Genome-wide screen of genes required for caffeine tolerance in fission yeast

Background: An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to atoxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.Methodology/Principal Findings: We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeinecontainingplates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 aresensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. Conclusions/Significance: With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we havedemonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.

The peroxiredoxin Tpx1 is essential as a H202-scavenger during aerobic growth in fission yeast

Peroxiredoxins are known to interact with hydrogen peroxide (H2O2) and to participate in oxidant scavenging, redox signal transduction, and heat-shock responses. The two-cysteine peroxiredoxin Tpx1 of Schizosaccharomyces pombe has been characterized as the H2O2 sensor that transduces the redox signal to the transcription factor Pap1. Here, we show that Tpx1 is essential for aerobic, but not anaerobic, growth. We demonstrate that Tpx1 has an exquisite sensitivity for its substrate, which explains its participation in maintaining low steady-state levels of H2O2. We also show in vitro and in vivo that inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid is always preceded by a sulfinic acid form in a covalently linked dimer, which may be important for understanding the kinetics of Tpx1 inactivation. Furthermore, we provide evidence that a strain expressing Tpx1.C169S, lacking the resolving cysteine, can sustain aerobic growth, and we show that small reductants can modulate the activity of the mutant protein in vitro, probably by supplying a thiol group to substitute for cysteine 169.

The quest for a message: budding yeast, a model organism to study the control of pre-mRNA splicing

Removal of introns during pre-mRNA splicing is a critical process in gene expression, and understanding its control at both single-gene and genomic levels is one of the great challenges in Biology. Splicing takes place in a dynamic, large ribonucleoprotein complex known as the spliceosome. Combining Genetics and Biochemistry, Saccharomyces cerevisiae provides insights into its mechanisms, including its regulation by RNA-protein interactions. Recent genome-wide analyses indicate that regulated splicing is broad and biologically relevant even in organisms with a relatively simple intronic structure, such as yeast. Furthermore, the possibility of coordination in splicing regulation at genomic level is becoming clear in this model organism. This should provide a valuable system to approach the complex problem of the role of regulated splicing in genomic expression.

Deciphering 3'ss selection in the yeast genome reveals an RNA thermosensor that mediates alternative splicing

Poor understanding of the spliceosomal mechanisms to select intronic 3' ends (3'ss) is a major obstacle to deciphering eukaryotic genomes. Here, we discern the rules for global 3'ss selection in yeast. We show that, in contrast to the uniformity of yeast splicing, the spliceosome uses all available 3'ss within a distance window from the intronic branch site (BS), and that in 70% of all possible 3'ss this is likely to be mediated by pre-mRNA structures. Our results reveal that one of these RNA folds acts as an RNA thermosensor, modulating alternative splicing in response to heat shock by controlling alternate 3'ss availability. Thus, our data point to a deeper role for the pre-mRNA in the control of its own fate, and to a simple mechanism for some alternative splicing.

Topological comparison of methods for predicting transcriptional cooperativity in yeast

Background: The cooperative interaction between transcription factors has a decisive role in the control of the fate of the eukaryotic cell. Computational approaches for characterizing cooperative transcription factors in yeast, however, are based on different rationales and provide a low overlap between their results. Because the wealth of information contained in protein interaction networks and regulatory networks has proven highly effective in elucidating functional relationships between proteins, we compared different sets of cooperative transcription factor pairs (predicted by four different computational methods) within the frame of those networks. Results: Our results show that the overlap between the sets of cooperative transcription factors predicted by the different methods is low yet significant. Cooperative transcription factors predicted by all methods are closer and more clustered in the protein interaction network than expected by chance. On the other hand, members of a cooperative transcription factor pair neither seemed to regulate each other nor shared similar regulatory inputs, although they do regulate similar groups of target genes. Conclusion: Despite the different definitions of transcriptional cooperativity and the different computational approaches used to characterize cooperativity between transcription factors, the analysis of their roles in the framework of the protein interaction network and the regulatory network indicates a common denominator for the predictions under study. The knowledge of the shared topological properties of cooperative transcription factor pairs in both networks can be useful not only for designing better prediction methods but also for better understanding the complexities of transcriptional control in eukaryotes.

In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease.

Cyclosporine A (CsA) has been demonstrated to be effective for the treatment of a variety of ophthalmological conditions, including ocular surface disorders such as the dry eye disease (DED). Since CsA is characterised by its low water solubility, the development of a topical ophthalmic formulation represents an interesting pharmaceutical question. In the present study, two different strategies to address this challenge were studied and compared: (i) a water-soluble CsA prodrug formulated within an aqueous solution and (ii) a CsA oil-in-water emulsion (Restasis, Allergan Inc., Irvine, CA). First, the prodrug formulation was shown to have an excellent ocular tolerance as well as no influence on the basal tear production; maintaining the ocular surface properties remained unchanged. Then, in order to allow in vivo investigations, a specific analytical method based on ultra high pressure liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was developed and optimised to quantify CsA in ocular tissues and fluids. The CsA ocular kinetics in lachrymal fluid for both formulations were found to be similar between 15 min and 48 h. The CsA ocular distribution study evidenced the ability of the prodrug formulation to penetrate into the eye, achieving therapeutically active CsA levels in tissues of both the anterior and posterior segments. In addition, the detailed analysis of the in vivo data using a bicompartmental model pointed out a higher bioavailability and lower elimination rate for CsA when it is generated from the prodrug than after direct application as an emulsion. The interesting in vivo properties displayed by the prodrug solution make it a safe and suitable option for the treatment of DED.

Role of myosin V in actin cable organization in fission yeast

Functional specialization is tightly linked to the ability of eukaryotic cells to acquire a particular shape. Cell morphogenesis, in turn, relies on the capacity to establish and maintain cell "polarity", which is achieved by orienting the trafficking of signaling molecules and organelles towards specific cellular locations and/or membrane domains. The "oriented" transport is based upon cytoskeletal polymers, microtubules and actin filaments, which serve as tracks for molecular motors. These latter generate motion that is translated either into pulling forces or directed transport. Fission yeast, a rod-like unicellular eukaryote, shapes itself by restricting growth at cell tips through the concerted activity of microtubules and actin cables. Microtubules, which assemble into 2-6 bundles and run parallel to the long axis of the cell, serve to orient growth to the tips. Growth is supported by the actin cytoskeleton, which provides tracks, the cables, for motor-based transport of secretory vesicles. The molecular motors, which bind cargos and deliver them to the tips along cables, are also known as type V myosins (hereafter indicated as myosin V). How the bundles of parallel actin filaments, i.e. the cables, extend from the tips through the cell and whether they serve any other purpose, besides providing tracks, is poorly understood. It is also unclear how the crosstalk between the two cytoskeletal systems is achieved. These are the basic questions I addressed during my PhD. The first part of the thesis work (Chapter two) suggests that the sole function of actin cables in polarized growth is to serve as tracks for motors. The data indicate that cells may have evolved two cytoskeletal systems to provide robustness to the polarization process but in principle a unique cytoskeleton might have been able to direct and support polarized growth. How actin cables are organized within the cell to optimize cargo transport is addressed later on (Chapter three). The major finding, based on the actin cable defect of cells lacking myosin Vs, is that actin filaments self-organize through the activity of the transport motors. In fact, by delivering cargos to cell tips and exerting physical pulling forces on actin filaments, Myosin Vs contribute not only to polarize cargo transport but also actin tracks. Among the cargos transported by Myosin V, which may be relevant to its function in organizing cables, there is likely the endoplasmic reticulum (ER). Actin cables, which run parallel to cortical ER, may serve as tracks for Myosin V. Myosin V-driven displacement, in turn, may account for the dynamic expansion and organization of ER during polarized growth as suggested in Chapter four. The last part of the work (Chapter five) highlights the existence of a crosstalk between actin and microtubules. In absence of myosin V, indeed, microtubules contribute to actin cable organization, likely playing a scaffolding/tethering function. Whether or not the kinesin 1, Klp3, plays any role in such process has to be demonstrated. In conclusion the work proposes a novel role for myosin Vs in actin organization, besides its transport function, and provides molecular tools to further dissect the role of this type of myosin in fission yeast. - La spécialisation fonctionnelle est étroitement connectée à la capacité des cellules eucaryotes d'acquérir une forme particulière. La morphogenèse cellulaire à son tour, est basée sur la capacité d'établir et de maintenir la polarité cellulaire, polarité réalisée en orientant le trafic des molécules signales et des organelles vers des zones cellulaires spécifiques. Ce transport directionnel dépend des polymères du cytosquelette, microtubules et microfilaments, qui servent comme des voies pour les moteurs moléculaires. Ces derniers engendrent du mouvement, traduit soit en force de traction soit en transport directionnel. La levure fissipare, un eucaryote unicellulaire en forme de bâtonnet, acquière sa forme en limitant sa croissance aux extrémités par l'action concertée des microtubules et de l'actine. Les microtubules, qui s'assemblent de façon antiparallèle et parcourent la cellule parallèlement à l'axe longitudinal, servent à orienter la croissance aux extrémités. Cette croissance est permise par le cytosquelette d'actine, fournissant des voies, les câbles, pour le transport actif des vésicules de sécrétion. Les moteurs moléculaires, responsables de ce transport actif sont aussi appelés myosines de type V (par la suite appelés myosines V). La manière dont ces câbles s'étendent depuis l'extrémité jusqu'à l'intérieur de la cellule est peu connue. De plus, on ignore également si ces câbles présentent une fonction autre que le transport. L'interaction entre les deux cytosquelettes est également obscure. Ce sont ces questions de base auxquelles j'ai tenté de répondre lors de ma thèse. La première partie de cette thèse (chapitre II) suggère que les câbles d'actine, pendant la croissance polarisée, fonctionnent uniquement comme des voies pour les moteurs moléculaires. Les données indiqueraient que les cellules ont fait évoluer deux systèmes de cytosquelette pour assurer plus de robustesse au processus de polarisation, bien que, comme nous le verrons, un système unique est suffisant. Au chapitre III, nous verrons comment les câbles d'actine sont organisés à l'intérieur de la cellule afin d'optimiser le transport des cargo. La découverte majeure, réalisée en observant des cellules dont la myosine V fait défaut, est que ces filaments d'actine s'auto organisent grâce au passage des moteurs moléculaires le long de ces voies. En réalité, en délivrant les cargos aux extrémités de la cellule et en exerçant des forces de traction sur les câbles, les myosines V contribuent non seulement à polariser le transport mais également à polariser les voies elles mêmes. Nous verrons également au chapitre IV, que parmi les cargos importants pour l'organisation des câbles, il y aurait le réticulum endoplasmique (RE). En effet, les câbles d'actine, qui s'étalent parallèlement au RE cortical, pourraient servir comme voie pour la myosine V. Cette dernière en retour pourrait être responsable de l'expansion dynamique et de l'organisation du RE pendant la croissance polarisée.

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.

Gall inducing arthropods from a seasonally dry tropical forest in Serra do Cipó, Brazil

Highly diverse forms of galling arthropods can be identified in much of southeastern Brazil's vegetation. Three fragments of a Seasonally Dry Tropical Forest (SDTF) located in the southern range of the Espinhaço Mountains were selected for study in the first survey of galling organisms in such tropical vegetation. Investigators found 92 distinct gall morphotypes on several organs of 51 host plant species of 19 families. Cecidomyiidae (Diptera) was the most prolific gall-inducing species, responsible for the largest proportion of galls (77%) observed. Leaves were the most frequently galled plant organ (63%), while the most common gall morphotype was of a spherical shape (30%). The two plant species, Baccharis dracunculifolia (Asteraceae) and Celtis brasiliensis (Cannabaceae), presented the highest number of gall morphtypes, displaying an average of 5 gall morphotypes each. This is the first study of gall-inducing arthropods and their host plant species ever undertaken in a Brazilian SDTF ecosystem. Given the intense human pressure on SDTFs, the high richness of galling arthropods, and implied floral host diversity found in this study indicates the need for an increased effort to catalogue the corresponding flora and fauna, observe their intricate associations and further understand the implications of such rich diversity in these stressed and vulnerable ecosystems.