Intra- and intersexual selection and their potential consequences on fitness-relevant traits in several fish species

Résumé : Les mécanismes de sélection sexuelle, en particulier la compétition entre mâles (sélection inter-sexuelle) et le choix des femelles (sélection intra-sexuelle), peuvent fortement influencer le succès reproducteur d'un individu, c'est-à-dire son nombre de descendants. On observe ainsi que les mâles dominants et les mâles élaborant des caractères sexuels secondaires marqués ont un succès reproducteur élevé. Toutefois, le succès reproducteur ne suffit pas pour garantir une contribution génétique élevée, parce que la fitness dépend également de la performance des descendants (c'est-à-dire de leur survie et de leur propre succès reproducteur). Si cette performance dépend en partie des gènes paternels, les males ont un avantage certain à signaler leur qualité aux femelles afin d'atteindre des taux de reproduction élevé. Ce mécanisme de signalisation est connu sous le nom de 'good genes hypothesis', toutefois très peu d'études ont clairement démontré le lien entre la qualité génétique des individus et la signalisation. De plus, la performance des descendants peut aussi dépendre des effets génétiques de compatibilité entre mâles et femelles ('compatible genes'). C'est-à-dire que certains allèles paternels n'apporteraient un avantage aux descendants qu'en combinaison avec certains allèles maternels. Nous avons déterminé, durant la période de reproduction, le statut de dominance des mâles pour deux espèces de poissons d'eau douce : la truite (Salmo trotta) et le vairon (Phoxinus phoxinus), puis nous avons évalué la relation entre le succès reproducteur et le statut de dominance et/ou la quantité de signalisation des caractères sexuels secondaires. Nous avons également fécondés artificiellement des oeufs de truites et de corégones (Coregonus palaea), en croisant chaque mâle avec chaque femelle (full-factorial breeding design). Ce type de design autorise la quantification précise des effets génétiques et permet de séparer les effets de 'good genes' et de 'compatible genes'. Cela a été fait sous différentes intensités de stress bactérien, ainsi que dans des conditions naturelles, et nous avons pu ainsi tester si certains indicateurs de qualité génétique des mâles ('good genes') étaient liés a) à la dominance et/ou b) à l'expression des caractères sexuels secondaires des mâles comme l'intensité mélanique ou la taille des tubercules sexuels. En outre, nous cherchons à savoir si la survie des descendants est liée à certaines combinaison des gènes du complexe d'histocompatibilité majeur (MHC) et/ou à la parenté génétique des parents, les deux traits étant soupçonnés d'avoir des influences génétique de compatibilité (`compatible genes') à la performance des descendants. Nous avons constaté que la dominance des mâles est directement liée à la taille et au poids des mâles (truites, vairons), mais également aux caractères sexuels secondaires (tubercules). De plus, les mâles vairons dominant ont eu un succès de fécondation plus élevés que les mâles subordonnés. Nous montrons que les truites et corégones mâles diffèrent dans leur qualité génétique, qui a été mesurée avéc la survie embryonnaire, le temps avant l'éclosion et enfin la croissance juvénile. Contrairement aux prédictions, la dominance (ou les traits indicatifs de dominance) n'était liée à la qualité génétique, dans aucun des traitements, et ne fonctionne donc pas comme indicateur de qualité. Par contre, la qualité génétique était liée aux caractères sexuels secondaires, particulièrement par la teinte mélanique chez les truites. Les embryons de truites issus de pères sombres survivaient mieux que ceux issus de pères clairs dans des environnements difficiles, de plus leur croissance était plus élevée lors de leur première année dans des conditions naturelles. La taille des juvéniles lors de leur première année est un trait important lié au succès dans la compétition pour des ressources telles qu'abri ou nourriture. De plus, les femelles truites peuvent augmenter la survie de leurs descendants en choisissant des mâles selon leur type de MHC ou selon leur degré de parenté. En outre, chez les corégones, la morphologie des tubercules sexuels ne semble pas signaler la qualité génétique. Nous avons également remarqué que l'exposition à des pathogènes non-létaux pouvait influencer la performance des alevins à court et long terme, probablement en affaiblissant leur système immunitaire. Cette thèse montre que les mâles diffèrent dans leur qualité génétique et que différents mécanismes de sélection inter- ou intra-sexuelle (par exemple la préférence pour des mâles sombres, pour des génotypes MHC ou pour des couples avec degré de parenté basse) pouvait avoir un effet positif sur la qualité des descendants, bien que cet effet génétique pouvait changer au cours du temps et entre différents environnements. Contrairement à nos attentes, le résultat de la compétition intra-sexuelle (la hiérarchie de dominance entre mâles) n'était pas lié à la qualité génétique individuelle ('good genes'). Dans ce sens, ce travail permet également de contribuer à l'explication du fait que la sélection sexuelle, de par sa forte sélection directionnelle, ne conduit pas à la diminution de la variance génétique, mais plutôt à la maintenance du polymorphisme génétique. Summary : Sexual selection mechanisms, especially male-male competition (inteasexual selection) and female mate choice (inteasexual selection), can strongly influence individual mating success, often resulting in dominant males and males with elaborate secondary sexual characters having higher fertilisation success. However, siring a high number of offspring alone does not guarantee high individual fitness, as fitness does also strongly depend on offspring performance (i.e. survival, fecundity). If this superiority in offspring performance depends on paternally inherited genes, the fathers are expected to signal this potential indirect benefit to females in order to attain high mating rates. This mechanism is also known as the 'good genes' hypothesis of sexual selection but until now most studies failed to conclusively show the relation of an individual genetic quality and its potential signalling traits. Further, offspring performance could also depend on compatible gene effects. These are alleles that increase offspring performance only in combination with other specific alleles. We first determined male dominance status from intrasexual competition during mating season for brown trout (Salmo trutta) and European minnows (Phoxinus phoxinus). For minnows we additionally checked if dominance and/or secondary sexual traits were linked to fertilisation success. Further, we artificially fertilised brown trout and alpine whitefish (Coregonus palaea) eggs, following full factorial breeding designs, enabling to properly measure `good gene' and `compatible gene' effects on offspring performance. This was done under different intensities of natural stressors, as well as under natural conditions. This procedure allowed us to test if the obtained male genetic quality measures (good genes effects) were indicated by a) dominance or lay traits linked to dominance and/or by b) secondary sexual characteristics such as melanin-based male skin darkness or breeding tubercles. Further, we investigated if offspring survival was linked to the MHC (major histocompatibility complex) gene combinations and/or to the parental genetic relatedness, as both traits were shown to have 'compatible gene' effects that may influence offspring performance. We found that male dominance in intrasexual competition was positively linked to body size, body weight (brown trout, minnows) but also to elaborate secondary sexual characteristics (breeding tubercles in minnows). Further, dominant minnow males did have an increased fertilisation success compared to subordinate ones. We show that brown trout and whitefish males do usually differ in their genetic quality, which was measured as embryo survival, hatching timing and finally as juvenile growth. Contrary to prediction male dominance or dominance indicating traits do not function as a quality signal as they were not linked to genetic quality. This result was constant when measuring genetic quality under different levels of natural stressors and under natural conditions (brown trout). On the other hand genetic quality seemed to be indicated by secondary sexual characteristics, specifically by melanin-based skin darkness in brown trout as brown trout embryos sired by darker fathers had increased survival rates when raised under harsh conditions and. they grew larger as juveniles after one year of growth in a natural stream, which is an important trait influencing success of juveniles in competition for hidings, food and other resources. Furthermore, brown trout females may increase the survival of their embryos when choosing males according to their MHC genotypes or to the general genetic relatedness between themselves and their potential mates. In whitefish on the other hand breeding tubercle morphology did not seem to signal genetic quality. Eventually, we saw that anon-lethal exposure to pathogens might influence short term and long term offspring performance probably by weakening an exposed individual's immune system. This thesis shows that males usually differ in their genetic quality and that different inter- or intrasexual selection mechanisms (e.g. mate selection favouring dark males, preference for MHC genotype combinations or for unrelated mates) may have strong positive effects on genetically dependent offspring performance but that such genetìc effects can change over time and environments. In contrast to our a priori expectations, the outcome of intrasexual selection, namely male dominance hierarchies, with dominant males often having high fertilisation success, was not linked to individual genetic quality (`good genes'). In this sense the present thesis may also be a helpful contribution to understand why sexual selection does not lead to rapid loss of genetic variation by strong directional selection but could even lead to the maintenance of genetic variation in natural populations.

Modulation of phosphorylation of neuronal cytoskeletal proteins by neuronal depolarization.

1. The neuronal cytoskeletal protein tau and the carboxy tails of cytoskeletal proteins neurofilament-M (NF-M) and neurofilament-H (NF-H) are phosphorylated on serine residues by the cyclin-dependent kinase cdk-5. 2. In aggregating neuronal-glial cultures we show that veratridine-mediated cation influx causes dephosphorylation of tau, NF-M and NF-H. Dephosphorylation was blocked specifically by cyclosporine A but not by okadiac acid at concentrations up to 200 nM. 3. These results suggest that veratridine-triggered cation influx causes activation of PP-2B (calcineurin) leading to dephosphorylation of these cytoskeletal proteins.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Differential expression and modification of neurofilament triplet proteins during cat cerebellar development.

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton.

Characterization of the immunological properties of " Plasmodium falciparum " and " Plasmodium berghei " circumsporozoite proteins : antibody responses and recognition by specific CD8+ T cells

SUMMARY Interest in developing intervention strategies against malaria by targeting the liver stage of the Plasmodium life cycle has been fueled by studies which show that sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites. Anti-malarial drugs and insecticides have been widely used to control the disease, but in the hope of developing a more cost-effective intervention strategy, vaccine development has taken centre stage in malaria research. There is currently no vaccine against malaria. Attenuated sporozoite-induced immunity is achieved by antibodies and T cells against malaria liver stage antigens, the most abundant being the circumsporozoite protein (CSP), and many vaccine formulations aim at mimicking this immunity. However, the mechanisms by which the antibody and T cell immune responses are generated after infection by sporozoites, or after immunization with different vaccine formulations are still not well understood. The first part of this work aimed at determining the ability of primary hepatocytes from BALB/c mice to process and present CSP-derived peptides after infection with P. berghei sporozoites. Both infected hepatocytes and those traversed by sporozoites during migration were found to be capable of processing and presenting the CSP to specific CD8+ T cells in vitro. The pathway of processing and presentation involved the proteasome, aspartic proteases and transport through a post-Endoplasmic Reticulum (ER) compartment. These results suggest that in vivo, infected hepatocytes contribute to the elicitation and expansion of a T cell response. In the second part, the antibody responses of CB6F1 mice to synthetic peptides corresponding to the N- and C-terminal domains of P. berghei and P. falciparum CS proteins were characterized. Mice were immunized with single peptides or a combination of N- and C-terminal peptides. The peptides were immunogenic in mice and the antisera generated could recognize the native CSP on the sporozoite surface. Antisera generated against the N-terminal peptides or against the combinations inhibited sporozoite invasion of hepatocytes in vitro. In vivo, more mice immunized with single P. berghei peptides were protected from infection upon a challenge with P. berghei sporozoites, than mice immunized with a combination of N- and C-terminal peptides. Furthermore, P. falciparum N-terminal peptides were recognized by serum samples from people living in malaria-endemic areas. Importantly, recognition of a peptide from the N-terminal fragment of the P. falciparum CSP by sera from children living in a malaria-endemic region was associated with protection from disease. These results underline the potential of using such peptides as malaria vaccine candidates. RESUME L'intérêt de développer des stratégies d'intervention contre la malaria ciblant le stade pré-erythrocytaire a été alimenté par des études qui montrent qu'il est possible d'obtenir une immunité par l'injection de sporozoites irradiés. Les médicaments et les insecticides anti-paludiques ont été largement utilisés pour contrôler la maladie, mais dans l'espoir de développer une stratégie d'intervention plus rentable, le développement de vaccins a été placé au centre des recherches actuelles contre la malaria. A l'heure actuelle, il n'existe aucun vaccin contre la malaria. L'immunité induite par les sporozoites irradiés est due à l'effet combiné d'anticorps et de cellules T qui agissent contre les antigènes du stade hépatique dont le plus abondant est la protéine circumsporozoite (CSP). Beaucoup de formulations de vaccin visent à imiter l'immunité induite par les sporozoites irradiés. Cependant, les mécanismes par lesquels les anticorps et les cellules T sont génerés après infection par les sporozoites ou après immunisation avec des formulations de vaccin ne sont pas bien compris. La première partie de ce travail a visé à déterminer la capacité de hépatocytes primaires provenant de souris BALB/c à "processer" et à présenter des peptides dérivés de la CSP, après infection par des sporozoites de Plasmodium berghei. Nous avons montré que in vitro, les hépatocytes infectés et ceux traversés par les sporozoites pendant leur migration étaient capables de "processer" et de présenter la CSP aux cellules T CD8+ spécifiques. La voie de présentation implique le protéasome, les protéases de type aspartique et le transport à travers un compartiment post-reticulum endoplasmique. Ces résultats suggèrent que in vivo, les hépatocytes infectés contribuent à l'induction et à l'expansion d'une réponse immunitaire spécifique aux cellules T. Dans la deuxième partie, nous avons caractérisé les réponses anticorps chez les souris de la souche CB6F1 face aux peptides N- et C-terminaux des protéines circumsporozoites de Plasmodium berghei et Plasmodium falciparum. Les souris ont été immunisées avec les peptides individuellement ou en combinaison. Les peptides utilisés étaient immunogéniques chez les souris, et les anticorps produits pouvaient reconnaître la protéine CSP native à la surface des sporozoites. In vitro, les sera contre les peptides N-teminaux et les combinaisons étaient capables d'inhiber l'invasion de hépatocytes par les sporozoites. In vivo, plus de souris immunisées avec les peptides individuels de la CSP de P. berghei étaient protégées contre la malaria que les souris immunisées avec une combinaison de peptides N- et C-terminaux. De plus, les peptides N-terminaux de la CSP de P. falciparum ont été reconnus par les sera de personnes vivant dans des régions endémiques pour la malaria. Il est intéressant de voir que la reconnaissance d'un peptide N-terminal de P. falciparum par des sera d'enfants habitant dans des régions endémiques était associé à la protection contre la maladie. Ces résultats soulignent le potentiel de ces peptides comme candidats-vaccin contre la malaria.

Molecular basis of interaction between Na,K-ATPase and FXYD proteins.

Résumé au large public Notre corps est constitué de différents types de cellules. La condition minimale ou primordiale pour la survie des cellules est d'avoir de l'énergie. Cette tâche est assumée en partie par une protéine qui se situe dans la membrane de chaque cellule. Nommé Na, K¬ATPase ou pompe à sodium, c'est une protéine pressente dans toutes les cellules chez les mammifères est composée de deux sous-unités, α et β. En transportant 3 ions de sodium hors de la cellule et 2 ions de potassium à l'intérieur de la cellule, elle transforme l'énergie chimique sous forme de l'ATP en énergie motrice, qui permet aux cellules par la suite d'échanger des matériaux entre l'espace intracellulaire et extracellulaire ainsi que d'ingérer des nutriments provenant de son environnement. Le manque de cette protéine chez la souris entraîne la mort de l'embryon. Des défauts fonctionnels de cette protéine sont responsables de plusieurs maladies humaines comme par exemple, un type de migraine. En dehors de sa fonction vitale, cette protéine est également engagée dans diverses activités physiologiques comme la contractilité musculaire, l'activité nerveuse et la régulation du volume sanguin. Vue l'importance de cette protéine, sa découverte par Jens C. Skou en 1957 a été honorée d'un Prix Noble de chimie quarante ans plus tard. Depuis lors, nous connaissons de mieux en mieux les mécanismes de fonctionnement de la Na, K-ATPase. Entre autre, sa régulation par une famille de protéines appelées protéines FXYD. Cette famille contient 7 membres (FXYD 1-7). L'un d'entre eux nommé FXYD 2 est lié à une maladie héréditaire connue sous le nom de hypomagnesemia. Nous disposons actuellement d'informations concernant les conséquences de la régulation par les protéines FXYD sur activité de la Na, K-ATPase, mais nous savons très peu sur le mode d'interaction entre les protéines FXYD et la Na, K-ATPase. Dans ce travail de thèse, nous avons réussi à localiser des zones d'interaction dans la sous- unité a de la Na, K-ATPase et dans FXYD 7. En même temps, nous avons déterminé un 3ème site de liaison spécifique au sodium de la Na, K-ATPase. Une partie de ce site se situe à l'intérieur d'un domaine protéique qui interagit avec les protéines FXYD. De plus, ce site a été démontré comme responsable d'un mécanisme de transport de la Na, K-ATPase caractérisé par un influx ionique. En conclusion, les résultats de ce travail de thèse fournissent de nouvelles preuves sur les régions d'interaction entre la Na, K-ATPase et les protéines FXYD. La détermination d'un 3ème site spécifique au sodium et sa relation avec un influx ionique offrent la possibilité 1) d'explorer les mécanismes avec lesquels les protéines FXYD régulent l'activité de la Na, ATPase et 2) de localiser un site à sodium qui est essentielle pour mieux comprendre l'organisation et le fonctionnement de la Na, K-ATPase. Résumé Les gradients de concentration de Na+ et de K+ à travers la membrane plasmatique des cellules animales sont cruciaux pour la survie et l'homéostasie de cellules. De plus, des fonctions cellulaires spécifiques telles que la reabsorption de Na dans le rein et le côlon, la contraction musculaire et l'excitabilité nerveuse dépendent de ces gradients. La Na, K¬ATPase ou pompe à sodium est une protéine membranaire ubiquitaire. Elle crée et maintient ces gradients en utilisant l'énergie obtenu par l'hydrolyse de l'adénosine triphosphate. L'unité fonctionnelle minimale de cette protéine se compose d'une sous-unité catalytique α et d'une sous-unité régulatrice β. Récemment, il a été montré que des membres de la famille FXYD, sont des régulateurs tissu-spécifiques de la Na, K-ATPase qui influencent ses propriétés de transport. Cependant, on connaît peu de chose au sujet de la nature moléculaire de l'interaction entre les protéines FXYD et la Na, K-ATPase. Dans cette étude, nous fournissons, pour la première fois, l'évidence directe que des résidus du domaine transmembranaire (TM) 9 de la sous-unité α de la Na, K-ATPase sont impliqués dans l'interaction fonctionnelle et structurale avec les protéines FXYD. De plus nous avons identifié des régions dans le domaine transmembranaire de FXYD 7 qui sont importantes pour l'association stable avec la Na, K-ATPase et une série de résidus responsables des régulations fonctionnelles. Nous avons aussi montré les contributions fonctionnelles du TM 9 de la Na, K-ATPase à la translocation de Na + en déterminant un 3ème site spécifique au Na+. Ce site se situe probablement dans un espace entre TM 9, TM 6 et TM 5 de la sous-unité α de la pompe à sodium. De plus, nous avons constaté que le 3ème site de Na + est fonctionnellement lié à un courant entrant de la pompe sensible à l'ouabaïne et activé par le pH acide. En conclusion, ce travail donne de nouvelles perspectives de l'interaction structurale et fonctionnelle entre les protéines FXYD et la Na, K-ATPase. En outre, les contributions fonctionnelles de TM 9 offrent de nouvelles possibilités pour explorer le mécanisme par lequel les protéines FXYD régulent les propriétés fonctionnelles de la Na, K-ATPase. La détermination du 3ème site au Na + fournit une compréhension avancée du site spécifique au Na + de la Na, K-ATPase et du mécanisme de transport de la Na, K-ATPase. Summary The Na+ and K+ gradients across the plasma membrane of animal cells are crucial for cell survival and homeostasis. Moreover, specific tissue functions such as Na+ reabsorption in kidney and colon, muscle contraction and nerve excitability depend on the maintenance of these gradients. Na, K-ATPase or sodium pump, an ubiquitous membrane protein, creates and maintains these gradients by using the energy from the hydrolysis of ATP. The minimal functional unit of this protein is composed of a catalytic α subunit and a regulatory β subunit. Recently, members of the FXYD family, have been reported to be tissue-specific regulators of Na, K-ATPase by influencing its transport properties. However, little is known about the molecular nature of the interaction between FXYD proteins and Na, K-ATPase. In this study, we provide, for the first time, direct evidence that residues from the transmembrane (TM) domain 9 of the α subunit of Na, K-ATPase are implicated in the functional and structural interaction with FXYD proteins. Moreover, we have identified regions in the TM domain of FXYD 7 important for the stable association with Na, K-ATPase and a stretch of residues responsible for the functional regulations. We have further revealed the functional contributions of TM 9 of the Na, K-ATPase α subunit to the Na+ translocation by determining a 3rd Na+-specific cation binding site. This site is likely in a space between TM 9, TM 6 and TM 5 of the a subunit of the sodium pump. Moreover, we have found that the 3rd Na+ binding site is functionally linked to an acidic pH- activated ouabain-sensitive inward pump current. In conclusion, this work gives new insights into the structural and functional interaction between FXYD proteins and Na, K-ATPase. Functional contributions of TM 9 offer new possibilities to explore the mechanism by which FXYD proteins regulate functional properties of Na, K-ATPase. The determination of the 3rd Na+ binding site provides an advanced understanding concerning the Na+ -specific binding site of Na, K-ATPase and the 3rd Na+ site related transport mechanism.

A novel neuro-muscular marker for the heavy subunit of neurofilament proteins, NF-H, and striated muscle myosin of Xenopus laevis.

A novel monoclonal antibody, M7, is described, that reacts on Western blots with the large subunit of the neurofilament triplet proteins (NF-H) and with striated muscle myosin of Xenopus laevis. Enzymatically digested neurofilament and myosin proteins revealed different immunoreactive peptide fragments on Western blots. Therefore, the antibody must react with immunologically related epitopes common to both proteins. Immunohistochemistry showed staining of large and small axons in CNS and PNS, and nerves could be followed into endplate regions of skeletal muscles. These muscles were characterized by a striated immunostaining of the M-lines. Despite the crossreactivity of M7 with NF-H and muscle myosin, this antibody may be a tool to study innervation of muscle fibers, and to define changes in the neuromuscular organization during early development and metamorphosis of tadpoles.

Use of fluorescent proteins to monitor the regulation of antifungal activity in root-associated Pseudomonas fluorescens CHA0

SUMMARY Roots of crop plants are the target of soil-borne pathogens, mainly fungi that cause considerable damage to plant health. By antagonizing these pathogens, some root-colonizing pseudomonads provide plants with efficient biological protection from disease. Pseudomonas fluorescens CHAO is a soil bacterium with the ability to suppress a considerable range of root diseases. A major characteristic conferring biocontrol capacity to this strain is the production of antifungal compounds, in particular 2,4-diacetyphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the biosyntheses of these metabolites is complex and involves several regulatory systems responding to multiple environmental signals. In the present work, we have developed reporter systems based on green (GFP) and red fluorescent (DsRed) proteins to monitor regulation of antifungal gene expression in vitro and on plant roots. Stable and unstable GFP-based reporter fusions to the DAPG and PLT biosynthetic genes allowed us to demonstrate that P. fluorescens CHAO keeps the two antifungal compounds at a fine-tuned balance that can be affected by environmental signals. A GFP-based screening technique helped us to identify two novel regulators of balanced antibiotic production, i.e. MvaT and MvaV that are functionally and structurally related to the nucleoid-binding protein H-NS. They act in concert as global regulators of DAPG and PLT production and other biocontrol-related traits in P. fluorescens CHAO, and are essential for the bacterium's capacity to control a root disease caused by Pythium. The combined use of autofluorescent reporters, flow cytometry, and epifluorescence microscopy permitted us to visualize and quantify the expression of DAPG and PLT biosynthetic genes on roots. A GFP- and DsRed-based two-color approach was then developed to further improve the sensitivity of the flow cytometric quantitation method. The findings of this study shed more light on the complex regulatory mechanisms controlling antifungal activity of P. filuorescens in the rhizosphere. RESUME 4 e Les racines de plantes de culture sont la cible de divers pathogènes, principalement des champignons, qui nuisent gravement à la santé des plantes. Certains pseudomonades colonisant les racines peuvent avoir un effet antagoniste sur les pathogènes et protéger ainsi les plantes de manière efficace. Pseudomonas fluorescens CHAO est une bactérie du sol ayant la capacité de supprimer une gamme considérable de maladies racinaires. Une des caractéristiques principales conférant la capacité de biocontrôle à cette souche, est la production de composés antifongiques, en particulier le 2,4-diacétyphloroglucinol (DAPG) et la pyolutéorine (PLT). La régulation de la biosynthèse de ces métabolites est complexe et implique plusieurs systèmes régulateurs répondant à de multiples signaux environnementaux. Dans ce travail, nous avons développé des systèmes rapporteurs basés sur des protéines fluorescentes verte (GFP) et rouge (DsRed), afin d'étudier la régulation de l'expression des gènes d'antifongiques in vitro et sur les racines des plantes. Des fusions GFP stables et instables rapportrices de l'expression des gènes de biosynthèse du DAPG et de la PLT nous ont permis de démontrer que P. fluorescens CHAO gère les deux antifongiques dans une balance finement régulée pouvant être affectée par des signaux environnementaux. Une technique de criblage basée sur la GFP nous a permis d'identifier deux nouveaux régulateurs de la production d'antibiotiques, MvaT et MvaV, apparentés à la protéine H-NS liant l'ADN, Elles agissent de concert en tant que régulateurs globaux sur la production de DAPG et de PLT, ainsi que sur d'autres éléments relatifs au biocontrôle chez P. fluorescens CHAO. De plus, elles sont essentielles à la bactérie pour contrôler une maladie racinaire causée par Pythium. L'utilisation combinée de rapporteurs autofluorescents, de cytométrie de flux et de microscopie à épifluorescence nous a permis de visualiser et de quantifier l'expression des gènes de biosynthèse du DAPG et de la PLT sur les racines. Une approche utilisant simultanément la GFP et la DsRed a ensuite été développée afin d'améliorer la sensibilité de la méthode de quantification par cytométrie de flux. Les résultats de cette étude ont apporté plus de lumière sur les mécanismes régulateurs complexes contrôlant l'activité antifongique de P. fluorescens dans la rizosphère.

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex.

The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

In vitro heat exposure induces a redistribution of nuclear matrix proteins in human K562 erythroleukemia cells.

By using both conventional and confocal laser scanning microscopy with three monoclonal antibodies recognizing nuclear matrix proteins we have investigated by means of indirect fluorescence whether an incubation of isolated nuclei at the physiological temperature of 37 degrees C induces a redistribution of nuclear components in human K562 erythroleukemia cells. Upon incubation of isolated nuclei for 45 min at 37 degrees C, we have found that two of the antibodies, directed against proteins of the inner matrix network (M(r) 125 and 160 kDa), gave a fluorescent pattern different from that observed in permeabilized cells. By contrast, the fluorescent pattern did not change if nuclei were kept at 0 degrees C. The difference was more marked in case of the 160-kDa polypeptide. The fluorescent pattern detected by the third antibody, which recognizes the 180-kDa nucleolar isoform of DNA topoisomerase II, was unaffected by heat exposure of isolated nuclei. When isolated nuclear matrices prepared from heat-stabilized nuclei were stained by means of the same three antibodies, it was possible to see that the distribution of the 160-kDa matrix protein no longer corresponded to that observable in permeabilized cells, whereas the fluorescent pattern given by the antibody to the 125-kDa polypeptide resembled that detectable in permeabilized cells. The 180-kDa isoform of topoisomerase II was still present in the matrix nucleolar remnants. We conclude that a 37 degrees C incubation of isolated nuclei induces a redistribution of some nuclear matrix antigens and cannot prevent the rearrangement in the spatial organization of one of these antigens that takes place during matrix isolation in human erythroleukemia cells. The practical relevance of these findings is discussed.

Differential distribution of tau proteins in developing cat cerebral cortex and corpus callosum.

During the postnatal development of cat visual cortex and corpus callosum the molecular composition of tau proteins varied with age. In both structures, they changed between postnatal days 19 and 39 from a set of two juvenile forms to a set of at least two adult variants with higher molecular weights. During the first postnatal week, tau proteins were detectable with TAU-1 antibody in axons of corpus callosum and visual cortex, and in some perikarya and dendrites in the visual cortex. At later ages, tau proteins were located exclusively within axons in all cortical layers and in the corpus callosum. Dephosphorylation of postnatal day 11 cortical tissue by alkaline phosphatase strongly increased tau protein immunoreactivity on Western blots and in numerous perikarya and dendrites in all cortical layers, in sections, suggesting that some tau forms had been unmasked. During postnatal development the intensity of this phosphate-dependent somatodendritic staining decreased, but remained in a few neurons in cortical layers II and III. On blots, the immunoreactivity of adult tau to TAU-1 was only marginally increased by dephosphorylation. Other tau antibodies (TAU-2, B19 and BR133) recognized two juvenile and two adult cat tau proteins on blots, and localized tau in axons or perikarya and dendrites in tissue untreated with alkaline phosphatase. Tau proteins in mature tissue were soluble and not associated with detergent-resistant structures. Furthermore, dephosphorylation by alkaline phosphatase resulted in the appearance of more tau proteins in soluble fractions. Therefore tau proteins seem to alter their degree of phosphorylation during development. This could affect microtubule stability as well as influence axonal and dendritic differentiation.

Subknots in ideal knots, random knots, and knotted proteins.

We introduce disk matrices which encode the knotting of all subchains in circular knot configurations. The disk matrices allow us to dissect circular knots into their subknots, i.e. knot types formed by subchains of the global knot. The identification of subknots is based on the study of linear chains in which a knot type is associated to the chain by means of a spatially robust closure protocol. We characterize the sets of observed subknot types in global knots taking energy-minimized shapes such as KnotPlot configurations and ideal geometric configurations. We compare the sets of observed subknots to knot types obtained by changing crossings in the classical prime knot diagrams. Building upon this analysis, we study the sets of subknots in random configurations of corresponding knot types. In many of the knot types we analyzed, the sets of subknots from the ideal geometric configurations are found in each of the hundreds of random configurations of the same global knot type. We also compare the sets of subknots observed in open protein knots with the subknots observed in the ideal configurations of the corresponding knot type. This comparison enables us to explain the specific dispositions of subknots in the analyzed protein knots.