Caveolin proteins drive formation of caveolae, specialized cell-surface microdomains that influence cell signaling. Signaling proteins are proposed to use conserved caveolin-binding motifs (CBMs) to associate with caveolae via the caveolin scaffolding domain (CSD). However, structural and bioinformatic analyses argue against such direct physical interactions: In the majority of signaling proteins, the CBM is buried and inaccessible. Putative CBMs do not form a common structure for caveolin recognition, are not enriched amongst caveolin-binding proteins, and are even more common in yeast, which lack caveolae. We propose that CBM/CSD-dependent interactions are unlikely to mediate caveolar signaling, and the basis for signaling effects should therefore be reassessed.
Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics.
Cells in some tissues acquire a polarisation in the plane of the tissue in addition to apical-basal polarity. This polarisation is commonly known as planar cell polarity and has been found to be important in developmental processes, as planar polarity is required to define the in-plane tissue coordinate system at the cellular level.
We have built an in-silico functional model of cellular polarisation that includes cellular asymmetry, cell-cell signalling and a response to a global cue. The model has been validated and parameterised against domineering non-autonomous wing hair phenotypes in Drosophila.
We have carried out a systematic comparison of in-silico polarity phenotypes with patterns observed in vivo under different genetic manipulations in the wing. This has allowed us to classify the specific functional roles of proteins involved in generating cell polarity, providing new hypotheses about their specific functions, in particular for Pk and Dsh. The predictions from the model allow direct assignment of functional roles of genes from genetic mosaic analysis of Drosophila wings.
Planar polarity; PCP; Mathematical modelling; Self organisation; Drosophila; In-silico phenotyping
The Ras GTPases comprising three main isoforms H-, N- and K-Ras operate at the plasma membrane as molecular switches in essential signaling pathways. Active concentration of the minor phospholipid phosphatidylserine in the inner leaflet of the plasma membrane contributes to the electrostatic potential that is required for K-Ras anchoring to the plasma membrane. We recently observed that staurosporine and related analogs: 7-oxostaurosporine, UCN-01 and UCN-02, long known as relatively non-specific protein kinase inhibitors, block endosomal sorting and recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine to endosomes and endomembranes with concomitant mislocalization of K-Ras. Staurosporines are therefore a new tool to study phosphatidylserine trafficking. We discuss whether the mechanism of action of UCN-01, an FDA-approved staurosporine analog used as an anti-cancer therapeutic, is related to effects on phosphatidylserine subcellular distribution. Given the high prevalence of expression of constitutively active K-Ras in human cancers, we ask whether inhibitors of phosphatidylserine trafficking may have important therapeutic applications.
Ras GTPase; phosphatidylserine; lipid recycling; staurosporine; UCN-01
Motivation: The identification of nucleosomes along the chromatin is key to understanding their role in the regulation of gene expression and other DNA-related processes. However, current experimental methods (MNase-ChIP, MNase-Seq) sample nucleosome positions from a cell population and contain biases, making thus the precise identification of individual nucleosomes not straightforward. Recent works have only focused on the first point, where noise reduction approaches have been developed to identify nucleosome positions.
Results: In this article, we propose a new approach, termed NucleoFinder, that addresses both the positional heterogeneity across cells and experimental biases by seeking nucleosomes consistently positioned in a cell population and showing a significant enrichment relative to a control sample. Despite the absence of validated dataset, we show that our approach (i) detects fewer false positives than two other nucleosome calling methods and (ii) identifies two important features of the nucleosome organization (the nucleosome spacing downstream of active promoters and the enrichment/depletion of GC/AT dinucleotides at the centre of in vitro nucleosomes) with equal or greater ability than the other two methods.
Availability: The R code of NucleoFinder, an example datafile and instructions are available for download from https://sites.google.com/site/beckerjeremie/
Supplementary information: Supplementary data are available at Bioinformatics online.
Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes.
palmitoylation; GTPase; isoforms; organelle; plasma membrane; microdomains; nanoclusters
Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation) in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.
This paper describes an approach to providing computer-interpretable logical definitions for the terms of the Human Phenotype Ontology (HPO) using PATO, the ontology of phenotypic qualities, to link terms of the HPO to the anatomic and other entities that are affected by abnormal phenotypic qualities. This approach will allow improved computerized reasoning as well as a facility to compare phenotypes between different species. The PATO mapping will also provide direct links from phenotypic abnormalities and underlying anatomic structures encoded using the Foundational Model of Anatomy, which will be a valuable resource for computational investigations of the links between anatomical components and concepts representing diseases with abnormal phenotypes and associated genes.
Ras isoform-specific signaling from the plasma membrane appears to be regulated by interactions with distinct functional microdomains. We have developed protocols allowing the generation of 2-D spatial maps describing cell surface microdomain distributions. The combined electron microscopic (EM)-statistics approach provides nanometer scale resolution allowing both inner and outer leaflet domains to be visualized and cross-correlated with each other or with a protein of interest. In particular, the technique has allowed the interaction of Ras isoforms with signaling microdomains and proteins regulating these compartments to be screened. By allowing detailed monitoring of cell surface organization and compartmentalization the approach has widespread potential for studies of plasma membrane-dependent cell biology, including regulated signaling and membrane trafficking.
Hydrogen sulfide (H2S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H2S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H2S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H2S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsicum anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.
abscisic acid; GYY4137; hydrogen sulfide; nitric oxide; stomatal aperture
Hydroxmethylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) lower serum cholesterol but exhibit pleiotropic biological effects that are difficult to ascribe solely to cholesterol depletion. Here, we investigated the effect of lovastatin on protein prenylation and cell signaling. We show that high concentrations (50 μM) of lovastatin inhibit Ras, Rho, and Rap prenylation but that therapeutic levels of lovastatin (50 nM to 500 nM) do not. In contrast, depletion of cellular cholesterol by therapeutic levels of lovastatin increased Ras GTP loading and mitogen-activated protein kinase (MAPK) activation in human umbilical vein endothelial cells and rodent fibroblasts. Elevated Ras signaling was not seen in statin-treated cells if cholesterol levels were maintained by supplementation. Activation of Ras-MAPK signaling was a consequence of, and dependent on, activation of phospholipase D2 (PLD2). Expression of dominant interfering PLD2 or biochemical inhibition of PLD2 abrogated Ras and MAPK activation induced by lovastatin. In contrast, ectopic expression of wild-type PLD2 enhanced Ras and MAPK activation in response to therapeutic levels of lovastatin. Statin-induced cholesterol depletion also modestly activated the epidermal growth factor receptor (EGFR), resulting in downregulation of EGFR expression. These results suggest that statins modulate key cell signaling pathways as a direct consequence of cholesterol depletion and identify the EGFR-PLD2-Ras-MAPK axis as an important statin target.
To evaluate how well current anatomical ontologies fit the way real-world users apply anatomy terms in their data annotations.
Annotations from three diverse multi-species public-domain datasets provided a set of use cases for matching anatomical terms in two major anatomical ontologies (the Foundational Model of Anatomy and Uberon), using two lexical-matching applications (Zooma and Ontology Mapper).
Approximately 1500 terms were identified; Uberon/Zooma mappings provided 286 matches, compared to the control and Ontology Mapper returned 319 matches. For the Foundational Model of Anatomy, Zooma returned 312 matches, and Ontology Mapper returned 397.
Our results indicate that for our datasets the anatomical entities or concepts are embedded in user-generated complex terms, and while lexical mapping works, anatomy ontologies do not provide the majority of terms users supply when annotating data. Provision of searchable cross-products for compositional terms is a key requirement for using ontologies.
Although we have amassed extensive catalogues of signalling network components, our understanding of the spatiotemporal control of emergent network structures has lagged behind. Dynamic behaviour is starting to be explored throughout the genome, but analysis of spatial behaviours is still confined to individual proteins. The challenge is to reveal how cells integrate temporal and spatial information to determine specific biological functions. Key findings are the discovery of molecular signalling machines such as Ras nanoclusters, spatial activity gradients and flexible network circuitries that involve transcriptional feedback. They reveal design principles of spatiotemporal organization that are crucial for network function and cell fate decisions.
Cellular ATP levels are generated by glucose-stimulated mitochondrial metabolism and determine metabolic responses, such as glucose-stimulated insulin secretion (GSIS) from the β-cells of pancreatic islets. We describe an analysis of the evolutionary processes affecting the core enzymes involved in glucose-stimulated insulin secretion in mammals. The proteins involved in this system belong to ancient enzymatic pathways: glycolysis, the TCA cycle and oxidative phosphorylation.
We identify two sets of proteins, or protein coalitions, in this group of 77 enzymes with distinct evolutionary patterns. Members of the glycolysis, TCA cycle, metabolite transport, pyruvate and NADH shuttles have low rates of protein sequence evolution, as inferred from a human-mouse comparison, and relatively high rates of evolutionary gene duplication. Respiratory chain and glutathione pathway proteins evolve faster, exhibiting lower rates of gene duplication. A small number of proteins in the system evolve significantly faster than co-pathway members and may serve as rapidly evolving adapters, linking groups of co-evolving genes.
Our results provide insights into the evolution of the involved proteins. We find evidence for two coalitions of proteins and the role of co-adaptation in protein evolution is identified and could be used in future research within a functional context.
The laboratory mouse is the pre-eminent model organism for the dissection of human disease pathways. With the advent of a comprehensive panel of gene knockouts, projects to characterise the phenotypes of all knockout lines are being initiated. The range of genotype-phenotype associations can be represented using the Mammalian Phenotype ontology. Using publicly available data annotated with this ontology we have constructed gene and phenotype networks representing these associations. These networks show a scale-free, hierarchical and modular character and community structure. They also exhibit enrichment for gene coexpression, protein-protein interactions and Gene Ontology annotation similarity. Close association between gene communities and some high-level ontology terms suggests that systematic phenotyping can provide a direct insight into underlying pathways. However some phenotypes are distributed more diffusely across gene networks, likely reflecting the pleiotropic roles of many genes. Phenotype communities show a many-to-many relationship to human disease communities, but stronger overlap at more granular levels of description. This may suggest that systematic phenotyping projects should aim for high granularity annotations to maximise their relevance to human disease.
Quantitative ultrastructural analysis and proteomics detail CLIC structure, composition, and function.
Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.
Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.
The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots.
maize; maize silk; plant productivity; pollen; proline; proline dehydrogenase; Δ1-pyrroline-5-carboxylate synthetase; Zea mays L.
The present article proposes the adoption of a community-defined, uniform, generic description of the core attributes of biological databases, BioDBCore. The goals of these attributes are to provide a general overview of the database landscape, to encourage consistency and interoperability between resources; and to promote the use of semantic and syntactic standards. BioDBCore will make it easier for users to evaluate the scope and relevance of available resources. This new resource will increase the collective impact of the information present in biological databases.
The present article proposes the adoption of a community-defined, uniform, generic description of the core attributes of biological databases, BioDBCore. The goals of these attributes are to provide a general overview of the database landscape, to encourage consistency and interoperability between resources and to promote the use of semantic and syntactic standards. BioDBCore will make it easier for users to evaluate the scope and relevance of available resources. This new resource will increase the collective impact of the information present in biological databases.
H-Ras is a binary switch that is activated by multiple co-factors and triggers several key cellular pathways one of which is MAPK. The specificity and magnitude of downstream activation is achieved by the spatio-temporal organization of the active H-Ras in the plasma membrane. Upon activation, the GTP bound H-Ras binds to Galectin-1 (Gal-1) and becomes transiently immobilized in short-lived nanoclusters on the plasma membrane from which the signal is propagated to Raf. In the current study we show that stabilizing the H-Ras-Gal-1 interaction, using bimolecular fluorescence complementation (BiFC), leads to prolonged immobilization of H-Ras.GTP in the plasma membrane which was measured by fluorescence recovery after photobleaching (FRAP), and increased signal out-put to the MAPK module. EM measurements of Raf recruitment to the H-Ras.GTP nanoclusters demonstrated that the enhanced signaling observed in the BiFC stabilized H-Ras.GTP nanocluster was attributed to increased H-Ras immobilization rather than to an increase in Raf recruitment. Taken together these data demonstrate that the magnitude of the signal output from a GTP-bound H-Ras nanocluster is proportional to its stability.
In recent years, progress in the study of the lateral organization of the plasma membrane has led to the proposal that mammalian cells use two different organelles to store lipids: intracellular lipid droplets (LDs) and plasma membrane caveolae. Experimental evidence suggests that caveolin (CAV) may act as a sensitive lipid-organizing molecule that physically connects these two lipid-storing organelles. Here, we determine the sequences necessary for efficient sorting of CAV to LDs. We show that targeting is a process cooperatively mediated by two motifs. CAV's central hydrophobic domain (Hyd) anchors CAV to the endoplasmic reticulum (ER). Next, positively charged sequences (Pos-Seqs) mediate sorting of CAVs into LDs. Our findings were confirmed by identifying an equivalent, non-conserved but functionally interchangeable Pos-Seq in ALDI, a bona fide LD-resident protein. Using this information, we were able to retarget a cytosolic protein and convert it to an LD-resident protein. Further studies suggest three requirements for targeting via this mechanism: the positive charge of the Pos-Seq, physical proximity between Pos-Seq and Hyd and a precise spatial orientation between both motifs. The study uncovers remarkable similarities with the signals that target proteins to the membrane of mitochondria and peroxisomes
ALDI; caveolin; endoplasmic reticulum; lipid droplets; sorting signal
The recent explosion of biological data and the concomitant proliferation of distributed databases make it challenging for biologists and bioinformaticians to discover the best data resources for their needs, and the most efficient way to access and use them. Despite a rapid acceleration in uptake of syntactic and semantic standards for interoperability, it is still difficult for users to find which databases support the standards and interfaces that they need. To solve these problems, several groups are developing registries of databases that capture key metadata describing the biological scope, utility, accessibility, ease-of-use and existence of web services allowing interoperability between resources. Here, we describe some of these initiatives including a novel formalism, the Database Description Framework, for describing database operations and functionality and encouraging good database practise. We expect such approaches will result in improved discovery, uptake and utilization of data resources.
Database URL: http://www.casimir.org.uk/casimir_ddf
The laboratory mouse has become the organism of choice for discovering gene function and unravelling pathogenetic mechanisms of human diseases through the application of various functional genomic approaches. The resulting deluge of data has led to the deployment of numerous online resources and the concomitant need for formalized experimental descriptions, data standardization, database interoperability and integration, a need that has yet to be met. We present here the Mouse Resource Browser (MRB), a database of mouse databases that indexes 217 publicly available mouse resources under 22 categories and uses a standardised database description framework (the CASIMIR DDF) to provide information on their controlled vocabularies (ontologies and minimum information standards), and technical information on programmatic access and data availability. Focusing on interoperability and integration, MRB offers automatic generation of downloadable and re-distributable SOAP application-programming interfaces for resources that provide direct database access. MRB aims to provide useful information to both bench scientists, who can easily navigate and find all mouse related resources in one place, and bioinformaticians, who will be provided with interoperable resources containing data which can be mined and integrated.
Database URL: http://bioit.fleming.gr/mrb