S), is phosphorylated in S2R+ cells. HOW proteins were immunoprecipitated using an anti-HOW antibody and reacted with anti-pTP (top panel) and anti-HOW (middle panel) antibodies. HOW proteins in the crude extract are shown in the bottom panel. From the left: HOW(S)^WT, HOW(L)^WT and HOW(L)^TTAA. C. Treatment with the MAPKK/MEK inhibitor U0126 decreases HOW phosphorylation. HOW proteins were immunoprecipitated with an anti-HOW antibody from cells transfected with <i>how(l)^WT</i> treated (or not) with U0126, or with <i>how(l)^TTAA</i> and reacted with anti-pTP antibody (top panel) or with anti-HOW antibody (2^nd from the top). The crude extract was reacted with anti-pERK antibody (2^nd from the bottom), to confirm the extent of U0126 inhibition, and anti-Actin (bottom panel) as a loading control. C′. Quantification of the reduction of phosphorylation following treatment with U0126. For each sample, the ratio between the pTP band measurement and the total HOW band was calculated, and normalized to the HOW(L)^WT (non-treated sample) ratio. Results shown are the average of three experiments; error bars indicate SEM. Following U0126 treatment, HOW phosphorylation was reduced to 0.47±0.07 relative to non-treated cells (P = 0.0021, unpaired t-test, n = 3). D. Same as in C, except cells were treated with TPA/PMA. A representative experiment is presented. D′. Quantification of three TPA treatment experiments, in which HOW phosphorylation was increased by 1.28±0.06 (P = 0.0076, unpaired t-test, n = 3).</p

RNA-Seq of early follicle cells – EGFRact Rep1 Read1

Paired-end RNA-Sequencing data from early follicle cells Genotype 109-30-Gal4, UAS-mCD8::GFP, UAS-EGFR[lambda]top Replicate #1, Read

Phosphorylated HOW is detected in the nuclei of somatic muscles and cardioblasts.

<p>A. Anti-pHOW (pT64) antibody reacts with HOW, in a phosphorylation-dependent manner. HOW protein was immunoprecipitated with an anti-HOW antibody from S2R+ cells transfected with either HOW(L)^WT, treated (or not) with U0126 (lanes 3, 2 respectively), with HOW(L)^TTAA (lane 4), or no HOW (lane 1). The IP was reacted with anti-pHOW (pT64) antibody (top) or with anti-HOW antibody (middle). The crude extract was reacted with anti-pERK antibody (bottom), confirming U0126 inhibition. Note specific reactivity of the anti-pHOW antibody. B. Anti-pHOW (pT64) antibody reacts with HOW protein overexpressed in embryonic muscles and heart. Transgenic UAS-HOW(L)^WT (lane 2) and UAS-HOW(L)^TTAA (lane 3) flies or wild-type controls (lane 1) were crossed to the <i>mef2</i>-GAL4 driver line. Embryos were collected, and protein extracts were subjected to IP with an anti-HA antibody followed by Western with anti-pHOW (pT64) antibody (top), and anti-HOW antibody (2^nd from top). Crude extracts were reacted with anti-HOW (2^nd from bottom), and anti-Actin (bottom) as a loading control. C-F. <i>Drosophila</i> embryos stained with anti-pHOW (pT64) exhibit specific staining in somatic muscles and heart cardioblasts. Stage 16 WT (C,D) and <i>how ^stru</i> mutant (E,F) embryos were stained with anti-pHOW (pT64) (red, C–F) and anti-HOW (green in merge D,F). Embryos are oriented dorsal to the top and anterior to the left. (C,D) Note the staining in muscles (hollow arrow), heart cardioblasts (white arrow) and the lack of pHOW staining in the muscle attachment sites (arrowhead). (E,F) Weak staining in cardioblasts in <i>how ^stru−/−</i> mutants. G–I. <i>Drosophila</i> embryos expressing CD8-GFP, which localizes at the plasma membrane, but also often concentrates in the ER surrounding the nuclei, in muscles under <i>mef2</i>-GAL4, were stained with anti-pHOW (red, G) shown merged with GFP (green, H) and with HOW (blue, I). J–M. HOW(L) overexpressed in embryonic tendon cells reacts with the anti-pHOW (pT64) antibody. Embryos expressing UAS-HOW(L)^WT-HA (J,K) or UAS-HOW(L)^TTAA–HA (L,M) in the tendon cells under <i>sr</i>–GAL4 were stained with anti-pHOW (pT64) (red, J–M) and anti-HA (green, K, M, merged with the anti-pHOW staining). Arrows mark tendon cells reactive with anti-pHOW upon expression of HOW(L)^WT (J,K), or non-reactive upon expression of the phospho-mutant (L,M).</p

HOW(L) phosphorylation correlates with its homo-dimerization.

<p>A. Phospho-mutant HOW(L)^TTAA oligomerizes to a lesser extent than HOW(L)^WT. S2R+ cells were co-transfected with expression vectors for the following: <i>gfp- how(l)^WT</i> and <i>how(l)^WT-HA</i> (1) <i>gfp- how(l)^TTAA</i> with <i>how(l)^TTAA-HA</i> (2); <i>gfp-how(l)^EG</i> with <i>how(l)^EG-HA</i> (3); or <i>gfp</i> with <i>how(l)^WT-HA</i> (4) as a control. IP was carried out using anti-GFP antibody and Western blotting was performed with anti-HOW. Crude lysate was reacted with anti-HOW (2^nd panel from the top), anti-GFP (to visualize GFP-HOW (lanes 1–3) and the GFP control (lane 4)), as well as with anti-Actin. A′. Quantification of lanes 1–3 from three experiments as described in A. For each sample, the ratio between the Co-immunoprecipitated HA protein and its total crude level was normalized to the amount of the GFP protein that pulled it down. HOW^TTAA (2) exhibited partial dimerization (0.56±0.14, P = 0.039 unpaired t-test, n = 3). The HOW^EG (3) is almost completely non-dimerized (0.13±0.04, P<0.0001, unpaired t-test, n = 3). B. HOW(L) protein that is unable to homo-dimerize undergoes significantly less phosphorylation. S2R+ cells were transfected with either <i>how(l)^WT</i>, <i>how(l)^TTAA</i> or the <i>how(l)^EG</i> mutant construct. Proteins were immunoprecipitated using anti-HOW antibody. The amount of lysate used for the IP of the HOW(L)^EG protein was doubled, in order to obtain a comparable amount of immunoprecipitated HOW protein (since it does not dimerize, less protein precipitates). B′. Quantification of two of several experiments in which the total levels of the different proteins were comparable. For each sample, a ratio between the pTP band measurement and the total HOW band was calculated, and normalized to the HOW(L)^WT ratio. Error bars indicate SEM (HOW(L)^EG 0.12±0.10).</p

HOW phosphorylation strengthens its affinity to RNA, while not affecting its subcellular localization.

<p>A. <i>In vitro</i> RNA-protein binding assay. HA tagged HOW(L)^WT, HOW(L)^TTAA or HOW(L)^EG constructs were purified from S2R+ cells by immunoprecipitation of the HA tag, followed by elution with free HA peptide. HOW(L)^WT transfected cells were either treated with U0126 or not treated. Equal volumes of purified proteins were incubated with biotin labeled 12 nt oligomers that either bind HOW (top panel) or do not bind HOW (middle panel). The complexes were incubated with avidin beads, eluted by boiling in sample buffer and reacted with anti HOW in a Western blot. The amounts of HOW protein used for each reaction were comparable (bottom panel). B–C′. Localization of HOW(L) was not altered due to the mutations in the Thr residues. Cells expressing either HOW(L)^WT (B,B′) or HOW(L)^TTAA (C,C′) were stained for HOW (red, B–C). Merge images (B′–C′) also present GFP (green, marks transfected cells) and Lamin (blue).</p

Dorsal closure is defective in embryos deficient for Egfr signaling.

<p>(A-H) Cuticle preparations. (A) Wild-type cuticle; note the complete closure of the epidermis on the dorsal side. (B) <i>bsk</i> mutant embryo; note the characteristic dorsal-open phenotype (arrowhead). (C-H) Lack of functional Egfr signaling leads to the formation of dorsal-open holes (arrowheads), a phenotype typically associated with JNK pathway mutants (cf. B). Egfr pathway activity was compromised by <i>pnr>Gal4</i>-driven ectodermal expression of <i>Egfr</i>^<i>DN</i> (C) or <i>Ras</i>^<i>DN</i> (D), or in <i>rhomboid</i> (E), <i>spi</i> (F) and allelic <i>Egfr</i> (G-H) mutant embryos.</p

Egfr signaling is positively required for the full expression of the JNK pathway target gene <i>dpp</i> and for Mad phosphorylation.

<p>(A-F) Lateral (A, B, D, E) or dorsolateral (C, F) views of embryos hybridized using a digoxigenin-labeled RNA probe for <i>dpp</i> (blue). (A’-F’) Corresponding magnified views of the regions marked by black arrowheads in panels (A-F). (A”-F”) show embryos stained for pMad (red). (A-A”) Wild-type embryo showing the normal <i>dpp</i> (A-A’) and pMad (A”) patterns. Levels of <i>dpp</i> and pMad are reduced in <i>rhomboid</i> mutants (D-D”), as well as in embryo expressing <i>pnr>Egfr</i>^<i>DN</i> (E- E”). Conversely, both expand in embryo expressing <i>pnr>Ras</i>^<i>V12</i> (F- F”). These effects largely phenocopy loss- or gain-of-function JNK signaling (<i>bsk</i> mutant and <i>pnr>Hep</i>^<i>Act</i> embryo in B-B” and C-C”, respectively).</p