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Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1–3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4–7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI: http://dx.doi.org/10.7554/eLife.00792.001, eLife digest Humans and other animals can use the five senses—touch, taste, sight, smell, and hearing—to interpret the world around them. Single-celled organisms, however, must rely on molecular cues to understand their immediate surroundings. In particular, bacteria gather information about external conditions, including potential hosts nearby, by secreting protein sensors that can relay messages back to the cell. Bacteria export these sensors via secretion systems that enable the organism both to receive information about the environment and to invade a host cell. A total of seven separate secretion systems, known as types I–VII, have been identified. These different secretion systems handle distinct cargoes, allowing the bacterial cell to respond to a range of feedback from the external milieu. The type III secretion system, also known as the ‘injectisome’, is found in bacterial species that are enclosed by two membranes separated by a periplasmic space. The injectisome comprises different components that combine to form the basal body, which spans the inner and outer membranes, and a projection from the basal body, called the hollow needle, that mediates the export of cargo from a bacterium to its host or the local environment. The distance between the inner and outer membranes may vary across species or according to environmental conditions, so the basal body must be able to accommodate these changes. However, no mechanism has yet been established that might introduce such elasticity into the injectisome. Now, Kudryashev et al. have generated three-dimensional structures for the injectisomes of two species of bacteria, Shigella flexneri and Yersinia enterocolitica, and shown that the size of the basal body can fluctuate by up to 20%. Kudryashev et al. imaged whole injectisomes in these two species and found that the height of the basal body was proportional to the distance between the inner and outer membranes. To probe how this could occur, the properties of two proteins that are important components of the basal body were studied in greater detail. YscD, a protein that extends across the periplasmic space, was crystallized and its structure was then determined and used to develop a computer model to assess its compressibility: this model indicated that YscD could stretch or contract by up to 50% of its total length. The outer membrane component YscC also appeared elastic: when the protein was isolated and introduced into synthetic membranes, its length was reduced 30–40% relative to that observed in intact bacterial membranes. A further experiment confirmed the adaptability of the basal body: when the separation of the membranes was deliberately increased by placing bacteria in a high-salt medium, the basal body extended approximately 10% in length. Cumulatively, therefore, these experiments suggest that the in-built flexibility of the basal body of the injectisome allows bacteria to adjust to environmental changes while maintaining their sensory abilities and host-invasion potential. DOI: http://dx.doi.org/10.7554/eLife.00792.002

Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. We identified the abundant phosphoprotein F17, the dual-specificity phosphatase VH1, and the oxidoreductase G4 as bona fide LB components. After reaching the cytosol, F17 was degraded in a proteasome-dependent manner. Proteasome activity, and presumably LB disassembly, was required for the immediate immunomodulatory activity of VH1: dephosphorylation of STAT1 to prevent interferon-γ-mediated antiviral responses. These results reveal a mechanism used by poxviruses to deliver viral enzymes to the host cell cytosol and are likely to facilitate the identification of additional LB-resident viral effectors. ISSN:2666-3864 ISSN:2211-1247

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of high-mass proteins requires suitable calibration standards at high m/z ratios. Several possible candidates were investigated, and concatenated polyproteins based on recombinantly expressed maltodextrin-binding protein (MBP) are shown here to be well-suited for this purpose. Introduction of two specific recognition sites into the primary sequence of the polyprotein allows for the selective cleavage of MBP3 into MBP and MBP2. Moreover, these MBP2 and MBP3 oligomers can be dimerized specifically, such that generation of MPB4 and MBP6 is possible as well. With the set of calibrants presented here, the m/z range of 40-400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules.

Sphingolipids are membrane components and signaling molecules. Nutrient-sensitive TORC1 inhibition stimulates the kinase Npr1 to phosphorylate Orm proteins and thereby up-regulate complex sphingolipid synthesis. This appears to be a physiological response to starvation to up-regulate the nutrient-scavenging permease Gap1., The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.

Observations gained from model organisms are essential, yet it remains unclear to which degree they are applicable to distant relatives. For example, in the dicotyledon Arabidopsis thaliana (Arabidopsis), auxin biosynthesis via indole-3-pyruvic acid (IPA) is essential for root development and requires redundant TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and TAA1-RELATED (TAR) genes. A promoter T-DNA insertion in the monocotyledon Brachypodium distachyon (Brachypodium) TAR2-LIKE gene (BdTAR2L) severely down-regulates expression, suggesting reduced tryptophan aminotransferase activity in this mutant, which thus represents a hypomorphic Bdtar2l allele (Bdtar2lhypo). Counterintuitive however, Bdtar2lhypo mutants display dramatically elongated seminal roots because of enhanced cell elongation. This phenotype is also observed in another, stronger Bdtar2l allele and can be mimicked by treating wild type with L-kynerunine, a specific TAA1/TAR inhibitor. Surprisingly, L-kynerunine-treated as well as Bdtar2l roots display elevated rather than reduced auxin levels. This does not appear to result from compensation by alternative auxin biosynthesis pathways. Rather, expression of YUCCA genes, which are rate-limiting for conversion of IPA to auxin, is increased in Bdtar2l mutants. Consistent with suppression of Bdtar2lhypo root phenotypes upon application of the ethylene precursor 1-aminocyclopropane-1-carboxylic-acid (ACC), BdYUCCA genes are down-regulated upon ACC treatment. Moreover, they are up-regulated in a downstream ethylene-signaling component homolog mutant, Bd ethylene insensitive 2-like 1, which also displays a Bdtar2l root phenotype. In summary, Bdtar2l phenotypes contrast with gradually reduced root growth and auxin levels described for Arabidopsis taa1/tar mutants. This could be explained if in Brachypodium, ethylene inhibits the rate-limiting step of auxin biosynthesis in an IPA-dependent manner to confer auxin levels that are sub-optimal for root cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis., Author Summary The plant hormone auxin is pivotal for root system development. For instance, its local biosynthesis is essential for root formation and growth in the dicotyledon model Arabidopsis. Thus, increasing interference with auxin biosynthesis results in increasingly shorter roots, partly because of reduced cell elongation. In this study, we isolated a hypomorphic mutant in an auxin biosynthesis pathway enzyme in the monocotyledon model Brachypodium. Counterintuitive, this mutant displays a dramatically longer seminal root, because mature cells are thinner, more elongated and therefore more anisotropic than in wild type. Interestingly, this phenotype can be mimicked in wild type by pharmacological interference with production of a key auxin biosynthesis intermediate, but also by interference with the biosynthesis of another plant hormone, ethylene. The latter controls auxin biosynthesis in Arabidopsis roots. Surprisingly however, auxin levels in the Brachypodium mutant are elevated rather than reduced, because of a simultaneous up-regulation of the second, rate-limiting step of the pathway. Ethylene normally represses this second step, suggesting an inverted regulatory relation between the two hormones as compared to Arabidopsis. Our results point to a complex homeostatic crosstalk between auxin and ethylene in Brachypodium roots, which is fundamentally different from Arabidopsis and might be conserved in other monocotyledons.

We propose a novel acquisition scheme and non-parametric multi-image based method for correcting illumination in fluorescence images. Our approach measures changes in intensity of the subject by moving the microscope stage at regularly spaced intervals, and exploits this information to learn the correction function. The acquisition process and learning are performed prior to imaging, and take only a few minutes. Afterwards, images can be corrected for vignetting and photobleaching effects on the fly. Our approach can be implemented in any microscope with a motorized stage, and does not require a reference calibration slide. Experiments demonstrate that our method outperforms standard approaches to illumination correction.

The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix αinh that is provided by a cognate anti-toxin (class I), or is part of the N- or C-terminal part of the Fic protein itself (classes II and III). In vitro, inhibition is relieved by mutation of a conserved glutamate of αinh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain β-interactions with the β-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein.

4 páginas, 3 figuras -- PAGS nros. 587-590, Microtubule-stabilizing agents (MSAs) are efficacious chemotherapeutic drugs widely used for the treatment of cancer. Despite the importance of MSAs for medical applications and basic research, their molecular mechanisms of action on tubulin and microtubules remain elusive. We determined high-resolution crystal structures of αβ-tubulin in complex with two unrelated MSAs, zampanolide and epothilone A. Both compounds were bound to the taxane pocket of β-tubulin and used their respective side chains to induce structuring of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these findings explain how taxane-site MSAs promote microtubule assembly and stability. Further, our results offer fundamental structural insights into the control mechanisms of microtubule dynamics, This work was supported by grants from the Ministerio de Economía y Competitividad (BIO2010-16351) and the Comunidad Autónoma de Madrid (S2010/BMD-2457) (to J.F.D.), a Ph.D. fellowship from the Roche Research Foundation (to D.Z.), and grants from the Swiss National Science Foundation (310030B_138659) and the Swiss SystemsX.ch initiative (BIP-2011/122) (to M.O.S.). Coordinates have been deposited at the Protein Data Bank (PDB) under identification (ID) nos. 4I4T (T2R-TTL-Zampa), 4I50 (T2R-TTL-EpoA), and 4I55 (T2R-TTL)

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell’s plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions., Developmental Biology, 381 (1), ISSN:0012-1606, ISSN:1095-564X

Metabolome, proteome and physiology measurements were combined with mathematical modeling to unravel the temporal regulation of the metabolic fluxes during the diauxic shift in Saccharomyces cerevisiae., The diauxic shift involves three main events: a reduction in the glycolytic flux and the production of storage compounds before glucose depletion; the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation upon glucose exhaustion; and the shutting down of the pentose phosphate (PP) pathway with a change in the source of NADPH regeneration. The redistribution of fluxes toward the production of storage compounds prior glucose depletion drives glycolytic reactions closer to equilibrium, which is essential for the reversion of fluxes upon glucose exhaustion. The onset of the glyoxylate cycle is quantitatively more important than the activation of the tricarboxylic acid cycle for growth on ethanol. Flux through the PP pathway is halted in the later stages of the adaptation and NADPH regeneration is taken over by NADP-dependent enzymes in the glyoxylate cycle and ethanol metabolism., The diauxic shift in Saccharomyces cerevisiae is an ideal model to study how eukaryotic cells readjust their metabolism from glycolytic to gluconeogenic operation. In this work, we generated time-resolved physiological data, quantitative metabolome (69 intracellular metabolites) and proteome (72 enzymes) profiles. We found that the diauxic shift is accomplished by three key events that are temporally organized: (i) a reduction in the glycolytic flux and the production of storage compounds before glucose depletion, mediated by downregulation of phosphofructokinase and pyruvate kinase reactions; (ii) upon glucose exhaustion, the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation triggered by an increased expression of the enzymes that catalyze the malate synthase and cytosolic citrate synthase reactions; and (iii) in the later stages of the adaptation, the shutting down of the pentose phosphate pathway with a change in NADPH regeneration. Moreover, we identified the transcription factors associated with the observed changes in protein abundances. Taken together, our results represent an important contribution toward a systems-level understanding of how this adaptation is realized.

Plant cell expansion is controlled by a fine-tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in-depth knowledge of cell wall mechanics. Pollen tubes are tip-growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20–90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM-based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.

Ecol. Lett. ISI Document Delivery No.: 132OA Times Cited: 0 Cited Reference Count: 46 de Mazancourt, Claire Isbell, Forest Larocque, Allen Berendse, Frank De Luca, Enrica Grace, James B. Haegeman, Bart Polley, H. Wayne Roscher, Christiane Schmid, Bernhard Tilman, David van Ruijven, Jasper Weigelt, Alexandra Wilsey, Brian J. Loreau, Michel Wiley-blackwell Hoboken; International audience; As biodiversity is declining at an unprecedented rate, an important current scientific challenge is to understand and predict the consequences of biodiversity loss. Here, we develop a theory that predicts the temporal variability of community biomass from the properties of individual component species in monoculture. Our theory shows that biodiversity stabilises ecosystems through three main mechanisms: (1) asynchrony in species’ responses to environmental fluctuations, (2) reduced demographic stochasticity due to overyielding in species mixtures and (3) reduced observation error (including spatial and sampling variability). Parameterised with empirical data from four long-term grassland biodiversity experiments, our prediction explained 22–75% of the observed variability, and captured much of the effect of species richness. Richness stabilised communities mainly by increasing community biomass and reducing the strength of demographic stochasticity. Our approach calls for a re-evaluation of the mechanisms explaining the effects of biodiversity on ecosystem stability.

Simian Virus 40 (SV40) is a paradigm pathogen with multivalent binding sites for the sphingolipid GM1, via which it induces its endocytosis for infection. Here we report that SV40 also utilizes cell surface integrins to activate signaling networks required for infection, even in the absence of the previously implicated glycosphingolipids. We identify ILK, PDK1, the RhoGAP GRAF1 and RhoA as core nodes of the signaling network activated upon SV40 engagement of integrins. We show that integrin-mediated signaling through host SV40 engagement induces the de-phosphorylation of Ezrin leading to uncoupling of the plasma membrane and cortical actin. Our results provide functional evidence for a mechanism by which SV40 activates signal transduction in human epithelial cells via integrins in the context of clathrin-independent endocytosis., PLoS ONE, 8 (2), ISSN:1932-6203

Phototropism allows plants to align their photosynthetic tissues with incoming light. The direction of incident light is sensed by the phototropin family of blue light photoreceptors (phot1 and phot2 in Arabidopsis), which are light-activated protein kinases. The kinase activity of phototropins and phosphorylation of residues in the activation loop of their kinase domains are essential for the phototropic response. These initial steps trigger the formation of the auxin gradient across the hypocotyl that leads to asymmetric growth. The molecular events between photoreceptor activation and the growth response are only starting to be elucidated. In this review, we discuss the major steps leading from light perception to directional growth concentrating on Arabidopsis. In addition, we highlight links that connect these different steps enabling the phototropic response.

We have identified the zinc-finger transcription factor Kruppel-like factor 4 (Klf4) among the transcription factors that are significantly downregulated in their expression during epithelial-mesenchymal transition (EMT) in mammary epithelial cells and in breast cancer cells. Loss and gain of function experiments demonstrate that the down-regulation of Klf4 expression is required for the induction of EMT in vitro and for metastasis in vivo. In addition, reduced Klf4 expression correlates with shorter disease-free survival of subsets of breast cancer patients. Yet, reduced expression of Klf4 also induces apoptosis in cells undergoing TGFβ-induced EMT. Chromatin immunoprecipitation/deep-sequencing in combination with gene expression profiling reveals direct Klf4 target genes, including E-cadherin (Cdh1), N-cadherin (Cdh2), vimentin (Vim), β-catenin (Ctnnb1), VEGF-A (Vegfa), endothelin-1 (Edn1) and Jnk1 (Mapk8). Thereby, Klf4 acts as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. Specifically, increased expression of Jnk1 (Mapk8) upon down-regulation of its transcriptional repressor Klf4 is required for EMT cell migration and for the induction of apoptosis. The data demonstrate a central role of Klf4 in the maintenance of epithelial cell differentiation and the prevention of EMT and metastasis.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.

S-palmitoylation is post-translational modification, which consists in the addition of a C16 acyl chain to cytosolic cysteines and which is unique amongst lipid modifications in that it is reversible. It can thus, like phosphorylation or ubiquitination, act as a switch. While palmitoylation of soluble proteins allows them to interact with membranes, the consequences of palmitoylation for transmembrane proteins are more enigmatic. We briefly review the current knowledge regarding the enzymes responsible for palmitate addition and removal. We then describe various observed consequences of membrane protein palmitoylation. We propose that the direct effects of palmitoylation on transmembrane proteins, however, might be limited to four non-mutually exclusive mechanistic consequences: alterations in the conformation of transmembrane domains, association with specific membrane domains, controlled interactions with other proteins and controlled interplay with other post-translational modifications.

This essay provides an introduction to the terminology, concepts, methods, and challenges of image-based modeling in biology. Image-based modeling and simulation aims at using systematic, quantitative image data to build predictive models of biological systems that can be simulated with a computer. This allows one to disentangle molecular mechanisms from effects of shape and geometry. Questions like “what is the functional role of shape” or “how are biological shapes generated and regulated” can be addressed in the framework of image-based systems biology. The combination of image quantification, model building, and computer simulation is illustrated here using the example of diffusion in the endoplasmic reticulum.

International audience; Genome-scale metabolic network reconstructions are now routinely used in the study of metabolic pathways, their evolution and design. The development of such reconstructions involves the integration of information on reactions and metabolites from the scientific literature as well as public databases and existing genome-scale metabolic models. The reconciliation of discrepancies between data from these sources generally requires significant manual curation, which constitutes a major obstacle in efforts to develop and apply genome-scale metabolic network reconstructions. In this work, we discuss some of the major difficulties encountered in the mapping and reconciliation of metabolic resources and review three recent initiatives that aim to accelerate this process, namely BKM-react, MetRxn and MNXref (presented in this article). Each of these resources provides a pre-compiled reconciliation of many of the most commonly used metabolic resources. By reducing the time required for manual curation of metabolite and reaction discrepancies, these resources aim to accelerate the development and application of high-quality genome-scale metabolic network reconstructions and models.

A two step automated high throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated during virus assembly. After entering the cytosol of an uninfected cell the viral DNA was released from the core through the activity of the cell's proteasomes. Next a Cullin3 based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will moreover provide a resource for future virus host cell interaction studies and for the discovery of antivirals. Although smallpox among the most devastating pathogens in human history has been eradicated the spread of other poxvirus species and bioterrorism still poses a danger. To understand the replication cycle of these complex viruses Pelkmans Helenius and colleagues have now analyzed the cell biology of vaccinia virus infection. Using an RNAi based screen the authors identify host cell factors that the virus needs for infection. They find that the ubiquitin proteasome system is needed at two steps: DNA uncoating and DNA replication initiation. © 2012 The Authors.

Quantitative biology requires quantitative data. No high-throughput technologies exist capable of obtaining several hundred independent kinetic binding measurements in a single experiment. We present an integrated microfluidic device (k-MITOMI) for the simultaneous kinetic characterization of 768 biomolecular interactions. We applied k-MITOMI to the kinetic analysis of transcription factor (TF)—DNA interactions, measuring the detailed kinetic landscapes of the mouse TF Zif268, and the yeast TFs Tye7p, Yox1p, and Tbf1p. We demonstrated the integrated nature of k-MITOMI by expressing, purifying, and characterizing 27 additional yeast transcription factors in parallel on a single device. Overall, we obtained 2,388 association and dissociation curves of 223 unique molecular interactions with equilibrium dissociation constants ranging from 2 × 10 -6 M to 2 × 10 -9 M, and dissociation rate constants of approximately 6 s -1 to 8.5 × 10 -3 s -1 . Association rate constants were uniform across 3 TF families, ranging from 3.7 × 10 6 M -1 s -1 to 9.6 × 10 7 M -1 s -1 , and are well below the diffusion limit. We expect that k-MITOMI will contribute to our quantitative understanding of biological systems and accelerate the development and characterization of engineered systems.

We present a novel analytical platform for peptides quantitative assays in biological matrices based on microscale liquid chromatography fractionation and matrix-assisted laser desorption/ionization mass spectrometric detection using the selected reaction monitoring (SRM) mode. The MALDI source was equipped with a high frequency Nd:YAG laser (1000 Hz) and mounted on a triple quadrupole/linear ion trap mass spectrometer (MALDI-QqQ(LIT)). Compared to conventional LC-ESI-SRM/MS, the separated analytes are "time-frozen" onto the MALDI plate in fractions, and navigation through the LC chromatogram makes it possible to perform SRM experiments as well as enhanced product ion spectra acquisition for confirmatory analyses without time constraints. The LC spots were analyzed using different rastering speeds ranging from 0.25 to 4 mm/sec with the shortest analysis time of 425 ms/spot. Since the LC runs can be multiplexed and do not need a comprehensive investigation, the present platform offers a valuable alternative to LC-ESI-SRM/MS for high throughput proteomic analyses. In addition, the derivatization of the N-terminal α-amino group by sulfonation was found to be key for the fragmentation of singly charged peptides under low collision energy regime. Under such conditions, y-ion series were observed in the MS/MS spectra, and thus the design of SRM experiments was greatly simplified. The quantitative performance of the platform was compared to that of LC-ESI-SRM/MS by spiking yeast tryptic peptides in human plasma digests. Both platforms exhibited similar sensitivities, accuracy (within ±20%) and precision (under 20%) in the relative quantification mode. As a proof of principle, the relative and absolute quantification of proteins associated with glycolysis, glyoxylate and tricarboxylic acid (TCA) cycles over a growth time course of Saccharomyces cerevisiae on glucose media was successfully performed using isotopic dilution.

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7) phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions. STN8, which is mainly involved in phosphorylation of PSII core subunits, influences folding of the thylakoid membranes and repair of PSII after photodamage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases. In a reverse genetic screen, we identified the chloroplast PP2C phosphatase, PHOTOSYSTEM II CORE PHOSPHATASE (PBCP), which is required for efficient dephosphorylation of PSII proteins. Its targets, identified by immunoblotting and mass spectrometry, largely coincide with those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolated thylakoids. Thylakoid folding is affected in the absence of PBCP, while its overexpression alters the kinetics of state transitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity and physiological functions are distinct from those of STN7 and the counteracting phosphatase PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, but their activities may overlap to some degree.

Although protein expression is regulated both temporally and spatially, most proteins have an intrinsic, "typical" range of functionally effective abundance levels. These extend from a few molecules per cell for signaling proteins, to millions of molecules for structural proteins. When addressing fundamental questions related to protein evolution, translation and folding, but also in routine laboratory work, a simple rough estimate of the average wild type abundance of each detectable protein in an organism is often desirable. Here, we introduce a meta-resource dedicated to integrating information on absolute protein abundance levels; we place particular emphasis on deep coverage, consistent post-processing and comparability across different organisms. Publicly available experimental data are mapped onto a common namespace and, in the case of tandem mass spectrometry data, re-processed using a standardized spectral counting pipeline. By aggregating and averaging over the various samples, conditions and cell-types, the resulting integrated data set achieves increased coverage and a high dynamic range. We score and rank each contributing, individual data set by assessing its consistency against externally provided protein-network information, and demonstrate that our weighted integration exhibits more consistency than the data sets individually. The current PaxDb-release 2.1 (at http://pax-db.org/) presents whole-organism data as well as tissue-resolved data, and covers 85,000 proteins in 12 model organisms. All values can be seamlessly compared across organisms via pre-computed orthology relationships.

We present a discrete, unsupervised multi-region competition algorithm for image segmentation over different energy functionals. The number of regions present in an image does not need to be known a priori, nor their photometric properties. The algorithm jointly estimates the number of regions, their photometries, and their contours. The required regularization is provided by defining a region as a connected set of pixels. The evolving contours in the image are represented by computational particles that move as driven by an energy-minimization algorithm. We present an efficient discrete algorithm that allows minimizing a range of well-known energy functionals under the topological constraint of regions being connected components. The presented framework and algorithms are implemented in the open-source Insight Toolkit (ITK) image-processing library.

One of the most obvious phenotypes of a cell is its metabolic activity, which is defined by the fluxes in the metabolic network. Although experimental methods to determine intracellular fluxes are well established, only a limited number of fluxes can be resolved. Especially in eukaryotes such as yeast, compartmentalization and the existence of many parallel routes render exact flux analysis impossible using current methods. To gain more insight into the metabolic operation of S. cerevisiae we developed a new computational approach where we characterize the flux solution space by determining elementary flux modes (EFMs) that are subsequently classified as thermodynamically feasible or infeasible on the basis of experimental metabolome data. This allows us to provably rule out the contribution of certain EFMs to the in vivo flux distribution. From the 71 million EFMs in a medium size metabolic network of S. cerevisiae, we classified 54% as thermodynamically feasible. By comparing the thermodynamically feasible and infeasible EFMs, we could identify reaction combinations that span the cytosol and mitochondrion and, as a system, cannot operate under the investigated glucose batch conditions. Besides conclusions on single reactions, we found that thermodynamic constraints prevent the import of redox cofactor equivalents into the mitochondrion due to limits on compartmental cofactor concentrations. Our novel approach of incorporating quantitative metabolite concentrations into the analysis of the space of all stoichiometrically feasible flux distributions allows generating new insights into the system-level operation of the intracellular fluxes without making assumptions on metabolic objectives of the cell., PLoS Computational Biology, 8 (3), ISSN:1553-734X, ISSN:1553-7358

Wartlick et al . (Research Articles, 4 March 2011, p. 1154) reported that growth rates in the Drosophila wing disc correlate with increasing Dpp signaling levels, suggesting that the rate of Dpp increase determines the cell-cycle length. Contradicting their model, we found that cells in which the increase of Dpp signaling levels was genetically abrogated grew at rates comparable to those of wild-type cells.

Many nucleic acid stains show a strong fluorescence enhancement upon binding to double-stranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of ∼14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures.

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective degeneration of dopaminergic neurons in the nigrostriatal pathway. Several lines of evidence indicate that mitochondrial dysfunction contributes to its etiology. Other studies have suggested that alterations in sterol homeostasis correlate with increased risk for PD. Whether these observations are functionally related is, however, unknown. In this study, we used a toxin-induced mouse model of PD and measured levels of nine sterol intermediates. We found that lanosterol is significantly (∼50%) and specifically reduced in the nigrostriatal regions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, indicative of altered lanosterol metabolism during PD pathogenesis. Remarkably, exogenous addition of lanosterol rescued dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in culture. Furthermore, we observed a marked redistribution of lanosterol synthase from the endoplasmic reticulum to mitochondria in dopaminergic neurons exposed to MPP+, suggesting that lanosterol might exert its survival effect by regulating mitochondrial function. Consistent with this model, we find that lanosterol induces mild depolarization of mitochondria and promotes autophagy. Collectively, our results highlight a novel sterol-based neuroprotective mechanism with direct relevance to PD.

Distinguishing subpopulations in group behavioral experiments can reveal the impact of differences in genetic, pharmacological and life-histories on social interactions and decision-making. Here we describe Fluorescence Behavioral Imaging (FBI), a toolkit that uses transgenic fluorescence to discriminate subpopulations, imaging hardware that simultaneously records behavior and fluorescence expression, and open-source software for automated, high-accuracy determination of genetic identity. Using FBI, we measure courtship partner choice in genetically mixed groups of Drosophila.

Phyllotaxis, the regular arrangement of leaves and flowers around the stem, is a key feature of plant architecture. Current models propose that the spatiotemporal regulation of organ initiation is controlled by a positive feedback loop between the plant hormone auxin and its efflux carrier PIN-FORMED1 (PIN1). Consequently, pin1 mutants give rise to naked inflorescence stalks with few or no flowers, indicating that PIN1 plays a crucial role in organ initiation. However, pin1 mutants do produce leaves. In order to understand the regulatory mechanisms controlling leaf initiation in Arabidopsis (Arabidopsis thaliana) rosettes, we have characterized the vegetative pin1 phenotype in detail. We show that although the timing of leaf initiation in vegetative pin1 mutants is variable and divergence angles clearly deviate from the canonical 137° value, leaves are not positioned at random during early developmental stages. Our data further indicate that other PIN proteins are unlikely to explain the persistence of leaf initiation and positioning during pin1 vegetative development. Thus, phyllotaxis appears to be more complex than suggested by current mechanistic models.

Adaptive behavior often exploits generalizations from past experience by applying them judiciously in new situations. This requires a means of quantifying the relative importance of prior experience and current information, so they can be balanced optimally. In this study, we ask whether the brain generalizes in an optimal way. Specifically, we used Bayesian learning theory and fMRI to test whether neuronal responses reflect context-sensitive changes in ambiguity or uncertainty about experience-dependent beliefs. We found that the hippocampus expresses clear ambiguity-dependent responses that are associated with an augmented rate of learning. These findings suggest candidate neuronal systems that may be involved in aberrations of generalization, such as over-confidence., Author Summary Intelligent behavior requires flexible responses to new situations, which exploit learned principles or abstractions. When no such principles exist, the imperative is to learn quickly from scratch. Behaviorally, we show that subjects learn action-reward relationships in a manner that enables them to generalize rules to new situations. Our fMRI results show that when subjects have no evidence that such a rule exists, medial temporal lobe responses (that reflect uncertainty) predict their augmented learning.

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria., PLoS Pathogens, 8 (7), ISSN:1553-7374, ISSN:1553-7366

Auxotrophic markers - mutations within genes encoding enzymes in pathways for the biosynthesis of metabolic building blocks, such as an amino acid or nucleotide, are used as selection markers in the vast majority of yeast genetics and genomics experiments 1-3. The nutritional deficiency caused by the mutation (auxotrophy) can be compensated by supplying the required nutrient in the growth medium. This compensation, however, is not necessarily quantitative because such mutations influence a number of physiological parameters and may act in combination 2,4,5 . The construction of a prototrophic derivative of the parent strain of the widely used genome-scale yeast deletion collection1 has confirmed the need to remove auxotrophic markers in order to reduce bias in physiological and metabolic studies 2 . Moreover, flux balance analyses using a genome-wide metabolic model (Yeast 5)6 indicate that the activity status of some 200-300 reactions changes between different auxotrophic strains and the wild type. To alleviate this bias we have constructed a version of the haploid deletant collection restoring prototrophy in the genetic background, such that influence of auxotrophy to the phenotype of a given gene deletion is prevented. This new deletant library facilitates the exploitation of yeast in both functional genomics and quantitative systems biology. The physiological impact of auxotrophy was assessed by monitoring the growth of 16 yeast strains carrying all possible combinations of the markers (histidine (his3Δ1), leucine (leu2Δ), methionine (met15Δ) and uracil (ura3Δ) used in the MATa version of the yeast deletion collection1. All markers and their combinations affected yeast growth, but without altering the adenylate (ATP, ADP, AMP) energy charge (Fig 1 a). As the most critical phenotypic quantity, the maximum specific growth rate (μmax) varied between 0.125 h−1 (leu2Δ) and 0.20 h−1 (his3Δmet15Δura3Δ), rendering quantitative comparisons between these strains impossible (Fig 1 a, Suppl. Table 1). These growth differences were not explained by the different media supplementations, as i) prototrophic yeast exhibited a different and substantially less diverse growth pattern in the 16 minimal media (Fig 1c, left panel; media recipes are given in the Supplementary material); and ii) growth differences where altered, but not abrogated, when other proteogenic amino acids were supplemented as well (synthetic complete (SC) medium; Fig 1b). Importantly, on both types of media, complex interactions between all auxotrophic mutations were observed. For instance, restoring MET15 in leu2Δura3Δhis3Δmet15Δ (0.185 h−1 → 0.164 h−1) or leu2Δura3Δmet15Δ (0.162 h−1 → 0.149 h−1) had a negative effect on μmax, but surprisingly promoted growth in leu2Δhis3Δmet15Δ (0.136 h−1 → 0.173 h−1) (Fig 1a); restoring LEU2 in leu2Δura3Δhis3Δ (0.164 h−1 → 0.185 h−1) or leu2Δura3Δmet15Δ (0.136 h−1 → 0.161 h−1) had a positive effect, but not in leu2Δura3Δhis3Δmet15Δ (0.185 h−1 → 0.186 h−1) (Fig 1a; Suppl. Table 1). Thus, although blocking different pathways, all markers influence each other, indicating that they have a wide-ranging and combinatorial influence on the metabolic network. Figure 1 The combinatorial impact of yeast auxotrophic markers In batch culture experiments, further problems arise from the unequal consumption of amino acid supplements resulting in cultivation phase-dependent starvation. Growth of BY4741 (the auxotrophic parent of the standard yeast gene-deletion collection 1) in SC media depleted nutrients in a way they became first limiting for met15Δ, then for leu2Δ, his3Δ1 and finally for ura3Δ auxotrophic yeast (Fig 1d). This effect could not be compensated by increasing amino acid supplementation(s), as this inhibited cell growth (Fig 1c, right panel). Chronological lifespan (CLS) is a phenotype that is profoundly influenced by both nutrient supplementation and growth rate. Indeed, we observed an increase in stationary phase survival in YPD media upon restoring prototrophy. In a competitive growth experiment, auxotrophic cells had lost their colony-forming capacities within 10 days, but their prototrophic counterparts were perfectly viable for more than 20 days (Fig 1e). Longer CLS of prototrophic versus auxotrophic yeast was also reported for other backgrounds, and in synthetic media nutrient starvation shortened the lifespan of auxotrophic cells 7,8. Restoring protrotrophy is thus, to our knowledge, one of the most powerful genetic modifications for extending CLS. Hence, as auxotrophic markers have substantial and combinatorial influences on fundamental biological parameters such as growth and ageing, auxotrophic genome resources introduce bias for analyzing physiological parameters and even more to quantitative studies addressing the metabolic network. We would thus encourage the yeast community to switch, where possible, to prototrophic yeast for experiments in transcriptomics, proteomics, and metabolomics. To create a prototrophic resource for genome-scale experiments, we re-introduced auxotrophic markers into the MATa versions of the S288c-based deletion collection (5185 strains)1 and the titratable promoter essential collection (839 strains)3. These strains were transformed with a centromere-containing single-copy vector (minichromosome), containing the chromosome VI centromere, the autonomous replication sequence of HHF1 (ARSH4)9, and the marker genes HIS3, URA3, LEU2, and MET15 under control of their endogenous promoter sequences (pHLUM; Suppl Fig 1, Addgene ID 40276). Under non-selective conditions, the vector was transmitted in 99.15% of cell divisions (0.85% segregation mean over 20 generations). After 20 days, all cells were found prototrophic due to their positive selection (Fig 1e), facilitating screens on both selective and non-selective media. Furthermore, pHLUM- transformed BY4741 derivates wild type for HIS3, LEU2, MET15 or URA3 grew similar as BY4741 pHLUM (Suppl. Fig 2), indicating that the minichromosome fully restored prototrophy. The titratable-promoter essential collection3 was exploited to demonstrate screening capacities. By replicating original and prototrophic strains onto doxycycline-containing media, we found that 13 of the 370 lethal phenotypes were compensated (Fig 1 f, Suppl. Table 2). Thus, auxotrophic markers do not only influence physiological parameters, they are also responsible for a number of essential phenotypes. Since all strains possess a native metabolic network, the new library reduces bias from the use of auxotrophic markers in functional genomics and metabolic systems biology. Based on the pHLUM minichromosome, which is counterselectable, the new resource retains full compatibility with the popular S288c knock-out and essential collections 1,3. However, the use of a plasmid will introduce confounding factors to those mutants which have deficits in plasmid stability and segregation. The library is distributed as arrayed on 96-well plates (Euroscarf, Frankfurt), and contains a deep-red coloured and counter-selectable mutant (ade12Δ) on both universal and plate-specific positions, which simplifies plate orientation and identification, and can serve as replicate-control in quantitative metabolomics experiments (Suppl. Fig 3).

RNA viruses exist as genetically diverse populations displaying different phenotypes, including diverse degrees of virulence. The evolution of virulence in viral populations is, however, poorly understood. Based on the experimental observation of an RNA virus clone in cell culture diversifying into two subpopulations of different virulence, we study the dynamics of heterogeneous virus populations and the evolution of virulence. We introduce a competition-colonization trade-off into standard mathematical models of intra-host viral infection. Colonizers are fast spreading, virulent strains, whereas competitors are less virulent variants that are more successful within coinfected cells. We observe biphasic dynamics of the population: Early in the infection the population is dominated by colonizers, which later will be outcompeted by competitors. The simulations suggest the existence of a steady state where few low-virulence variants coexist. This equilibrium implies collective virulence attenuation in the population, in contrast to previous models predicting development of the population towards increased virulence. Nevertheless, the attenuation effect disappears if we include a highly simplified immune response in our models. Thus, the competition-colonization trade-off indicates a role for virulence in the modulation of the viral population diversity. The evolution of virulence is a dynamic feature of the population shaped by interactions between individuals and by the structure of the patchy habitat., Comment: This paper has been withdrawn by the authors. 17 pages, 5 figures. Submitted to journal, currently under review. Supplementary material can be obtained from the corresponding author upon request via email. Withdrawn due to issues with the journal we submitted the manuscript to

We have developed a single-cell assay platform that allows quantitative analysis of single cell chemotaxis by dynamic morphogenetic gradients, subcellular microscopic imaging and automated image analysis, and have applied these to measure cellular polarization of budding yeast. The computer-controlled microfluidic device regulates the gradient profile at any given time, and allows quantitative monitoring of cell morphology and the localization and expression of specific marker proteins during the dynamic polarization process. With this integrated experimental system, we compare the polarized signaling response of wild-type and far1-H7 mutant cells, which express a truncated Far1 protein unable to interact with Cdc24. Our results confirm that Far1 functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling. Moreover, by changing the gradient profile and estimating the number of bound surface receptors, we quantitatively address why surprisingly small differences in pheromone concentration across yeast cells can be amplified into a robust polarity axis. This integrated single cell experimental platform thus opens the possibility to quantitatively investigate the molecular regulatory mechanism of chemotaxis in yeast, which serves as a paradigm to understand the fundamental processes involved in cancer metastasis, angiogenesis and axon generation.