Urochordate Histoincompatible Interactions Activate Vertebrate-Like Coagulation System Components

The colonial ascidian Botryllus schlosseri expresses a unique allorecognition system. When two histoincompatible Botryllus colonies come into direct contact, they develop an inflammatory-like rejection response. A surprising high number of vertebrates' coagulation genes and coagulation-related domains were disclosed in a cDNA library of differentially expressed sequence tags (ESTs), prepared for this allorejection process. Serine proteases, especially from the trypsin family, were highly represented among Botryllus library ortholgues and its “molecular function” gene ontology analysis. These, together with the built-up clot-like lesions in the interaction area, led us to further test whether a vertebrate-like clotting system participates in Botryllus innate immunity. Three morphologically distinct clot types (points of rejection; POR) were followed. We demonstrated the specific expression of nine coagulation orthologue transcripts in Botryllus rejection processes and effects of the anti-coagulant heparin on POR formation and heartbeats. In situ hybridization of fibrinogen and von Willebrand factor orthologues elucidated enhanced expression patterns specific to histoincompatible reactions as well as common expressions not augmented by innate immunity. Immunohistochemistry for fibrinogen revealed, in naïve and immune challenged colonies alike, specific antibody binding to a small population of Botryllus compartment cells. Altogether, molecular, physiological and morphological outcomes suggest the involvement of vertebrates-like coagulation elements in urochordate immunity, not assigned with vasculature injury

Life-Cycle and Genome of OtV5, a Large DNA Virus of the Pelagic Marine Unicellular Green Alga Ostreococcus tauri

Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon

Ultraviolet radiation induces structural and chromatin damage in Mediterranean sea-urchin spermatozoa.

International audienceThere is growing concern about the effects of enhanced levels of solar ultraviolet radiation on the living components of the biosphere (i.e. cancer, loss of biodiversity and productivity, etc.). In shallow coastal environments, many benthic species release their gametes directly in the water column where fertilisation occurs and the planktonic larvae remain for several weeks. Any effects on these early life stages could significantly impair reproductive input or alter the fitness of the community. The purpose of this paper is to provide new insights into the mechanisms of UV toxicity on sea-urchin spermatozoa in a cytological context, and to address the question of the potential ecological consequences of the damage. The Mediterranean sea-urchin Sphaerechinus granularis (Lamarck) was chosen as a model to study the effects of ecologically relevant doses of UV-R on the spermatozoa of marine invertebrates. Structural damage was visualised by use of transmission electron microscopy and the single-cell gel electrophoresis (Comet) assay was used to assess chromatin integrity in spermatozoa. The present results provide experimental evidence that irradiation with UV induces structural and chromatin damage in sea-urchin sperm. Almost 90% of spermatozoa exhibited morphological alterations and DNA strand breakage increased 2-fold. The observed alterations of the acrosome, plasma membrane and mitochondria can explain the concomitant impairment of fertilisation (23% decrease of fertilisation rate), which in turn may affect reproductive success. On the other hand, how DNA damage and fertilisation rate correlate remains unclear; however, when not repaired genetic lesions can lead to abnormal development and/or the transmission of heritable damage. The 3-fold decrease of the frequency of 2-celled embryos indicates a delay or inhibition of the first cell division, which may be ascribed to impairment of nuclear chromatin and/or other cellular targets

Integration of light signals by the retinoblastoma pathway in the control of S phase entry in the picophytoplanktonic cell Ostreococcus.

Although the decision to proceed through cell division depends largely on the metabolic status or the size of the cell, the timing of cell division is often set by internal clocks such as the circadian clock. Light is a major cue for circadian clock entrainment, and for photosynthetic organisms it is also the main source of energy supporting cell growth prior to cell division. Little is known about how light signals are integrated in the control of S phase entry. Here, we present an integrated study of light-dependent regulation of cell division in the marine green alga Ostreococcus. During early G1, the main genes of cell division were transcribed independently of the amount of light, and the timing of S phase did not occur prior to 6 hours after dawn. In contrast S phase commitment and the translation of a G1 A-type cyclin were dependent on the amount of light in a cAMP-dependent manner. CyclinA was shown to interact with the Retinoblastoma (Rb) protein during S phase. Down-regulating Rb bypassed the requirement for CyclinA and cAMP without altering the timing of S phase. Overexpression of CyclinA overrode the cAMP-dependent control of S phase entry and led to early cell division. Therefore, the Rb pathway appears to integrate light signals in the control of S phase entry in Ostreococcus, though differential transcriptional and posttranscriptional regulations of a G1 A-type cyclin. Furthermore, commitment to S phase depends on a cAMP pathway, which regulates the synthesis of CyclinA. We discuss the relative involvements of the metabolic and time/clock signals in the photoperiodic control of cell division

Localization of Cyclins K and L in interphase.

<p>GFP-cyclin K and HA-cyclins L1 and L2 were expressed in HeLa cells and their localizations were analysed by fluorescence microscopy using respectively GFP-cyclin K (A) or immunofluorescence with anti-cyclin L1 (D) and L2 (G) antibodies. Localizations were compared with the one of endogenous PTB (B,E,H). Respective merge images (C,F,I) show an absence or a very poor co-localization of cyclins with CDK13 in PNC. An increased magnification of merge labelling is inserted in C and I. Scale bar: 5μm.</p

CDK13, a Kinase Involved in Pre-mRNA Splicing, Is a Component of the Perinucleolar Compartment

<div><p>The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human.</p></div

Distribution of CDK13 during cell cycle.

<p>Confocal images of HeLa (A-H) cells in which DNA is stained with SYBR green (A,C,E,G) and CDK13 was detected by immunolabeling using anti-CDK13 antibody (B,D,F,H). In interphase (A,B), arrows highlights brighter spots of CDK13 close to the nucleoli. CDK13 distribution is further described in metaphase (C,D), early (E,F) and late telophase (G,H). During mitosis, arrowheads highlight accumulation of CDK13 close to the chromatin (D) and show CDK13 localized in dots in late telophase (H). A co-labelling of CDK13 and SRSF2 (I-N) shows the enrichment of CDK13 in speckles (SRSF2 positive spots) and in dots negative for SFSF2 (arrows in K,N) in the nucleolar area of HeLa (I-K) and U2OS (L-N) cells. Scale bar: 5μm.</p

Clk2 is not present in PNC.

<p>Clk2, revealed with antibodies to the GST-tag (B,D) and GFP-CDK13 (A) localized in common foci in the nucleoplasm as shown in merge picture (C) while Clk2 is not present in PNC as visualized with PTB colabelling (D-F). Scale bar: 5μm.</p

Increased CDK13 complexes expression decreases PNC prevalence.

<p>(A) GFP-tagged cyclins were expressed with or without HA-CDK13 in HeLa cells and the level of protein expression was controlled by GFP fluorescence or immunofluorescence. PNC prevalence was measured by immunofluorescence labelling with anti-PTB antibodies. Percentages (± SD) of transfected (GFP-positive) and non-transfected (nt) cells containing nuclear PTB-positive dots were evaluated by counting for each condition 500 cultured cells in three different transfection experiments. In those experiments, 30% of non-transfected HeLa cells were PTB-positive. **P<0.001. (B) HeLa cells were treated with (or without) siRNAs targeting CDK13. SiRNA2 only faintly diminished CDK13 expression levels and did not significantly altered PNC prevalence. In contrast, siRNA5 strongly altered CDK13 expression, leading to a significant decrease in PNC prevalence. In this set of experiments, 50% of control cells (w/o siRNA) were PTB-positive. *P<0,05.</p

Localization of CDK13, nucleolin and fibrillarin during interphase.

<p>In HeLa cells, the localization of CDK13 (A,C,D,F), nucleolin (B,C) and fibrillarin (E,F) were detected using rabbit anti-CDK13 and mouse anti-nucleolin or anti-fibrillarin antibodies. A poor (C) or absent (F) colocalization of CDK13 with these nucleolar markers demonstrates that CDK13 is located in a perinucleolar structure. Scale bar: 5μm.</p