Protein levels of WAVE/SCAR complex subunits in different mutant contexts

<p><b>Copyright information:</b></p><p>Taken from "HSPC300 and its role in neuronal connectivity"</p><p>http://www.neuraldevelopment.com/content/2/1/18</p><p>Neural Development 2007;2():18-18.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2098765.</p><p></p> Immunoprecipitation experiments in S2 cytoplasmic cell extracts using the anti-HSPC300 antibody. From left to right: anti-HSPC300 immunoprecipitation, IgG immunoprecipitation, input (cytoplasmic extract). Proteins are indicated to the right, corresponding molecular weights to the left. Quantitative analysis of CYFIP, Kette, SCAR, Abi and HSPC300 protein levels by western blot on third instar larval extracts of the following genotypes: wild type (WT); zygotic null; zygotic null and maternal hypomorph; zygotic null. Proteins are indicated to the right, corresponding molecular weights to the left. β-tubulin represents a loading control

AMP expression extends lifespan.

<p>(A) <i>Dro</i> expression with the <i>tubulin</i>-GeneSwitch driver is dependent on the RU486 concentration. qPCR analysis of <i>Dro</i> in 12-day old female <i>Tub</i>^<i>GS</i><i>>Dro</i> flies. Expression was induced with 1 μg/ml (red) or 10 μg/ml (blue) RU. <i>n</i> = 3 replicates of four whole flies. (B–C) Ubiquitous expression of AMPs extends lifespan. Compared to controls fed without RU (–RU, black line), female <i>Tub</i>^<i>GS</i><i>>Dro</i> (B) or <i>Tub</i>^<i>GS</i><i>>CecA1</i> (C) flies fed with 1 μg/ml RU (+RU, red line) show 12.5% or 12.9% increased MLS, respectively. <i>n</i> ≥ 220 (B), <i>n</i> ≥ 191 (C) flies/condition. (D–E) <i>Dro</i> (D) and <i>CecA1</i> (E) transcription in the midgut of 14-day old female <i>Tub</i>^<i>GS</i><i>>Dro</i> (D) and <i>Tub</i>^<i>GS</i><i>>CecA1</i> (E) flies fed with 1 μg/ml RU (red). <i>n</i> = 4 (D), <i>n</i> = 5 (E) replicates of five midguts. (F–G) Gut specific expression of <i>Dro</i> extends lifespan. (F) <i>Dro</i> transcription in the midgut of 14-day old female <i>Ti</i>^<i>GS2</i><i>>Dro</i> flies fed with 10 μg/ml RU (blue). <i>n</i> = 4 replicates of five midguts. (G) Compared to controls (–RU, black line) female <i>Ti</i>^<i>GS2</i><i>>Dro</i> flies fed with 10 μg/ml RU (+RU, blue line) show 7.7% increased MLS. <i>n</i> ≥ 169 flies/condition. In (A) and (D–F) mean <i>log</i>_<i>10</i> of fold change is compared to controls (set to 0, not shown). Statistical tests: (A), (D–F) one-sample t-test, (B–C), (G) log-rank test (Kaplan-Meier analysis). * <i>P</i> ≤ 0.05, ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001. Error bars represent the standard error of the mean. <i>Dro</i>, <i>Drosocin</i>; <i>CecA1</i>, <i>Cecropin A1</i>; MLS, median lifespan; +RU, RU treatment. Genotypes were: <i>w/y</i>,<i>w;UAS-Dro/+;tubulin</i>^{<i>GeneSwitch</i>}<i>-gal4/+</i> (<i>Tub</i>^<i>GS</i><i>>Dro</i>), <i>w/y</i>,<i>w;+/+;tubulin</i>^{<i>GeneSwitch</i>}<i>-gal4/UAS-CecA1</i> (<i>Tub</i>^<i>GS</i><i>>CecA1</i>), <i>w/y</i>,<i>w;UAS-Dro/+;TiGS2</i>^{<i>GeneSwitch</i>}<i>-gal4/+ (Ti</i>^<i>GS2</i><i>>Dro)</i>.</p

<i>Dro</i> expression reduces intestinal stress response, immune and regeneration activity.

<p>(A) <i>Dro</i> transcription in the gut of female <i>Ti</i>^<i>GS2</i><i>>Dro</i> flies fed with 10 μg/ml RU at different ages. (B) Gut-specific expression of <i>Dro</i> reduces transcription of genes involved in stress response, immune and regeneration activity in the intestinal tract. Transcriptional analysis of different pathways in midguts of female <i>Ti</i>^<i>GS2</i><i>>Dro</i> flies fed with 10 μg/ml RU at different ages. For details of the tested genes and pathways see main text. (A–B) Mean l<i>og</i>_<i>10</i> of fold change is compared to controls of same age fed without RU (set to 0, not shown). <i>n</i> = number of replicates of five guts. X represents outliers (Grubbs test, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176689#sec007" target="_blank">Materials and methods</a>): –0.57 (<i>pirk</i>), –0.24 (<i>Socs36E</i>). Note that data in (A) and (B) originate from the same experiments. (C) <i>Dro</i> transcription in the gut reduces intestinal damage. Smurf analysis of female <i>Ti</i>^<i>GS2</i><i>>Dro</i> flies fed ± 10 μg/ml RU at different ages. <i>n</i> = number of flies. Statistical tests: (A–B) one sample t-test, (C) Fisher´s exact test. Error bars represent the standard error of the mean. <i>aos</i>, <i>argos</i>; <i>Dro</i>, <i>Drosocin</i>; EGF, Epidermal growth factor signaling; <i>Hsp70A</i>, <i>Heat-shock-protein-70A</i>; IMD, Immune Deficiency pathway; <i>Irc</i>, <i>Immune-regulated catalase</i>; JAK-STAT, Janus kinase / Signal Transducer and Activator of Transcription pathway; JNK, c-Jun N-terminal kinase signaling; <i>PGRP-LB</i>, <i>Peptidoglycan recognition protein LB</i>; <i>pirk</i>, <i>poor Imd response upon knock-in</i>; <i>puc</i>, <i>puckered</i>; <i>rho</i>, <i>rhomboid</i>; ROS, Reactive oxygen species production; <i>Socs36E</i>, <i>Suppressor of cytokine signaling at 36E</i>; <i>upd3</i>, <i>unpaired 3</i>. Genotype was: <i>w/y</i>,<i>w;UAS-Dro/+;TiGS2</i>^{<i>GeneSwitch</i>}<i>-gal4/+ (Ti</i>^<i>GS2</i><i>>Dro)</i>.</p

<i>Dro</i> enhances intestinal immunity and thereby extends lifespan.

<p>(A–B) Ubiquitous or gut-specific expression of <i>Dro</i> enhances intestinal immunity against pathogens. Persistence of <i>Pe</i> in female <i>Tub</i>^<i>GS</i><i>>Dro</i> (A) and <i>Ti</i>^<i>GS2</i><i>>Dro</i> (B) flies fed ± 0.5 or 1 μg/ml RU (A), or ± 10 μg/ml RU (B). <i>n</i> ≥ 15 replicates of one or two midguts (two biological repeats). (C–D) Flies kept on antibiotic-food live longer. Additional ubiquitous (C) or gut-specific (D) expression of <i>Dro</i> has no effect. (C) Lifespan analysis of antibiotic treated <i>Tub</i>^<i>GS</i><i>>Dro</i> flies fed ± 1 μg/ml RU (–RU/+AB, dashed black; +RU/+AB, dashed red) and control flies fed ± 1 μg/ml RU (–RU, solid black; +RU solid red). Note that–RU and +RU are the same cohorts as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176689#pone.0176689.g001" target="_blank">Fig 1B</a>. <i>n</i> ≥ 217 flies/condition. (D) Lifespan analysis of antibiotic treated flies fed ± 10 μg/ml RU (–RU/+AB, dashed black; +RU/+AB, dashed blue) and control flies fed ± 10 μg/ml RU (–RU, solid black; +RU solid blue). Note that –RU and +RU are the same cohorts as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176689#pone.0176689.g001" target="_blank">Fig 1G</a>. <i>n</i> ≥ 169 flies/condition. (E–F) <i>Dro</i> transcription in the midgut of 14-day old female <i>Tub</i>^<i>GS</i><i>>Dro</i> (E) and <i>Ti</i>^<i>GS2</i><i>>Dro</i> (F) flies kept on antibiotic-food with 1 (E) or 10 (F) μg/ml RU. Mean <i>log</i>_<i>10</i> of fold change is compared to –RU/+AB controls (set to 0, not shown). <i>n</i> = 3 (E), <i>n</i> = 4 (F) replicates of five midguts. (G) Overexpression of <i>Dro</i> has no effect on intestinal microbiota. CFU analysis of 14-day old female <i>Ti</i>^<i>GS2</i><i>>Dro</i> flies fed ± 10 μg/ml RU. Midgut homogenates were plated on lysogeny broth medium (LB) or on selective medium for <i>Acetobacteriaceae</i> (Ace). <i>n</i> = 3 replicates of ten midguts. (H) Microbiota analysis in the midgut of female <i>Ti</i>^<i>GS2</i><i>>Dro</i> flies fed ± 10 μg/ml RU on standard food. Statistical tests: (A) Fisher´s LSD test (ANOVA), (B), (G, right side) two-sample t-test, (C–D) log-rank test (Kaplan-Meier analysis), (E–F) one sample t-test, (G, left side) Mann-Whitney test. ** <i>P</i> ≤ 0.01. Error bars in (E–G) represent the standard error of the mean. AB, antibiotic treatment; <i>Dro</i>, <i>Drosocin</i>; <i>CecA1</i>, <i>CecropinA1</i>; CFU, colony-forming units; +RU, RU treatment. Genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176689#pone.0176689.g001" target="_blank">Fig 1</a>.</p

Antimicrobial peptides extend lifespan in <i>Drosophila</i>

<div><p>Antimicrobial peptides (AMPs) are important defense molecules of the innate immune system. High levels of AMPs are induced in response to infections to fight pathogens, whereas moderate levels induced by metabolic stress are thought to shape commensal microbial communities at barrier tissues. We expressed single AMPs in adult flies either ubiquitously or in the gut by using the inducible GeneSwitch system to tightly regulate AMP expression. We found that activation of single AMPs, including <i>Drosocin</i>, resulted in a significant extension of <i>Drosophila</i> lifespan. These animals showed reduced activity of immune pathways over lifetime, less intestinal regenerative processes, reduced stress response and a delayed loss of gut barrier integrity. Furthermore, intestinal <i>Drosocin</i> induction protected the animals against infections with the natural <i>Drosophila</i> pathogen <i>Pseudomonas entomophila</i>, whereas a germ-reduced environment prevented the lifespan extending effect of <i>Drosocin</i>. Our study provides new insights into the crosstalk of innate immunity, intestinal homeostasis and ageing.</p></div