Philippe Schatt

Use of plankton-derived vitamin B1 precursors, especially thiazole-related precursor, by key marine picoeukaryotic phytoplankton

Several cosmopolitan marine picoeukaryotic phytoplankton are B1 auxotrophs requiring exogenous vitamin B1 or precursor to survive. From genomic evidence, representatives of picoeukaryotic phytoplankton (Ostreococcus and Micromonas spp.) were predicted to use known thiazole and pyrimidine B1 precursors to meet their B1 demands, however, recent culture-based experiments could not confirm this assumption. We hypothesized these phytoplankton strains could grow on precursors alone, but required a thiazole-related precursor other the well-known and extensively tested 4-methyl-5-thiazoleethanol. This hypothesis was tested using bioassays and co-cultures of picoeukaryotic phytoplankton and bacteria. We found that specific B1-synthesizing proteobacteria and phytoplankton are sources of a yet-to-be chemically identified thiazole-related precursor(s) that, along with pyrimidine B1 precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine, can support growth of Ostreococcus spp. (also Micromonas spp.) without B1. We additionally found that the B1-synthesizing plankton do not require contact with picoeukaryotic phytoplankton cells to produce thiazole-related precursor(s). Experiments with wild-type and genetically engineered Ostreococcus lines revealed that the thiazole kinase, ThiM, is required for growth on precursors, and that thiazole-related precursor(s) accumulate to appreciable levels in the euphotic ocean. Overall, our results point to thiazole-related B1 precursors as important micronutrients promoting the survival of abundant phytoplankton influencing surface ocean production and biogeochemical cycling.

Central role for ferritin in the day/night regulation of iron homeostasis in marine phytoplankton

Significance Phytoplankton transforms large amounts of inorganic to organic carbon, a critical step in the uptake of atmospheric CO2 in the ocean. Although iron is essential for this process, its bioavailability is often low. Phytoplankton has evolved strategies to cope with low environmental iron concentrations. We discovered that the iron storage protein ferritin is strongly regulated by the day/night cycle in the coastal California upwelling region dominated by the picophytoplanktonic genus Ostreococcus. Using genetic approaches, we showed that ferritin is used for short-term recycling of iron over diurnal cycles rather than for long-term iron storage. This work revealing the importance of ferritin in diurnal and circadian regulations of iron homeostasis should have implications for understanding carbon uptake by the ocean. In large regions of the open ocean, iron is a limiting resource for phytoplankton. The reduction of iron quota and the recycling of internal iron pools are among the diverse strategies that phytoplankton have evolved to allow them to grow under chronically low ambient iron levels. Phytoplankton species also have evolved strategies to cope with sporadic iron supply such as long-term storage of iron in ferritin. In the picophytoplanktonic species Ostreococcus we report evidence from observations both in the field and in laboratory cultures that ferritin and the main iron-binding proteins involved in photosynthesis and nitrate assimilation pathways show opposite diurnal expression patterns, with ferritin being maximally expressed during the night. Biochemical and physiological experiments using a ferritin knock-out line subsequently revealed that this protein plays a central role in the diel regulation of iron uptake and recycling and that this regulation of iron homeostasis is essential for cell survival under iron limitation.

Probing entrainment of Ostreococcus tauri circadian clock by green and blue light through a mathematical modeling approach

Most organisms anticipate daily environmental variations and orchestrate cellular functions thanks to a circadian clock which entrains robustly to the day/night cycle, despite fluctuations in light intensity due to weather or seasonal variations. Marine organisms are also subjected to fluctuations in light spectral composition as their depth varies, due to differential absorption of different wavelengths by sea water. Studying how light input pathways contribute to circadian clock robustness is therefore important. Ostreococcus tauri, a unicellular picoplanktonic marine green alga with low genomic complexity and simple cellular organization, has become a promising model organism for systems biology. Functional and modeling approaches have shown that a core circadian oscillator based on orthologs of Arabidopsis TOC1 and CCA1 clock genes accounts for most experimental data acquired under a wide range of conditions. Some evidence points at putative light input pathway(s) consisting of a two-component signaling system (TCS) controlled by the only two histidine kinases (HK) of O. tauri. LOV-HK is a blue light photoreceptor under circadian control, that is required for circadian clock function. An involvement of Rhodopsin-HK (Rhod-HK) is also conceivable since rhodopsin photoreceptors mediate blue to green light input in animal circadian clocks. Here, we probe the role of LOV-HK and Rhod-HK in mediating light input to the TOC1-CCA1 oscillator using a mathematical model incorporating the TCS hypothesis. This model agrees with clock gene expression time series representative of multiple environmental conditions in blue or green light, characterizing entrainment by light/dark cycles, free-running in constant light, and resetting. Experimental and theoretical results indicate that both blue and green light can reset O. tauri circadian clock. Moreover, our mathematical analysis suggests that Rhod-HK is a blue-green light receptor and drives the clock together with LOV-HK.

Evolution of Cyclin B3 Shows an Abrupt Three-Fold Size Increase, due to the Extension of a Single Exon in Placental Mammals, Allowing for New Protein–Protein Interactions

Cyclin B3 evolution has the unique peculiarity of an abrupt 3-fold increase of the protein size in the mammalian lineage due to the extension of a single exon. We have analyzed the evolution of the gene to define the modalities of this event and the possible consequences on the function of the protein. Database searches can trace the appearance of the gene to the origin of metazoans. Most introns were already present in early metazoans, and the intron-exon structure as well as the protein size were fairly conserved in invertebrates and nonmammalian vertebrates. Although intron gains are considered as rare events, we identified two cases, one at the prochordate-chordate transition and one in murids, resulting from different mechanisms. At the emergence of mammals, the gene was relocated from chromosome 6 of platypus to the X chromosome in marsupials, but the exon extension occurred only in placental mammals. A repetitive structure of 18 amino acids, of uncertain origin, is detectable in the 3,000-nt mammalian exon-encoded sequence, suggesting an extension by multiple internal duplications, some of which are still detectable in the primate lineage. Structure prediction programs suggest that the repetitive structure has no associated three-dimensional structure but rather a tendency for disorder. Splice variant isoforms were detected in several mammalian species but without conserved pattern, notably excluding the constant coexistence of premammalian-like transcripts, without the extension. The yeast two-hybrid method revealed that, in human, the extension allowed new interactions with ten unrelated proteins, most of them with specific three-dimensional structures involved in protein-protein interactions, and some highly expressed in testis, as is cyclin B3. The interactions with activator of cAMP-responsive element modulator in testis (ACT), germ cell-less homolog 1, and chromosome 1 open reading frame 14 remain to be verified in vivo since they may not be expressed at the same stages of spermatogenesis as cyclin B3.

CDK5 is present in sea urchin and starfish eggs and embryos and can interact with p35, cyclin E and cyclin B3

While most cyclin‐dependent kinases (CDKs) are involved in cell cycle control, CDK5 is mostly known for crucial functions in neurogenesis. However, we cloned sea urchin CDK5 from a two‐cell stage cDNA library and found that the protein is present in eggs and embryos, up to the pluteus stage, but without associated kinase activity. To investigate the potential for nonneuronal roles, we screened a starfish cDNA library with the yeast two‐hybrid system, for possible CDK5 partners. Interactions with clones expressing part of cyclin B3 and cyclin E proteins were found and the full‐length cyclins were cloned. These interactions were verified in vitro but not in extracts of starfish oocytes and embryos, at any stages, despite the presence of detectable amounts of CDK5, cyclin B3, and cyclin E. We then looked for p35, the CDK5‐specific activator, and cloned the sea urchin ortholog. A sea urchin‐specific anomaly in the amino acid sequence is the absence of N‐terminal myristoylation signal, but nucleotide environment analysis suggests a much higher probability of translation initiation on the second methionine(Met44), that is associated with a conserved myristoylation signal. p35 was found to associate with CDK5 and, when bacterially produced, to confer protein kinase activity to CDK5 immunoprecipitated from sea urchin eggs and embryos. However, p35 mRNA expression was found to begin only at the end of the blastula stage, and the protein was undetectable at any embryonic stage, suggesting a neuronal role beginning in late larval stages. Mol. Reprod. Dev. 77: 449–461, 2010.

Cybip, a starfish cyclin B-binding protein, is involved in meiotic M-phase exit.

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219-231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.

Skeletogenesis in sea urchin larvae under modified gravity conditions.

From many points of view, skeletogenesis in sea urchins has been well described. Based on this scientific background and considering practical aspects of sea urchin development (i.e. availability of material, size of larvae, etc.), we wanted to know whether orderly skeletogenesis requires the presence of gravity. The objective has been approached by three experiments successfully performed under genuine microgravity conditions (in the STS-65 IML-2 mission of 1994; in the Photon-10 IBIS mission of 1995 and in the STS-76 S/MM-03 mission of 1996). Larvae of the sea urchin Sphaerechinus granularis were allowed to develop in microgravity conditions for several days from blastula stage onwards (onset of skeletogenesis). At the end of the missions, the recovered skeletal structures were studied with respect to their mineral composition, architecture and size. Live larvae were also recovered for post-flight culture. The results obtained clearly show that the process of mineralisation is independent of gravity: that is, the skeletogenic cells differentiate correctly in microgravity. However, abnormal skeleton architectures were encountered, particularly in the IML-2 mission, indicating that the process of positioning of the skeletogenic cells may be affected, directly or indirectly, by environmental factors, including gravity. Larvae exposed to microgravity from blastula to prism/early pluteus stage for about 2 weeks (IBIS mission), developed on the ground over the next 2 months into normal metamorphosing individuals.

Comparative Analysis of Culture Conditions for the Optimization of Carotenoid Production in Several Strains of the Picoeukaryote Ostreococcus

Microalgae are promising sources for the sustainable production of compounds of interest for biotechnologies. Compared to higher plants, microalgae have a faster growth rate and can be grown in industrial photobioreactors. The microalgae biomass contains specific metabolites of high added value for biotechnology such as lipids, polysaccharides or carotenoid pigments. Studying carotenogenesis is important for deciphering the mechanisms of adaptation to stress tolerance as well as for biotechnological production. In recent years, the picoeukaryote Ostreococcus tauri has emerged as a model organism thanks to the development of powerful genetic tools. Several strains of Ostreococcus isolated from different environments have been characterized with respect to light response or iron requirement. We have compared the carotenoid contents and growth rates of strains of Ostreococcus (OTTH595, RCC802 and RCC809) under a wide range of light, salinity and temperature conditions. Carotenoid profiles and productivities varied in a strain-specific and stress-dependent manner. Our results also illustrate that phylogenetically related microalgal strains originating from different ecological niches present specific interests for the production of specific molecules under controlled culture conditions.

SGEBP, a giant protein from starfish oocytes able to interact with ERK

The mitogen‐activated protein kinase (MAPK) pathway is a key regulator of animal meiotic divisions. It involves cascades of kinases whose specificity has been shown to depend on binding proteins acting as scaffolds. We searched for proteins interacting with starfish extracellular signal‐regulated kinase (ERK) using the yeast two‐hybrid system. An interacting clone was found to encode the 5′ region of a giant 16.7‐kb transcript encoded by an intronless gene. The corresponding 630‐kDa protein could not be detected by Western blot, but the meiotic spindle was labelled by immunolocalization with an antibody against the ERK‐binding domain. A related gene was found in the genome of another starfish species, and similarities were also found to a 42.9‐kb open reading frame in the sea urchin genome. Yet, no conserved protein‐binding domain was detected in the amino acid sequence(s) compared to all the known motifs. Structure prediction software indicated that the encoded proteins are probably disordered while a query of the disordered protein database indicated some similarity with vertebrates microtubule‐associated protein 2 (MAP2). This predicts that SGEBP may function as a space‐filling polymer, having a role in both cytoskeleton organization and ERK targeting. Mol. Reprod. Dev. 80: 816–825, 2013.