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The plant hormone auxin is fundamental for plant growth, and its spatial distribution in plant tissues is critical for plant morphogenesis. We consider a leading model of the polar auxin flux, and study in full detail the stability of the possible equilibrium configurations. We show that the critical states of the auxin transport process are composed of basic building blocks, which are isolated in a background of auxin depleted cells, and are not geometrically regular in general. The same model was considered recently through a continuous limit and a coupling to the von Karman equations, to model the interplay of biochemistry and mechanics during plant growth. Our conclusions might be of interest in this setting, since, for example, we establish the existence of Lyapunov functions for the auxin flux, proving in this way the convergence of pure transport processes toward the set of critical configurations., Comment: 27 pages

International Journal of Computer Vision, 140 (1), ISSN:0920-5691, ISSN:1573-1405

Although changes in chromatin are integral to transcriptional reprogramming during cellular differentiation, it is currently unclear how chromatin modifications are targeted to specific loci. To systematically identify transcription factors (TFs) that can direct chromatin changes during cell fate decisions, we model the relationship between genome-wide dynamics of chromatin marks and the local occurrence of computationally predicted TF binding sites. By applying this computational approach to a time course of Polycomb-mediated H3K27me3 marks during neuronal differentiation of murine stem cells, we identify several motifs that likely regulate the dynamics of this chromatin mark. Among these, the sites bound by REST and by the SNAIL family of TFs are predicted to transiently recruit H3K27me3 in neuronal progenitors. We validate these predictions experimentally and show that absence of REST indeed causes loss of H3K27me3 at target promoters in trans, specifically at the neuronal progenitor state. Moreover, using targeted transgenic insertion, we show that promoter fragments containing REST or SNAIL binding sites are sufficient to recruit H3K27me3 in cis, while deletion of these sites results in loss of H3K27me3. These findings illustrate that the occurrence of TF binding sites can determine chromatin dynamics. Local determination of Polycomb activity by REST and SNAIL motifs exemplifies such TF based regulation of chromatin. Furthermore, our results show that key TFs can be identified ab initio through computational modeling of epigenome data sets using a modeling approach that we make readily accessible.

In the mammalian neocortex, segregated processing streams are thought to be important for forming sensory representations of the environment(1,2), but how local information in primary sensory cortex is transmitted to other distant cortical areas during behaviour is unclear. Here we show task-dependent activation of distinct, largely non-overlapping long-range projection neurons in the whisker region of primary somatosensory cortex (S1) in awake, behaving mice. Using two-photon calcium imaging, we monitored neuronal activity in anatomically identified S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice using their whiskers to perform a texture-discrimination task or a task that required them to detect the presence of an object at a certain location. Whisking-related cells were found among S2-projecting (S2P) but not M1-projecting (M1P) neurons. A higher fraction of S2P than M1P neurons showed touch-related responses during texture discrimination, whereas a higher fraction of M1P than S2P neurons showed touch-related responses during the detection task. In both tasks, S2P and M1P neurons could discriminate similarly between trials producing different behavioural decisions. However, in trials producing the same decision, S2P neurons performed better at discriminating texture, whereas M1P neurons were better at discriminating location. Sensory stimulus features alone were not sufficient to elicit these differences, suggesting that selective transmission of S1 information to S2 and M1 is driven by behaviour.

Mapping gene regulatory networks is a significant challenge in systems biology, yet only a few methods are currently capable of systems-level identification of transcription factors (TFs) that bind a specific regulatory element. We developed a microfluidic method for integrated systems-level interaction mapping of TF-DNA interactions, generating and interrogating an array of 423 full-length Drosophila TFs. With integrated systems-level interaction mapping, it is now possible to rapidly and quantitatively map gene regulatory networks of higher eukaryotes.

During vertebrate embryogenesis, the rhythmic and sequential segmentation of the body axis is regulated by an oscillating genetic network termed the segmentation clock. We describe a new dynamic model for the core pace-making circuit of the zebrafish segmentation clock based on a systematic biochemical investigation of the network's topology and precise measurements of somitogenesis dynamics in novel genetic mutants. We show that the core pace-making circuit consists of two distinct negative feedback loops, one with Her1 homodimers and the other with Her7:Hes6 heterodimers, operating in parallel. To explain the observed single and double mutant phenotypes of her1, her7, and hes6 mutant embryos in our dynamic model, we postulate that the availability and effective stability of the dimers with DNA binding activity is controlled in a "dimer cloud" that contains all possible dimeric combinations between the three factors. This feature of our model predicts that Hes6 protein levels should oscillate despite constant hes6 mRNA production, which we confirm experimentally using novel Hes6 antibodies. The control of the circuit's dynamics by a population of dimers with and without DNA binding activity is a new principle for the segmentation clock and may be relevant to other biological clocks and transcriptional regulatory networks. © 2012 Schröter et al. Fil: Schroter, Christian. University of Cambridge; Estados Unidos Fil: Ares, Saúl. Max Planck Institute For The Physics Of Complex Systems; Alemania Fil: Morelli, Luis Guillermo. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Isakova, Alina. Ecole Polytechnique Federale de Lausanne; Francia Fil: Hens, Korneel. Ecole Polytechnique Federale de Lausanne; Francia Fil: Soroldoni, Daniele. Max Planck Institute Of Molecular Cell Biology And Genetics; Alemania Fil: Gajewski, Martin. Universitat zu Köln; Alemania Fil: Jülicher, Frank. Max Planck Institute For The Physics Of Complex Systems; Alemania Fil: Maerkl, Sebastian J.. Ecole Polytechnique Federale de Lausanne; Francia Fil: Deplancke, Bart. Ecole Polytechnique Federale de Lausanne; Francia Fil: Oates, Andrew C.. Max Planck Institute Of Molecular Cell Biology And Genetics; Alemania

Understanding synaptic connectivity and plasticity within brain circuits and their relationship to learning and behavior is a fundamental quest in neuroscience. Visualizing the fine details of synapses using optical microscopy remains however a major technical challenge. Super resolution microscopy opens the possibility to reveal molecular features of synapses beyond the diffraction limit. With direct stochastic optical reconstruction microscopy, dSTORM, we image synaptic proteins in the brain tissue of the fruit fly, Drosophila melanogaster. Super resolution imaging of brain tissue harbors difficulties due to light scattering and the density of signals. In order to reduce out of focus signal, we take advantage of the genetic tools available in the Drosophila and have fluorescently tagged synaptic proteins expressed in only a small number of neurons. These neurons form synapses within the calyx of the mushroom body, a distinct brain region involved in associative memory formation. Our results show that super resolution microscopy, in combination with genetically labeled synaptic proteins, is a powerful tool to investigate synapses in a quantitative fashion providing an entry point for studies on synaptic plasticity during learning and memory formation.

With a targeted proteomic approach, we could quantify 90% of the enzymes involved in carbon and amino-acid metabolism in yeast, including complete isoenzyme families, throughout a set of different metabolic states. The data, interpreted through flux balance modeling, indicate that S. cerevisiae expresses enzymes, not necessarily used in a particular metabolic condition. For many isoenzymes our data suggest functional diversification, which might explain their parallel presence in the S. cerevisiae genome., The metabolic network in the yeast Saccharomyces cerevisiae has been very well characterized in terms of components and topology. The adaptation of metabolism to changing nutritional conditions, in contrast, is much less well understood. In this study, we exploited quantitative proteomic assays based on selected reaction monitoring (SRM) mass spectrometry to comprehensively analyze the set of enzymes involved in carbon and amino-acid metabolism in yeast (Figure 1), throughout a set of different metabolic states. To elucidate how this metabolic network of proteins adapts to environmental challenges, we chose five nutritional conditions resulting in maximal difference in magnitude and direction of metabolic fluxes. We could reproducibly detect and quantify across the different conditions, 90% of the targeted metabolic proteome, including complete families of isoenzymes, sharing up to 99.5% sequence identity and multi-subunit enzyme complexes. This yielded an information-rich proteomic data set that represents a nutritionally perturbed biological system with high coverage of its components. Interpreted through flux balance modeling, the data indicate that S. cerevisiae expresses—at least at a basal level—more proteins than are actually necessary for sustaining a given metabolic condition. One potential explanation for the presence of non-necessary proteins is that these enzymes could realize immediate basal metabolic fluxes upon a change to new environmental conditions. Next, we asked whether our data set could contribute to unravel the function of isoenzymes in the metabolic set. Previously proposed roles for isoenzymes include redundancy to buffer against mutations, a means to gene dosage or facilitation of evolutionary innovation and functional diversification. To address the role of isoenzymes in central metabolism, we used hierarchical clustering to analyze the abundance patterns of the metabolic proteins and their relationship to different functional classes and metabolic pathways. Interestingly, while subunits of the same protein complex preferably cluster in proximate branches, members of the same isoenzyme family often clustered in distant branches (Figure 5). The data therefore suggested functional diversification within most isoenzyme families and allowed to propose different functions of divergent isoenzymes. We expect that the comprehensive data set presented in this study will be an ideal blueprint for further developing models of yeast metabolism and for follow-up studies on the function of target metabolic proteins., Decades of biochemical research have identified most of the enzymes that catalyze metabolic reactions in the yeast Saccharomyces cerevisiae. The adaptation of metabolism to changing nutritional conditions, in contrast, is much less well understood. As an important stepping stone toward such understanding, we exploit the power of proteomics assays based on selected reaction monitoring (SRM) mass spectrometry to quantify abundance changes of the 228 proteins that constitute the central carbon and amino-acid metabolic network in the yeast Saccharomyces cerevisiae, at five different metabolic steady states. Overall, 90% of the targeted proteins, including families of isoenzymes, were consistently detected and quantified in each sample, generating a proteomic data set that represents a nutritionally perturbed biological system at high reproducibility. The data set is near comprehensive because we detect 95–99% of all proteins that are required under a given condition. Interpreted through flux balance modeling, the data indicate that S. cerevisiae retains proteins not necessarily used in a particular environment. Further, the data suggest differential functionality for several metabolic isoenzymes.

We performed microsecond long coarse-grained molecular dynamics simulations to elucidate the lateral structure and domain dynamics of a phospha- tidylethanolamine (PE)/phosphatidylglycerol (PG) mixed bilayer (7/3), mimicking the inner membrane of gram-negative bacteria. Specifically, we address the effect of surface bound antimicrobial peptides (AMPs) on the lateral organization of the membrane.Wefindthat, intheabsenceofthe peptides,theminor PGfractiononly forms small clusters, but that these clusters grow in size upon binding of the cationic AMPs. The presence of AMPs systematically affects the dynamics and induces long-range order in the structure of PG domains, stabilizing the separation between the two lipid fractions. Our results help in understanding the initial stages of destabilization of cytoplasmic bacterial membranes below the critical peptide concentration necessary for disruption, and provide a possible explanation for the multimodal character of AMP activity. SECTION Surfactants, Membranes

In yeast TOR and PKA pathways both control cell growth but how TORC1 and PKA signaling are linked is unknown. Here we show that TORC1 inhibition prevents the phosphorylation of some but not all PKA targets. We further demonstrate that TORC1 controls PKA by inhibiting the phosphorylation of the PKA regulatory subunit BCY1 by the MAP kinase MPK1., Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogenesis, translation, nutrient metabolism, and autophagy. In yeast, the protein kinases Target of Rapamycin (TOR) and protein kinase A (PKA) regulate these processes and are thereby the main activators of cell growth in response to nutrients. How TOR, PKA, and their corresponding signaling pathways are coordinated to control the same cellular processes is not understood. Quantitative analysis of the rapamycin-sensitive phosphoproteome combined with targeted analysis of PKA substrates suggests that TOR complex 1 (TORC1) activates PKA but only toward a subset of substrates. Furthermore, we show that TORC1 signaling impinges on BCY1, the negative regulatory subunit of PKA. Inhibition of TORC1 with rapamycin leads to BCY1 phosphorylation on several sites including T129. Phosphorylation of BCY1 T129 results in BCY1 activation and inhibition of PKA. TORC1 inhibits BCY1 T129 phosphorylation by phosphorylating and activating the S6K homolog SCH9 that in turn inhibits the MAP kinase MPK1. MPK1 phosphorylates BCY1 T129 directly. Thus, TORC1 activates PKA toward some substrates by preventing MPK1-mediated activation of BCY1.

In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant (mex1), which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme (DPE1) results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation.

Functional integration in the brain rests on anatomical connectivity (the presence of axonal connections) and effective connectivity (the causal influences mediated by these connections). The deployment of anatomical connections provides important constraints on effective connectivity, but does not fully determine it, because synaptic connections can be expressed functionally in a dynamic and context-dependent fashion. Although it is generally assumed that anatomical connectivity data is important to guide the construction of neurobiologically realistic models of effective connectivity; the degree to which these models actually profit from anatomical constraints has not yet been formally investigated. Here, we use diffusion weighted imaging and probabilistic tractography to specify anatomically informed priors for dynamic causal models (DCMs) of fMRI data. We constructed 64 alternative DCMs, which embodied different mappings between the probability of an anatomical connection and the prior variance of the corresponding of effective connectivity, and fitted them to empirical fMRI data from 12 healthy subjects. Using Bayesian model selection, we show that the best model is one in which anatomical probability increases the prior variance of effective connectivity parameters in a nonlinear and monotonic (sigmoidal) fashion. This means that the higher the likelihood that a given connection exists anatomically, the larger one should set the prior variance of the corresponding coupling parameter; hence making it easier for the parameter to deviate from zero and represent a strong effective connection. To our knowledge, this study provides the first formal evidence that probabilistic knowledge of anatomical connectivity can improve models of functional integration.

Pollen tubes are used as a model in the study of plant morphogenesis, cellular differentiation, cell wall biochemistry, biomechanics, and intra- and intercellular signaling. For a “systems-understanding” of the bio-chemo-mechanics of tip-polarized growth in pollen tubes, the need for a versatile, experimental assay platform for quantitative data collection and analysis is critical. We introduce a Lab-on-a-Chip (LoC) concept for high-throughput pollen germination and pollen tube guidance for parallelized optical and mechanical measurements. The LoC localizes a large number of growing pollen tubes on a single plane of focus with unidirectional tip-growth, enabling high-resolution quantitative microscopy. This species-independent LoC platform can be integrated with micro-/nano-indentation systems, such as the cellular force microscope (CFM) or the atomic force microscope (AFM), allowing for rapid measurements of cell wall stiffness of growing tubes. As a demonstrative example, we show the growth and directional guidance of hundreds of lily (Lilium longiflorum) and Arabidopsis (Arabidopsis thaliana) pollen tubes on a single LoC microscopy slide. Combining the LoC with the CFM, we characterized the cell wall stiffness of lily pollen tubes. Using the stiffness statistics and finite-element-method (FEM)-based approaches, we computed an effective range of the linear elastic moduli of the cell wall spanning the variability space of physiological parameters including internal turgor, cell wall thickness, and tube diameter. We propose the LoC device as a versatile and high-throughput phenomics platform for plant reproductive and development biology using the pollen tube as a model., PLoS ONE, 11 (12), ISSN:1932-6203

Computational Mechanics, 56 (5), ISSN:0178-7675, ISSN:1432-0924

Uneven illumination affects every image acquired by a microscope. It is often overlooked, but it can introduce considerable bias to image measurements. The most reliable correction methods require special reference images, and retrospective alternatives do not fully model the correction process. Our approach overcomes these issues for most optical microscopy applications without the need for reference images.

Bacteria are thought to cope with fluctuating environmental solute concentrations primarily by adjusting the osmolality of their cytoplasm. To obtain insights into the underlying metabolic adaptations, we analyzed the global metabolic response of Escherichia coli to sustained hyperosmotic stress using nontargeted mass spectrometry. We observed that 52% of 1,071 detected metabolites, including known osmoprotectants, changed abundance with increasing salt challenge. Unexpectedly, unsupervised data analysis showed a substantial increase of most intermediates in the ubiquinone-8 (Q8) biosynthesis pathway and a 110-fold accumulation of Q8 itself, as confirmed by quantitative lipidomics. We then demonstrated that Q8 is necessary for acute and sustained osmotic-stress tolerance using Q8 mutants and tolerance rescue through feeding nonrespiratory Q8 analogs. Finally, in vitro experiments with artificial liposomes showed that mechanical membrane stabilization is a principal mechanism of Q8-mediated osmoprotection. Thus, we find that besides regulating intracellular osmolality, E. coli enhances its cytoplasmic membrane stability to withstand osmotic stress.

Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments are widely used to determine, within entire genomes, the occupancy sites of any protein of interest, including, for example, transcription factors, RNA polymerases, or histones with or without various modifications. In addition to allowing the determination of occupancy sites within one cell type and under one condition, this method allows, in principle, the establishment and comparison of occupancy maps in various cell types, tissues, and conditions. Such comparisons require, however, that samples be normalized. Widely used normalization methods that include a quantile normalization step perform well when factor occupancy varies at a subset of sites, but may miss uniform genome-wide increases or decreases in site occupancy. We describe a spike adjustment procedure (SAP) that, unlike commonly used normalization methods intervening at the analysis stage, entails an experimental step prior to immunoprecipitation. A constant, low amount from a single batch of chromatin of a foreign genome is added to the experimental chromatin. This “spike” chromatin then serves as an internal control to which the experimental signals can be adjusted. We show that the method improves similarity between replicates and reveals biological differences including global and largely uniform changes.

Splicing reactions generally combine high speed with accuracy. However, some of the pre-mRNAs escape the nucleus with a retained intron. Intron retention can control gene expression and increase proteome diversity. We calculated the escape rate for the yeast PTC7 intron and pre-mRNA. This prediction was facilitated by the observation that splicing is a linear process and by deriving simple algebraic expressions from a model of co- and post-transcriptional splicing and RNA surveillance that determines the rate of the nonsense-mediated decay (NMD) of the pre-mRNAs with the retained intron. The escape rate was consistent with the observed threshold of splicing rate below which the mature mRNA level declined. When an mRNA contains multiple introns, the outcome of splicing becomes more difficult to predict since not only the escape rate of the pre-mRNA has to be considered, but also the possibility that the splicing of each intron is influenced by the others. We showed that the two adjacent introns in the SUS1 mRNA are spliced cooperatively, but this does not counteract the escape of the partially spliced mRNA. These findings will help to infer promoter activity and to predict the behavior of and to control splicing regulatory networks.

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.

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.

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.

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

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.

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.

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.

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.

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.