The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

The V-ATPase V(0) sector associates with the peripheral V(1) sector to form a proton pump. V(0) alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V(0) contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans-SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion-impairing proteolipid substitutions stabilize the interaction of V(0) with V(1). Deletion of the vacuolar v-SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V(0). Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE-dependent conformational change in V(0) proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.

Characterization of polyphosphate synthesis by isolated yeast vacuoles

Polyphosphate (iPOP) is a linear polymer of orthophosphate units linked together by high energy phosphoanhydride bonds. It is found in all organisms, localized in organelles called acidocalcisomes and ranges from a few to few hundred monomers in length. iPOP has been found to play a vast array of roles in all organisms, including phosphate and energy metabolism, regulation of enzymes, virulence, pathogenicity, bone remodelling and blood clotting, among many others. Recently it was found that iPOP levels were increased in myeloma cells. The growing interest in iPOP in human cell lines makes it an interesting molecule to study. However, not much is known about its metabolism in eukaryotes. Acidocalcisomes are electron dense, acidic organelles that belong to the group of Lysosome Related Organelles (LROs). The conservation of acidocalcisomes among all kingdoms of life is suggestive of their important roles for the organisms. However, they are difficult to analyse because of limited biochemical tools for investigation. Yeast vacuoles present remarkable similarities to acidocalcisomes in terms of their physiological and structural features, including synthesis and storage of iPOP, which make them an ideal candidate to study biological processes which are shared between vacuoles and acidocalcisomes. The availability of tools for genetic manipulation and isolation of vacuoles makes yeast a candidate of choice for the characterization of iPOP synthesis in eukaryotes. Our group has identified the Vacuolar Transporter Chaperone (VTC) complex as iPOP polymerase and identified the catalytic subunit (Vtc4). The goal of my study was to characterize the process of iPOP synthesis by isolated vacuoles and to reconstitute iPOP synthesis in liposomes. The first step was to develop a method for monitoring iPOP by isolated vacuoles over time and comparing it with previously known methods. Next, a detailed characterization was performed to determine the modulators of the process, both for intact as well as solubilized vacuoles. Finally, attempts were made to purify the VTC complex and reconstitute it in liposomes. A parallel line of study was the translocation and storage of synthesized iPOP in the lumen of the vacuoles. As a result of this study, it is possible to determine distinct pools of iPOP- inside and outside the vacuolar lumen. Additionally, I establish that the vacuolar lysate withstands harsh steps during reconstitution on liposomes and retains iPOP synthesizing activity. The next steps will be purification of the intact VTC complex and its structure determination by cryo-electron microscopy. - Les organismes vivants sont composés d'une ou plusieurs cellules responsables des processus biologiques élémentaires tels que la digestion, la respiration, la synthèse et la reproduction. Leur environnement interne est en équilibre et ils réalisent un très grand nombre de réactions chimiques et biochimiques pour maintenir cet équilibre. A différents compartiments cellulaires, ou organelles, sont attribuées des tâches spécifiques pour maintenir les cellules en vie. L'étude de ces fonctions permet une meilleure compréhension de la vie et des organismes vivants. De nombreux processus sont bien connus et caractérisés mais d'autres nécessitent encore des investigations détaillées. L'un de ces processus est le métabolisme des polyphosphates. Ces molécules sont des polymères linéaires de phosphate inorganique dont la taille peut varier de quelques dizaines à quelques centaines d'unités élémentaires. Ils sont présents dans tous les organismes, des bactéries à l'homme. Ils sont localisés principalement dans des compartiments cellulaires appelés acidocalcisomes, des organelles acides observés en microscopie électronique comme des structures denses aux électrons. Les polyphosphates jouent un rôle important dans le stockage et le métabolisme de l'énergie, la réponse au stress, la virulence, la pathogénicité et la résistance aux drogues. Chez l'homme, ils sont impliqués dans la coagulation du sang et le remodelage osseux. De nouvelles fonctions biologiques des polyphosphates sont encore découvertes, ce qui accroît l'intérêt des chercheurs pour ces molécules. Bien que des progrès considérables ont été réalisés afin de comprendre la fonction des polyphosphates chez les bactéries, ce qui concerne la synthèse, le stockage et la dégradation des polyphosphates chez les eucaryotes est mal connu. Les vacuoles de la levure Saccharomyces cerevisiae sont similaires aux acidocalcisomes des organismes supérieurs en termes de structure et de fonction. Les acidocalcisomes sont difficiles à étudier car il n'existe que peu d'outils génétiques et biochimiques qui permettent leur caractérisation. En revanche, les vacuoles peuvent être aisément isolées des cellules vivantes et manipulées génétiquement. Les vacuoles comme les acidocalcisomes synthétisent et stockent les polyphosphates. Ainsi, les découvertes faites grâce aux vacuoles de levures peuvent être extrapolées aux acidocalcisomes des organismes supérieurs. Le but de mon projet était de caractériser la synthèse des polyphosphates par des vacuoles isolées. Au cours de mon travail de thèse, j'ai mis au point une méthode de mesure de la synthèse des polyphosphates par des organelles purifés. Ensuite, j'ai identifié des composés qui modulent la réaction enzymatique lorsque celle-ci a lieu dans la vacuole ou après solubilisation de l'organelle. J'ai ainsi pu mettre en évidence deux groupes distincts de polyphosphates dans le système : ceux au-dehors de la vacuole et ceux en-dedans de l'organelle. Cette observation suggère donc très fortement que les vacuoles non seulement synthétisent les polyphosphates mais aussi transfère les molécules synthétisées de l'extérieur vers l'intérieur de l'organelle. Il est très vraisemblable que les vacuoles régulent le renouvellement des polyphosphates qu'elles conservent, en réponse à des signaux cellulaires. Des essais de purification de l'enzyme synthétisant les polyphosphates ainsi que sa reconstitution dans des liposomes ont également été entrepris. Ainsi, mon travail présente de nouveaux aspects de la synthèse des polyphosphates chez les eucaryotes et les résultats devraient encourager l'élucidation de mécanismes similaires chez les organismes supérieurs. - Les polyphosphates (iPOP) sont des polymères linéaires de phosphates inorganiques liés par des liaisons phosphoanhydres de haute énergie. Ces molécules sont présentes dans tous les organismes et localisées dans des compartiments cellulaires appelés acidocalcisomes. Elles varient en taille de quelques dizaines à quelques centaines d'unités phosphate. Des fonctions nombreuses et variées ont été attribuées aux iPOP dont un rôle dans les métabolismes de l'énergie et du phosphate, dans la régulation d'activités enzymatiques, la virulence, la pathogénicité, le remodelage osseux et la coagulation sanguine. Il a récemment été montré que les cellules de myélome contiennent une grande quantité de iPOP. Il y donc un intérêt croissant pour les iPOP dans les lignées cellulaires humaines. Cependant, très peu d'informations sur le métabolisme des iPOP chez les eucaryotes sont disponibles. Les acidocalcisomes sont des compartiments acides et denses aux électrons. Ils font partie du groupe des organelles similaires aux lysosomes (LROs pour Lysosome Related Organelles). Le fait que les acidocalcisomes soient conservés dans tous les règnes du vivant montrent l'importance de ces compartiments pour les organismes. Cependant, l'analyse de ces organelles est rendue difficile par l'existence d'un nombre limité d'outils biochimiques permettant leur caractérisation. Les vacuoles de levures possèdent des aspects structuraux et physiologiques très similaires à ceux des acidocalcisomes. Par exemple, ils synthétisent et gardent en réserve les iPOP. Ceci fait des vacuoles de levure un modèle idéal pour l'étude de processus biologiques conservés chez les vacuoles et les acidocalcisomes. De plus, la levure est un organisme de choix pour l'étude de la synthèse des iPOP compte-tenu de l'existence de nombreux outils génétiques et la possibilité d'isoler des vacuoles fonctionnelles. Notre groupe a identifié le complexe VTC (Vacuole transporter Chaperone) comme étant responsable de la synthèse des iPOP et la sous-unité Vtc4p comme celle possédant l'activité catalytique. L'objectif de cette étude était de caractériser le processus de synthèse des iPOP en utilisant des vacuoles isolées et de reconstituer la synthèse des iPOP dans des liposomes. La première étape a consisté en la mise au point d'un dosage permettant la mesure de la quantité de iPOP synthétisés par les organelles isolés en fonction du temps. Cette nouvelle méthode a été comparée aux méthodes décrites précédemment dans la littérature. Ensuite, la caractérisation détaillée du processus a permis d'identifier des composés modulateurs de la réaction à la fois pour des vacuoles intactes et des vacuoles solubilisées. Enfin, des essais de purification du complexe VTC et sa reconstitution dans des liposomes ont été entrepris. De façon parallèle, une étude sur la translocation et le stockage des iPOP dans le lumen des vacuoles a été menée. Il a ainsi été possible de mettre en évidence différents groupes de iPOP : les iPOP localisés à l'intérieur et ceux localisés à l'extérieur des vacuoles isolées. De plus, nous avons observé que le lysat vacuolaire n'est pas détérioré par les étapes de reconstitution dans les liposomes et conserve l'activité de synthèse des iPOP. Les prochaines étapes consisteront en la purification du complexe intact et de la détermination de sa structure par cryo-microscopie électronique.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH.

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.

Measuring limits of telomere movement on nuclear envelope.

The dynamic behavior of the decondensed chromatin can be monitored by real-time fluorescence confocal microscopy. It can be observed that different chromosomal sites enjoy different degrees of freedom during a certain period, exploring larger or smaller portions of nuclear volume. Here we measure the accessible surface for two chromosomal sites (yeast telomeres Tel3R and Tel6R) that both exhibit strong preferential association with the nuclear membrane in galactose-containing media, but differ significantly in gene activity. Telomere Tel6R, which harbors an inducible gene with high levels of transcription, explores a much larger surface than the telomere Tel3R, which is adjacent to inactive chromatin. Thus, our results distinguish two perinuclear movements characteristic of different transcriptional state, allowing for a better understanding of the correlation between activity of genes and chromatin dynamics.

The cAMP response element binding protein-2 (CREB-2) can interact with the C/EBP-homologous protein (CHOP).

cAMP response element binding protein-2 (CREB-2) is a basic leucine zipper (bZIP) factor that was originally described as a repressor of CRE-dependent transcription but that can also act as a transcriptional activator. Moreover, CREB-2 is able to function in association with the viral Tax protein as an activator of the human T-cell leukemia virus type I (HTLV-I) promoter. Here we show that CREB-2 is able to interact with C/EBP-homologous protein (CHOP), a bZIP transcription factor known to inhibit CAAT/enhancer-dependent transcription. Cotransfection of CHOP with CREB-2 results in decreased activation driven by the cellular CRE motif or the HTLV-I proximal Tax-responsive element, confirming that CREB-2 and CHOP can interact with each other in vivo.

Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover.

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi.

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.

Development and application of computational methodologies in qualitative modeling

La présente thèse s'intitule "Développent et Application des Méthodologies Computationnelles pour la Modélisation Qualitative". Elle comprend tous les différents projets que j'ai entrepris en tant que doctorante. Plutôt qu'une mise en oeuvre systématique d'un cadre défini a priori, cette thèse devrait être considérée comme une exploration des méthodes qui peuvent nous aider à déduire le plan de processus regulatoires et de signalisation. Cette exploration a été mue par des questions biologiques concrètes, plutôt que par des investigations théoriques. Bien que tous les projets aient inclus des systèmes divergents (réseaux régulateurs de gènes du cycle cellulaire, réseaux de signalisation de cellules pulmonaires) ainsi que des organismes (levure à fission, levure bourgeonnante, rat, humain), nos objectifs étaient complémentaires et cohérents. Le projet principal de la thèse est la modélisation du réseau de l'initiation de septation (SIN) du S.pombe. La cytokinèse dans la levure à fission est contrôlée par le SIN, un réseau signalant de protéines kinases qui utilise le corps à pôle-fuseau comme échafaudage. Afin de décrire le comportement qualitatif du système et prédire des comportements mutants inconnus, nous avons décidé d'adopter l'approche de la modélisation booléenne. Dans cette thèse, nous présentons la construction d'un modèle booléen étendu du SIN, comprenant la plupart des composantes et des régulateurs du SIN en tant que noeuds individuels et testable expérimentalement. Ce modèle utilise des niveaux d'activité du CDK comme noeuds de contrôle pour la simulation d'évènements du SIN à différents stades du cycle cellulaire. Ce modèle a été optimisé en utilisant des expériences d'un seul "knock-out" avec des effets phénotypiques connus comme set d'entraînement. Il a permis de prédire correctement un set d'évaluation de "knock-out" doubles. De plus, le modèle a fait des prédictions in silico qui ont été validées in vivo, permettant d'obtenir de nouvelles idées de la régulation et l'organisation hiérarchique du SIN. Un autre projet concernant le cycle cellulaire qui fait partie de cette thèse a été la construction d'un modèle qualitatif et minimal de la réciprocité des cyclines dans la S.cerevisiae. Les protéines Clb dans la levure bourgeonnante présentent une activation et une dégradation caractéristique et séquentielle durant le cycle cellulaire, qu'on appelle communément les vagues des Clbs. Cet évènement est coordonné avec la courbe d'activation inverse du Sic1, qui a un rôle inhibitoire dans le système. Pour l'identification des modèles qualitatifs minimaux qui peuvent expliquer ce phénomène, nous avons sélectionné des expériences bien définies et construit tous les modèles minimaux possibles qui, une fois simulés, reproduisent les résultats attendus. Les modèles ont été filtrés en utilisant des simulations ODE qualitatives et standardisées; seules celles qui reproduisaient le phénotype des vagues ont été gardées. L'ensemble des modèles minimaux peut être utilisé pour suggérer des relations regulatoires entre les molécules participant qui peuvent ensuite être testées expérimentalement. Enfin, durant mon doctorat, j'ai participé au SBV Improver Challenge. Le but était de déduire des réseaux spécifiques à des espèces (humain et rat) en utilisant des données de phosphoprotéines, d'expressions des gènes et des cytokines, ainsi qu'un réseau de référence, qui était mis à disposition comme donnée préalable. Notre solution pour ce concours a pris la troisième place. L'approche utilisée est expliquée en détail dans le dernier chapitre de la thèse. -- The present dissertation is entitled "Development and Application of Computational Methodologies in Qualitative Modeling". It encompasses the diverse projects that were undertaken during my time as a PhD student. Instead of a systematic implementation of a framework defined a priori, this thesis should be considered as an exploration of the methods that can help us infer the blueprint of regulatory and signaling processes. This exploration was driven by concrete biological questions, rather than theoretical investigation. Even though the projects involved divergent systems (gene regulatory networks of cell cycle, signaling networks in lung cells), as well as organisms (fission yeast, budding yeast, rat, human), our goals were complementary and coherent. The main project of the thesis is the modeling of the Septation Initiation Network (SIN) in S.pombe. Cytokinesis in fission yeast is controlled by the SIN, a protein kinase signaling network that uses the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this thesis, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN. Another cell cycle related project that is part of this thesis was to create a qualitative, minimal model of cyclin interplay in S.cerevisiae. CLB proteins in budding yeast present a characteristic, sequential activation and decay during the cell cycle, commonly referred to as Clb waves. This event is coordinated with the inverse activation curve of Sic1, which has an inhibitory role in the system. To generate minimal qualitative models that can explain this phenomenon, we selected well-defined experiments and constructed all possible minimal models that, when simulated, reproduce the expected results. The models were filtered using standardized qualitative ODE simulations; only the ones reproducing the wave-like phenotype were kept. The set of minimal models can be used to suggest regulatory relations among the participating molecules, which will subsequently be tested experimentally. Finally, during my PhD I participated in the SBV Improver Challenge. The goal was to infer species-specific (human and rat) networks, using phosphoprotein, gene expression and cytokine data and a reference network provided as prior knowledge. Our solution to the challenge was selected as in the final chapter of the thesis.

Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis.

Pneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.

Spontaneous cell polarization: Feedback control of Cdc42 GTPase breaks cellular symmetry.

Spontaneous polarization without spatial cues, or symmetry breaking, is a fundamental problem of spatial organization in biological systems. This question has been extensively studied using yeast models, which revealed the central role of the small GTPase switch Cdc42. Active Cdc42-GTP forms a coherent patch at the cell cortex, thought to result from amplification of a small initial stochastic inhomogeneity through positive feedback mechanisms, which induces cell polarization. Here, I review and discuss the mechanisms of Cdc42 activity self-amplification and dynamic turnover. A robust Cdc42 patch is formed through the combined effects of Cdc42 activity promoting its own activation and active Cdc42-GTP displaying reduced membrane detachment and lateral diffusion compared to inactive Cdc42-GDP. I argue the role of the actin cytoskeleton in symmetry breaking is not primarily to transport Cdc42 to the active site. Finally, negative feedback and competition mechanisms serve to control the number of polarization sites.

Enzymes of yeast polyphosphate metabolism: structure, enzymology and biological roles.

Inorganic polyphosphate (polyP) is found in all living organisms. The known polyP functions in eukaryotes range from osmoregulation and virulence in parasitic protozoa to modulating blood coagulation, inflammation, bone mineralization and cellular signalling in mammals. However mechanisms of regulation and even the identity of involved proteins in many cases remain obscure. Most of the insights obtained so far stem from studies in the yeast Saccharomyces cerevisiae. Here, we provide a short overview of the properties and functions of known yeast polyP metabolism enzymes and discuss future directions for polyP research.