Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments.

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.

High-precision U-Pb and 40Ar/39Ar dating of an Alpine ophiolite (Gets nappe, French Alps)

Coarse-grained gabbros from two different localities in the Gets nappe (Upper Prealps) have been dated by U-Pb and Ar-40/Ar-39 isotopic analyses. Zircons from both gabbros gave identical concordant U-Pb ages of 166 +/- 1 Ma (Fig. 4). Amphibole from one of them gave an Ar-40/Ar-39 plateau age of 165.9 +/- 2.2 Ma (Fig. 5). This concordance implies that 166 +/- 1 Ma is the age of magmatic crystallization of these gabbros. The Gets wildflysch with its mafic and ultramafic lenses is an ophiolitic melange, that we infer to come from a proximal part of the accretionary prism at the foot of the active SE margin of the Piemont ocean. In this position we can expect to find remnants of the oldest parts of the Piemont oceanic crust. These are the first high-precision dates using modern techniques from an Alpine ophiolite and are in excellent agreement with the following: 1) The few, somewhat younger, reliable ages on ophiolites from the probable continuation of the Piemont basin into the Apennines and Corsica; 2) Recent data on the age of the first supra-ophiolitic sediments (Late Bathonian to Early Callovian radiolarites); 3) The structural and stratigraphic evolution of the Brianconnais (s.s.) domain, the future NW margin of the Piemont ocean. We note a remarkable coincidence, in Late Bajocian time, between: (A) the end of tensile fracturing in the Brianconnais continental crust; (B) the beginning of its subsidence; (C) the age of the Gets ophiolites. This coincidence is consistent with an ocean opening mechanism based on a combination of subhorizontal extension and thermally driven vertical movements of the lithosphere.

Linking microtubules to the activation of CDC42 in fission yeast

Cell polarity is an essential property of most cell types and relies on a dynamic cytoskeleton of actin filaments and microtubules. In rod-shaped S. pombe cells microtubules are organized along the length of the cell and transport polarity factors to cell tips to regulate cell polarity. An important cell polarity factor is the protein Tea4, which is responsible for correct cell morphogenesis and bipolar growth. During my research I confirmed the known transport mechanism of Tea4 and I also showed alternative localization and anchoring mechanisms at the cell ends. Tea4 contains a conserved SH3 domain, the function of which was unknown and my results show that the SH3 domain of Tea4 is essential for Tea4 function in vivo. First, cells with tea4SH3 mutations show aberrant cell shapes and monopolar growth patterns similar to tea4A and in addition SH3 domain is important for proper localization of multiple cell polarity proteins. Second, I showed that Tea4 associates with Type 1 Phosphatase Dis2 through both its SH3 domain and an RVxF motif. Tea4 also binds the DYRK kinase Pomi through its SH3 domain. In addition Tea4 is proposed to promote the local dephosphorylation of Pomi by Dis2 to induce the formation of a cortical gradient from cell ends essential for cell size homeostasis. Polarized growth is also controlled by cell tip-localized Cdc42. This Rho- family GTPase is activated by the Guanine Exchange Factors Gef1 and Scd1 and inactivated by the Rho GTPase Activating Protein Rga4. In this study, I investigated the mechanisms of how Tea4 promotes Cdc42 activation. My work suggests that Tea4 promotes the local exclusion of Rga4, which in turn allows the accumulation of active Cdc42, which may result in growth. Exclusion of Rga4 by Tea4 is likely to be mediated by Dis2-dependent dephosphorylation. These results suggest a molecular pathway that links the microtubule- associated factor Tea4 with Cdc42 to promote cell polarization and morphogenesis. - La polarité cellulaire est une propriété essentielle de la plupart des types cellulaires et s'appuie sur une dynamique des cytosquelettes d'actine et de microtubules. Dans les cellules en forme de bâtonnet de S. pombe les microtubules sont alignés selon l'axe longitudinal de la cellule et les facteurs de polarité transportés aux extrémité cellulaires afin de réguler la polarité cellulaire. Un facteur important de polarité cellulaire est la protéine Tea4, qui est responsable de la morphogenèse des cellules et leur croissance bipolaire. Au cours de mes recherches, j'ai confirmé les mécanismes connus de transport de Tea4 et j'ai aussi mis en évidence d'autres mechanismes de localisation et d'ancrage de Tea4 aux extrémités cellulaires. Tea4 contient un domaine SH3 conservé, dont la fonction était inconnue et mes résultats montrent que le domaine SH3 est essentiel pour la fonction de Tea4 in vivo. Tout d'abord, les cellules avec des mutations tea4sm ont des formes aberrantes et leur croissance est monopolaire de manière similaire au mutant tea4A. De plus ce domaine SH3 est important pour la localisation correcte de plusieurs protéines de polarité cellulaire. Deuxièmement, j'ai montré que Tea4 s'associe avec la Phosphatase de Type-1 Dis2 par son domaine SH3 et un motif RVxF. Tea4 se lie également la kinase DYRK Pomi par son domaine SH3. De plus, Tea4 pourrait favoriser la déphosphorylation locale de Pomi par Dis2 afin d'induire la formation d'un gradient cortical de Pomi essentiel pour l'homéostasie de la longueur des cellules. La croissance polarisée est également contrôlée par la protéine Cdc42 localisée aux extrémités cellulaires. Cette GTPase de la famille de Rho GTPase est activée par les facteurs échange de guanine Gef1 et Scd1 et inactivée par la protéine "Rho GTPase activating" Rga4. Dans cette étude, j'ai étudié les mécanismes d' activation de Cdc42 par Tea4. Mes résultats suggèrent que Tea4 favorise l'exclusion locale de Rga4, ce qui permet l'accumulation de Cdc42 active, nécessaire à la croissance. L' exclusion de Rga4 par Tea4 est vraisemblablement médiée par une déphosphorylation Dis2- dépendente. Ces résultats suggèrent une voie moléculaire qui lie le facteur associé aux microtubules Tea4 à Cdc42 pour promouvoir la polarisation cellulaire et la morphogenèse. - Cell polarity is important for several essential biological functions such as generation of distinct cell fates during development and function of differentiated cells. Defective cell polarity has been related to uncontrolled cell division and subsequently to cancer initiation. Cell polarity depends on a functional cytoskeleton that consists of actin filaments and microtubules, which maintains cell shape, helps cellular motion, enables intracellular protein transport and plays a vital role in cell division. A component of cytoskeleton is microtubules that regulate cell polarization in diverse cell types. During my research, I worked with Schizosaccharomyces pombe, also named fission yeast, a powerful unicellular model organism that allows combination of genetic, biochemical and microscopic analysis for the proper study of cell polarity. Microtubule-associated protein Tea4 is transported to cell tips where it is thought to organize polarized growth. I showed that Tea4 and its evolutionarily conserved SH3 domain play an important role for maintenance of fission yeast cells shape and growth. Furthermore, Tea4 is responsible for the proper localization of multiple polarity proteins and acts as a mediator to control the local activity of an essential polarity regulator called Cdc42. Thus, my results provide a better understanding of the molecular mechanisms that regulate cell polarity. - La polarité cellulaire est importante pour plusieurs fonctions biologiques essentielles telles que la différenciation cellulaires au cours du développement et de la fonction de cellules différenciées. Les défauts de la polarité cellulaire ont été liés à des divisions cellulaires incontrôlées et à l'initiation de tumeur. La polarité cellulaire dépend d'un cytosquelette fonctionnel, qui maintient la forme des cellules, aide à la migration cellulaire, permet le transport intracellulaire des protéines et joue un rôle essentiel dans la division cellulaire. Un composant du cytosquelette est constitué de microtubules qui régissent la polarisation cellulaire dans divers types cellulaires. Au cours de mes recherches, j'ai travaillé avec Schizosaccharomyces pombe, appelé également levure fissipare, un modèle unicellulare puissant qui permet la combinaison de différentes d'approches expérimentales: génétiques, biochimiques et microscopiques pour l'étude de la polarité cellulaire. La protéine Tea4 associée aux microtubules est transportée aux extrémités cellulaires où elle organise la croissance polarisée. J'ai montré que Tea4 et son domaine conservé SH3 jouent un rôle important pour le maintien de la forme des cellules de levure et leur croissance. De plus, Tea4 est responsable de la localisation correcte de multiples facteurs de polarité et agit comme un médiateur pour contrôler l'activité locale d'un régulateur de polarité essentiel appelé Cdc42. Ainsi, mes résultats permettent de mieux comprendre les mécanismes moléculaires qui régulent la polarité cellulaire.

Dating human skeletal remains using a radiometric method: Biogenic versus diagenetic (90)Sr and (210)Pb in vertebrae.

In forensic science, there is a strong interest in determining the post-mortem interval (PMI) of human skeletal remains up to 50 years after death. Currently, there are no reliable methods to resolve PMI, the determination of which relies almost exclusively on the experience of the investigating expert. Here we measured (90)Sr and (210)Pb ((210)Po) incorporated into bones through a biogenic process as indicators of the time elapsed since death. We hypothesised that the activity of radionuclides incorporated into trabecular bone will more accurately match the activity in the environment and the food chain at the time of death than the activity in cortical bone because of a higher remodelling rate. We found that determining (90)Sr can yield reliable PMI estimates as long as a calibration curve exists for (90)Sr covering the studied area and the last 50 years. We also found that adding the activity of (210)Po, a proxy for naturally occurring (210)Pb incorporated through ingestion, to the (90)Sr dating increases the reliability of the PMI value. Our results also show that trabecular bone is subject to both (90)Sr and (210)Po diagenesis. Accordingly, we used a solubility profile method to determine the biogenic radionuclide only, and we are proposing a new method of bone decontamination to be used prior to (90)Sr and (210)Pb dating.

Shaping fission yeast cells by rerouting actin-based transport on microtubules.

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other. In this organism, microtubules and actin cables both contribute to polarized growth by transporting cargos to cell poles, but with distinct roles: microtubules transport landmarks to label cell poles for growth and actin assembly but do not directly contribute to the growth process [1]. Actin cables serve as tracks for myosin V delivery of growth vesicles to cell poles [2-4]. We engineered a chimera between the motor domain of the kinesin 7 Tea2 and the globular tail of the myosin V Myo52, which we show transports Ypt3, a myosin cargo receptor, to cell poles along microtubules. Remarkably, this chimera restores polarized growth and viability to cells lacking actin cables. It also bypasses the normal microtubule-dependent marking of cell poles for polarized growth, but not for other functions. Thus, a synthetic motor protein successfully redirects cargos along a distinct cytoskeletal route.

Lifetime of an ocean island volcano feeder zone: constraints from U-Pb dating on coexisting zircon and baddeleyite, and 40Ar/39Ar age determinations, Fuerteventura, Canary Islands

High-precision isotope dilution - thermal ionization mass spectrometry (ID-TIMS) U-Pb zircon and baddeleyite ages from the PX1 vertically layered mafic intrusion Fuerteventura, Canary Islands, indicate initiation of magma crystallization at 22.10 +/- 0.07 Ma. The magmatic activity lasted a minimum of 0.52 Ma. Ar-40/Ar-39 amphibole dating yielded ages from 21.9 +/- 0.6 to 21.8 +/- 0.3, identical within errors to the U-Pb ages, despite the expected 1% theoretical bias between Ar-40/Ar-39 and U-Pb dates. This overlap could result from (i) rapid cooling of the intrusion (i. e., less than the 0.3 to 0.6 Ma 40Ar/39Ar age uncertainties) from closure temperatures (T-c) of zircon (699-988 degrees C) to amphibole (500-600 degrees C); (ii) lead loss affecting the youngest zircons; or (iii) excess argon shifting the plateau ages towards older values. The combination of the Ar-40/Ar-39 and U/Pb datasets implies that the maximum amount of time PX1 intrusion took to cool below amphibole T-c is 0.8 Ma, suggesting PX1 lifetime of 520 000 to 800 000 Ma. Age disparities among coexisting baddeleyite and zircon (22.10 +/- 0.07/0.08/0.15 Ma and 21.58 +/- 0.15/0.16/0.31 Ma) in a gabbro sample from the pluton margin suggest complex genetic relationships between phases. Baddeleyite is found preserved in plagioclase cores and crystallized early from low silica activity magma. Zircon crystallized later in a higher silica activity environment and is found in secondary scapolite and is found close to calcite veins, in secondary scapolite that recrystallised from plagioclase. close to calcite veins. Oxygen isotope delta O-18 values of altered plagioclase are high (+7.7), indicating interaction with fluids derived from host-rock carbonatites. The coexistence of baddeleyite and zircon is ascribed to interaction of the PX1 gabbro with CO2-rich carbonatite-derived fluids released during contact metamorphism.

Documents : Dating a document

The determination of the age of a document is a very frequent query, however it is also one of the most challenging and controversial areas of questioned document examination. Several approaches were defined to address this problem. The first is based on the introduction date of raw constituents (like paper or ink) on the market. The second approach is based on the aging of documents, which is unfortunately not only influenced by passing time, but also by storage conditions and document composition. The third approach considers the relative age of documents and aims at reconstructing their chronology. The three approaches are equally complex to develop and encounter quantity of problems which are not negligible. This article aims to expose the potential applications and limitations of current ink dating methods. Method development and validation and the interpretation of evidence prove to be essential criteria for the dating of documents.

How the visual brain encodes and keeps track of time.

Time is embedded in any sensory experience: the movements of a dance, the rhythm of a piece of music, the words of a speaker are all examples of temporally structured sensory events. In humans, if and how visual cortices perform temporal processing remains unclear. Here we show that both primary visual cortex (V1) and extrastriate area V5/MT are causally involved in encoding and keeping time in memory and that this involvement is independent from low-level visual processing. Most importantly we demonstrate that V1 and V5/MT come into play simultaneously and seem to be functionally linked during interval encoding, whereas they operate serially (V1 followed by V5/MT) and seem to be independent while maintaining temporal information in working memory. These data help to refine our knowledge of the functional properties of human visual cortex, highlighting the contribution and the temporal dynamics of V1 and V5/MT in the processing of the temporal aspects of visual information.

Réhabilitation accélérée multimodate postcésarienne, la somme de toutes les astuces [Fast-track multimodal rehabilitation after cesarean, the sum of all tricks].

Fast-track multimodal rehabilitation after cesarean, the sum of all tricks Fast-track multimodal rehabilitation after caesarean is an interdisciplinary concept allowing an accelerated return to normal physiology. Fast-track rehabilitation combines minimising surgical trauma, regional anaesthesia and active management of pain control, minimally invasive postoperative care while promoting return to autonomy.

Analysis of the mechanisms controlling the activity of flp1, a chromosomal passenger protein in the fission Schizosaccharomyces pombe

ABSTRACT In S. cerevisiae, the protein phosphatase Cdc14pwt is essential far mitotic exit through its contribution to reducing mitotic CDK activity. But Cdc14pwt also acts as a mare general temporal coordinator of mid and late mitotic events by controlling the partitioning of DNA, microtubule stability and cytokinesis. Cdc14pwt orthologs are well conserved from yeasts to humans, and sequence comparison revealed the presence of three domains, A, B and C, of which A and B form the catalytic domain. Cdc14pwt orthologs are regulated (in part) through cell cycle dependent changes in their localization. Some of them are thought to be kept inactive by sequestration in the nucleolus during interphase. This is the case for flp1pwt, the single identified Cdc14pwt ortholog in the fission yeast S. pombe. In early mitosis, flp1pwt leaves the nucleolus and localizes to the kinetochores, the contractile ring and the mitotic spindle, suggesting that it has multiple substrates and regulates many mitotic processes. flp1D cells show a high chromosome loss rate and septation defects, suggesting a role for flp1wt in the fidelity of chromosome transmission and cytokinesis. The aim of this study is to characterize the mechanisms underlying flp1pwt functions and the control of its activity. A structure-function analysis has revealed that the presence of both A and B domains is required for biological function and for proper flp1pwt mitotic localization. In contrast, the C domain of flp1pwt is responsible for its proper nucleolar localization in G2/interphase. My data suggest that dephosphorylation of substrates by flp1pwt is not necessary for any changes in localization of flp1pwt except that at the medial ring. In that particular case, the catalytic activity of flp1pwt is required for efficient localization, therefore revealing an additional level of regulation. All the functions of flp1pwt assayed to date require its catalytic activity, emphasizing the importance of further identification of its substrates. As described for other orthologs, the capability of selfinteraction and phosphorylation status might help to control flp1pwt activity. My data suggest that flp1pwt forms oligomers in vivo and that phosphorylation is not essential far localization changes of the protein. In addition, the hypophosphorylated form of flp1pwt might be specifically involved in the promotion of cytokinesis. The results of this study suggest that multiple modes of regulation including localization, selfassociation and phosphorylation allow a fine-tuning regulation of flp1pwt phosphatase activity, and more generally that of Cdc14pwt family of phosphatases. RESUME Chez la levure S. cerevisiae, la protéine phosphatase Cdc14pwt est essentielle pour la sortie de mitose du fait de sa contribution dans la réduction d'activité des CDK mitotiques. Comme elle contrôle également le partage de l'ADN, la stabilité des microtubules et la cytokinèse, Cdc14pwt est en fait considérée comme un coordinateur temporel général des évènements de milieu et de fin de mitose. Les orthologues de Cdc14pwt sont bien conservés, des levures jusqu'à l'espèce humaine. Des comparaisons de séquence ont révélé la présence de trois domaines A, B et C, les deux premiers constituant le domaine catalytique. Ils sont régulés (en partie) via des changements dans leur localisation, eux-mêmes dépendants du cycle cellulaire. Plusieurs de ces orthologues sont supposés inactivés par séquestration dans le nucléole en interphase, ce qui est le cas de flp1pwt le seul orthologue de Cdc14pwt identifié chez la levure fissipare S, pombe. En début de mitose, flp1pwt quitte le nucléole et localise au niveau des kinetochores, de l'anneau contractile d'actine et du fuseau mitotique, ce qui laisse supposer de multiples substrats et fonctions. Comme les cellules délétées pour le gène flp1wt présentent un taux élevé de perte de chromosome et des défauts de septation, flp1pwt semble jouer un rôle dans la fidélité de la transmission du matériel génétique et la cytokinèse. Le but de cette étude est de caractériser les mécanismes impliqués dans les fonctions assurées par flp1pwt d'une part, et dans le contrôle de son activité d'autre part. Une analyse structure-fonction a révélé que la présence simultanée des deux domaines A et B est requise pour la fonction biologique de flp1pwt et sa localisation correcte pendant la mitose. Par contre, le domaine C de flp1pwt confère une localisation nucléolaire adéquate en G2/interphase. Mes données suggèrent que la déphosphorylation de substrats par flp1pwt est dispensable pour sa localisation correcte excepté celle à l'anneau médian, qui requiert dans ce cas, l'activité catalytique de flp1pwt, révélant ainsi un niveau de régulation supplémentaire. Toutes les fonctions de flp1 pwt testées jusqu'à présent nécessitent également son activité catalytique, ce qui accentue l'importance de l'identification future de ses substrats. Comme cela a déjà été décrit pour d'autres orthologues, la capacité d'auto-intéraction et le niveau de phosphorylation pourraient contrôler l'activité de flp1pwt. En effet, mes données suggèrent que flp1pwt forme des oligomères in vivo et que la phosphorylation n'est pas essentielle pour les changements de localisation observés pour la protéine. De plus, la forme hypophosphorylée de flp1pwt pourrait être spécifiquement impliquée dans la promotion de la cytokinèse. De multiples modes de régulation incluant la localisation, l'auto-association et la phosphorylation semblent permettre un contrôle fin et subtil de l'activité de la phosphatase flp1pwt, et plus généralement celle des protéines de la famille de Cdc14pwt.

Mutual control of membrane fission and fusion proteins.

Membrane fusion and fission are antagonistic reactions controlled by different proteins. Dynamins promote membrane fission by GTP-driven changes of conformation and polymerization state, while SNAREs fuse membranes by forming complexes between t- and v-SNAREs from apposed vesicles. Here, we describe a role of the dynamin-like GTPase Vps1p in fusion of yeast vacuoles. Vps1p forms polymers that couple several t-SNAREs together. At the onset of fusion, the SNARE-activating ATPase Sec18p/NSF and the t-SNARE depolymerize Vps1p and release it from the membrane. This activity is independent of the SNARE coactivator Sec17p/alpha-SNAP and of the v-SNARE. Vps1p release liberates the t-SNAREs for initiating fusion and at the same time disrupts fission activity. We propose that reciprocal control between fusion and fission components exists, which may prevent futile cycles of fission and fusion.