Global proteomics analysis of the response to starvation in <i>C. elegans</i>

Periodic starvation of animals induces large shifts in metabolism but may also influence many other cellular systems and can lead to adaption to prolonged starvation conditions. To date, there is limited understanding of how starvation affects gene expression, particularly at the protein level. Here, we have used mass-spectrometry-based quantitative proteomics to identify global changes in the Caenorhabditis elegans proteome due to acute starvation of young adult animals. Measuring changes in the abundance of over 5,000 proteins, we show that acute starvation rapidly alters the levels of hundreds of proteins, many involved in central metabolic pathways, highlighting key regulatory responses. Surprisingly, we also detect changes in the abundance of chromatin-associated proteins, including specific linker histones, histone variants, and histone posttranslational modifications associated with the epigenetic control of gene expression. To maximize community access to these data, they are presented in an online searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/)

If you don&apos;t want them shed them

Seminal studies in C. elegans contributed to our general understanding of programmed cell death conferred by apoptosis. A recent study unravelled a new form of cell death in the worm and provided insights into its regulation. Affected cells are shed from intact tissues, a modality of death likely to be conserved and relevant to cancer

The Opportunistic Pathogen Serratia marcescens Utilizes Type VI Secretion To Target Bacterial Competitors

The type VI secretion system (T6SS) is the most recently described and least understood of the protein secretion systems of Gram-negative bacteria. It is widely distributed and has been implicated in the virulence of various pathogens, but its mechanism and exact mode of action remain to be defined. Additionally there have been several very recent reports that some T6SSs can target bacteria rather than eukaryotic cells. Serratia marcescens is an opportunistic enteric pathogen, a class of bacteria responsible for a significant proportion of hospital-acquired infections. We describe the identification of a functional T6SS in S. marcescens strain Db10, the first report of type VI secretion by an opportunist enteric bacterium. The T6SS of S. marcescens Db10 is active, with secretion of Hcp to the culture medium readily detected, and is expressed constitutively under normal growth conditions from a large transcriptional unit. Expression of the T6SS genes did not appear to be dependent on the integrity of the T6SS. The S. marcescens Db10 T6SS is not required for virulence in three nonmammalian virulence models. It does, however, exhibit dramatic antibacterial killing activity against several other bacterial species and is required for S. marcescens to persist in a mixed culture with another opportunist pathogen, Enterobacter cloacae. Importantly, this antibacterial killing activity is highly strain specific, with the S. marcescens Db10 T6SS being highly effective against another strain of S. marcescens with a very similar and active T6SS. We conclude that type VI secretion plays a crucial role in the competitiveness, and thus indirectly the virulence, of S. marcescens and other opportunistic bacterial pathogens

Dynamic SUMO modification regulates mitotic chromosome assembly and cell cycle progression in Caenorhabditis elegans

The small ubiquitin-like modifier (SUMO), initially characterized as a suppressor of a mutation in the gene encoding the centromeric protein MIF2, is involved in many aspects of cell cycle regulation. The dynamics of conjugation and deconjugation and the role of SUMO during the cell cycle remain unexplored. Here we used Caenorhabditis elegans to establish the contribution of SUMO to a timely and accurate cell division. Chromatin-associated SUMO conjugates increase during metaphase but decrease rapidly during anaphase. Accumulation of SUMO conjugates on the metaphase plate and proper chromosome alignment depend on the SUMO E2 conjugating enzyme UBC-9 and SUMO E3 ligase PIAS(GEI-17). Deconjugation is achieved by the SUMO protease ULP-4 and is crucial for correct progression through the cell cycle. Moreover, ULP-4 is necessary for Aurora B(AIR-2) extraction from chromatin and relocation to the spindle mid-zone. Our results show that dynamic SUMO conjugation plays a role in cell cycle progression

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways

Site of action of NDK-1 in the analogous processes of engulfment and DTC migration.

<p><b>A, B</b>: Cell corpse engulfment and DTC migration are similar processes. In each case, the surface membrane of a cell (black) extends along the surface of another cell (hatched). The small arrows near the black cells indicate the directions of cell-surface extension. <b>B</b>: Only the relevant parts of body muscles are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Wu1" target="_blank">[19]</a>. <b>C</b>: Schematic review of DTC migration (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Meighan1" target="_blank">[21]</a>). DTCs are located on the distal edges of the gonad primordium and start to migrate in L2. They migrate along the ventral surface (dashed line) of the hermaphrodite in L2 (first or ventral phase). Then they turn to the dorsal side during L3 (second or ventral to dorsal phase). A second turn redirects migration along the dorsal surface toward the center of the nematode during L4 (third or dorsal phase). The end of the migration is dorsal to the vulva, resulting in the mirror image U-shaped gonad of the adult. The developmental stage is indicated at right of each diagram. <b>D</b>: Signaling pathways in engulfment and DTC migration (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Meighan1" target="_blank">[21]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Hurwitz1" target="_blank">[22]</a>. Common genes are blue, green colour indicates the factors involved only in DTC migration, genes in purple boxes play a role only in engulfment. We suggest that NDK-1/NM23 acts downstream of CED-10/Rac in the processes of DTC migration and engulfment of apoptotic corpses. NDK-1 shows a genetic interaction with DYN-1/Dynamin.</p

<i>ndk-1</i>(<i>ok314</i>);<i>dyn-1</i>(<i>ky51</i>) double mutants are lethal.

<p><b>A</b>: In the F1 progeny of <i>ndk-1</i>(-)<i>/+</i> heterozygotes only 9.2% homozygous Pvl, Ste adults can be observed instead of the expected 25%, since 15.8% of <i>ndk-1</i>(<i>ok314</i>) homozygotes die as embryos at 25°C. At the restrictive temperature (25°C) 50% of <i>dyn-1</i>(<i>ky51</i>) single mutants die as embryos. At 25°C, in the F1 progeny of <i>dyn-1(ky51);ndk-1(ok314)/+</i> animals we got decreased brood size and we did not notice any Pvl, Ste animals, suggesting that the double mutants are not viable. <b>B</b>: 3-fold stage homozygous <i>ndk-1</i>(<i>ok314</i>) embryo shows late embryonic lethality with persistent cell corpses. Arrows indicate apoptotic corpses.</p

Western blot analysis and migration assay of transfected MDA-MB-231T cells.

<p>MDA-MB-231T cells were stably transfected with, pcDNA3 (K1 and K2), pcDNA3/FLAG-<i>nm23-H1</i> (HA1 and HA2), pcDNA3/FLAG-<i>ndk-1</i> (CE1 and CE2) and pcDNA3/MYC-<i>nm23-H2</i> (HB1 and HB2). <b>A</b>: Western blot with anti-α-tubulin antibodies (loading control). <b>B</b>: Western blot with anti-FLAG- antibodies, visible band in HA1, HA2, CE1 and CE2 proves stable overexpression of introduced transgenes. <b>C</b>: Western blot with anti-MYC- antibodies, visible band in HB1 and HB2 (overexpression of NM23-H2). <b>D</b>: Migration assay. MDA-MB-231T cells stably transfected with one of the following constructs: pcDNA3 (K1 and K2), pcDNA3FLAG/<i>nm23</i>-H1 (HA1 and HA2), pcDNA3FLAG/<i>ndk-1</i> (CE1 and CE2) and pcDNA3/MYC-<i>nm23-H2</i> (HB1 and HB2) were tested for migration potential. The cells were stained with crystal violet and counted (the number of migrated cells were counted in four representative microscopic fields per each clone). The CE1 and CE2 clones as well as HA1, HA2, HB1 and HB2 exhibited significantly diminished migration potential compared to control (K1 and K2) clones (Student's t-test, p<0.05). The results are presented as an absolute number of migrated cells in 4 representative fields for every clone (±SD).</p

<i>ndk-1</i>(<i>ok314</i>);<i>abi-1</i>(<i>ok640</i>) double mutants show additive Ced phenotye.

<p><i>P_lim-7ced-1::gfp</i> transgenic worms treated by <i>ndk-1</i>(<i>RNAi</i>) (<b>D</b>) show an excess of apoptotic corpses in the germline compared to worms carrying the same transgene treated by control RNAi (<b>B</b>). <b>A</b>, <b>C</b> are corresponding DIC images of <b>B</b>, <b>D</b> respectively. Arrows indicate apoptotic germ cells. <b>E</b>: Increase of apoptotic germ cell death in <i>ndk-1</i>(<i>RNAi</i>) animals compared to the control, where p<i>_lim-7ced-1::gfp</i> transgenic worms were grown on control RNAi (e.g. <i>E. coli</i> HT115(DE3) carrying an empty vector). <b>F–K</b>: Monitoring apoptotic corpses in wild-type embryos (<b>F</b>), <i>ndk-1</i>(-) (<b>G</b>), <i>abi-1</i>(-) (<b>H</b>), <i>ced-10</i>(-) (<b>J</b>) single mutants and <i>ndk-1</i>(-);<i>abi-1</i>(-) (<b>I</b>), <i>ndk-1</i>(-);<i>ced-10</i>(-) (<b>K</b>) double mutants using DIC optics. Embryos slightly before or at the comma stage were scored. Each panel shows two focal planes (<b>F–K</b>). Arrowheads indicate apoptotic corpses. Panel <b>L</b> shows a summary of apoptotic corpses scored in <i>ndk-1</i>(<i>ok314</i>), <i>abi-1</i>(<i>ok640</i>), <i>ced-10</i>(<i>n1993</i>) single mutant and <i>ndk-1(ok314);abi-1(ok640)</i> and <i>ndk-1(ok314);ced-10(n1993)</i> double mutant embryos. <i>ndk-1</i>(-);<i>abi-1</i>(-) double mutants display an enhanced Ced phenotype compared to single mutants, however <i>ndk-1</i>(-);<i>ced-10</i>(-) doubles are reminiscent of <i>ced-10</i>(-) single mutants. Panel <b>M</b> is the graphic representation of panel <b>L</b>. <i>p</i>-values refer to comparisons of apoptotic cell corpse numbers between single and double engulfment mutants. * * * means <i>p</i><0.001; n.s. means not significant (<i>p</i> = 0.627).</p