Differential expression of proteins and phosphoproteins during larval metamorphosis of the polychaete Capitella sp. I

<p>Abstract</p> <p>Background</p> <p>The spontaneous metamorphosis of the polychaete <it>Capitella </it>sp. I larvae into juveniles requires minor morphological changes, including segment formation, body elongation, and loss of cilia. In this study, we investigated changes in the expression patterns of both proteins and phosphoproteins during the transition from larvae to juveniles in this species. We used two-dimensional gel electrophoresis (2-DE) followed by multiplex fluorescent staining and MALDI-TOF mass spectrometry analysis to identify the differentially expressed proteins as well as the protein and phosphoprotein profiles of both competent larvae and juveniles.</p> <p>Results</p> <p>Twenty-three differentially expressed proteins were identified in the two developmental stages. Expression patterns of two of those proteins were examined at the protein level by Western blot analysis while seven were further studied at the mRNA level by real-time PCR. Results showed that proteins related to cell division, cell migration, energy storage and oxidative stress were plentifully expressed in the competent larvae; in contrast, proteins involved in oxidative metabolism and transcriptional regulation were abundantly expressed in the juveniles.</p> <p>Conclusion</p> <p>It is likely that these differentially expressed proteins are involved in regulating the larval metamorphosis process and can be used as protein markers for studying molecular mechanisms associated with larval metamorphosis in polychaetes.</p

Gel-Based and Gel-Free Identification of Proteins and Phosphopeptides during Egg-to-Larva Transition in Polychaete Neanthes arenaceodentata

The polychaete Neanthes arenaceodentata- is cosmopolitan in distribution-, has been used as a laboratory test animal. Life history of this species has several unique features; the female dies after spawning and the male incubates the fertilized eggs through the 21-segmented stage. The larvae leave the tube and commence feeding. Changes in protein abundance and phosphorylation were examined during early development of N. arenaceodentata. A gel-based approach and gel-free enrichment of phosphopeptides coupled with mass spectrometry were used to identify proteins and phosphopeptides in fertilized ova and larval stages. Patterns of proteins and phosphoproteins changed from fertilized ova to larval stages. Twelve proteins occurred in phosphorylated form and nine as stage specific proteins. Cytoskeletal proteins have exhibited differential phosphorylation from ova to larval stages; whereas, other proteins exhibited stage-specific phosphorylation patterns. Ten phosphopeptides were identified that showed phosphorylation sites on serine or threonine residues. Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment. The abundance and distribution of two cytoskeleton proteins were examined further by 2-DE Western blot analysis. This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata. The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species

Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

<p>Abstract</p> <p>Background</p> <p>The metamorphosis of the spionid polychaete <it>Pseudopolydora vexillosa </it>includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of <it>P. vexillosa </it>suggested that little or no <it>de novo </it>protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles.</p> <p>Results</p> <p>We reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1) development, (2) cell differentiation and integrity, (3) transcription and translation, (4) metabolism, (5) protein-protein interaction and proteolysis, and (6) receptors and enzymes.</p> <p>Conclusion</p> <p>This is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete <it>P. vexillosa</it>. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with previous studies showing a high percentage of phosphorylation during competency in the barnacle <it>Balanus amphitrite </it>and the bryozoan <it>Bugula neritina</it>. The identified phosphoproteins may play an important role during metamorphosis, and further studies on the location and functions of important proteins during metamorphosis are warranted.</p

Acute Toxicity of the Antifouling Compound Butenolide in Non-Target Organisms

Butenolide [5-octylfuran-2(5H)-one] is a recently discovered and very promising anti-marine-fouling compound. In this study, the acute toxicity of butenolide was assessed in several non-target organisms, including micro algae, crustaceans, and fish. Results were compared with previously reported results on the effective concentrations used on fouling (target) organisms. According to OECD's guideline, the predicted no effect concentration (PNEC) was 0.168 µg l−1, which was among one of the highest in representative new biocides. Mechanistically, the phenotype of butenolide-treated Danio rerio (zebrafish) embryos was similar to the phenotype of the pro-caspase-3 over-expression mutant with pericardial edema, small eyes, small brains, and increased numbers of apoptotic cells in the bodies of zebrafish embryos. Butenolide also induced apoptosis in HeLa cells, with the activation of c-Jun N-terminal kinases (JNK), Bcl-2 family proteins, and caspases and proteasomes/lysosomes involved in this process. This is the first detailed toxicity and toxicology study on this antifouling compound

A close view of stage specific total proteins (A) and phosphoproteins (B) expressed in fertilized ova, and larvae of <i>Neanthes arenaceodentata.</i>

<p>A close view of stage specific total proteins (A) and phosphoproteins (B) expressed in fertilized ova, and larvae of <i>Neanthes arenaceodentata.</i></p

Western blot analysis of tubulin (A), actin (B) and HSP-90 (C) in Ova, early larvae (EL) and old larval (OL) stages of <i>Neanthes arenaceodentata</i>.

<p>Western blot analysis of tubulin (A), actin (B) and HSP-90 (C) in Ova, early larvae (EL) and old larval (OL) stages of <i>Neanthes arenaceodentata</i>.</p

The number of protein and phosphoproteinspots reproducibly detected in fertilized ova, and larvae of <i>Neanthes arenaceodentata.</i>

<p>The number of protein and phosphoproteinspots reproducibly detected in fertilized ova, and larvae of <i>Neanthes arenaceodentata.</i></p

Identification of abundant proteins during early development in <i>Neanthes arenaceoentata.</i>

a)<p>Accession numbers are from NCBI nr and Swiss-Prot database.</p>b)<p>significance threshold level for positive identification was p<0.05.</p>c)<p>observed MW and p<i>I</i> values were estimated from 2-DE gels.</p>d)<p>Theoretical MW and p<i>I</i> values were derived from a database search by MASCOT. PM: number of peptides matched to protein sequence; SC: sequence coverage.</p>*<p>phosphopeptides and phosphorylation sites are underlined.</p

Early developmental stages of the polychaete <i>Neanthes arenaceodentata</i>.

<p>Three developmental stages were chosen for proteomic analysis: Fertilized ova, 3–4 segmented early larva and 10–12 segmented old larva. Drawings: Three segmented larva and twelve segmented larva.</p

A close view of differential phosphorylation of cytoskeleton proteins (spots marked in circle) in fertilized ova and larvae of <i>Neanthes arenaceodentata.</i>

<p>A close view of differential phosphorylation of cytoskeleton proteins (spots marked in circle) in fertilized ova and larvae of <i>Neanthes arenaceodentata.</i></p