Frommers California 2008 (Frommers Complete)

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Travel experts Arthur Frommer and daughter Pauline Frommer provide the most immediate deals, reliable reviews and unusual destinations for savvy travelers. His books, articles and speeches have profoundly changed the travel habits of Americans and the products of the travel industry. Like Spock, he wrote with the authority of experience; like Kinsey, he opened up a whole new world. And while the work of those two men has been eclipsed by others, as well as by changing times and fashions, Frommer remains a powerful, and indefatigable, force in his field.

Pauline Frommer started traveling before she could speak, seeing the world at a young age with her guidebook-writing parents, Arthur Frommer and Hope Arthur. Following that, she returned to her roots, creating the Pauline Frommer Guidebooks , an award-winning series aimed at adult budget travelers. For the past three years, Pauline has hosted a nationally syndicated radio show on travel, now heard on more than stations. Currently, Pauline works with Weight Watchers, contributing a daily blog on staying fit on the road.

Pauline is blessed to be married to physical therapist Mahlon Stewart, and is the mother of two very well-traveled daughters, Beatrix and Veronica. Knowledge of the in vivo levels, distribution and flux of ions and metabolites is crucial to our understanding of physiology in both healthy and diseased states. The quantitative analysis of the dynamics of ions and metabolites with subcellular resolution in vivo poses a major challenge for the analysis of metabolic processes. FRET sensor proteins, for example, for glucose, can be targeted genetically to any cellular compartment, or even to subdomains e.

The sensors can be used for analyses in individual mammalian cells in culture, in tissue slices and in intact organisms. Applications include gene discovery, high-throughput drug screens or systematic analysis of regulatory networks affecting uptake, efflux and metabolism. Quantitative analyses obtained with the help of FRET sensors for glucose or other ions and metabolites provide valuable data for modeling of flux.

Here we provide a detailed protocol for monitoring glucose levels in the cytosol of mammalian cell cultures through the use of FRET glucose sensors; moreover, the protocol can be used for other ions and metabolites and for analyses in other organisms, as has been successfully demonstrated in bacteria, yeast and even intact plants. Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. FRET glucose sensors with different glucose affinities K d covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments.

Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a well plate using a fluorimeter. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup.

By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in View details for DOI Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales.

Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution.

In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation. We report here the genome sequence of the lycophyte Selaginella moellendorffii Selaginella , the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages.

Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes. Quantitative and dynamic analysis of metabolites and signalling molecules is limited by technical challenges in obtaining temporally resolved information at the cellular and compartmental level. FRET nanosensors represent powerful tools for gene discovery, and analysis of regulatory networks, for example by screening mutants.

This drawback was overcome here by expressing the nanosensors in RNA silencing mutants. However, the use of silencing mutants requires the generation of homozygous lines deficient in RNA silencing as well as the mutation of interest and co-expression of the nanosensor. Here it is shown that dynamic changes in cytosolic glucose levels can readily be quantified in wild-type Arabidopsis plants at early stages of development d before silencing had a major effect on fluorescence intensity.

A detailed protocol for screening mutant seedlings per day is provided. The detailed imaging protocol provided here is suitable for analysing sugar flux in young wild-type plants as well as mutants affected in sugar signalling, metabolism, or transport using a wide spectrum of FRET nanosensors. AMTs form trimers and use intermolecular interaction between subunits to regulate activity. So far, binding forces that stabilize AMT protein complexes are not well characterized.

High temperature or reducing agents released mono- and dimeric forms from trimeric complexes formed by AMT1;1 from Arabidopsis and tomato.

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However, in the paralogue LeAMT1;3, trimeric complexes were not detected. Truncation of the N-terminus in LeAMT1;1 destabilized the trimer and led to loss of functionality when expressed in yeast. C3S or C27S variants of LeAMT1;1 showed reduced complex stability, which coincided with lower transport capacity for the substrate analogue methylammonium. Both cysteine-substituted LeAMT1;1 variants showed weaker interactions with the wildtype as determined by a quantitative analysis of the complex stability using the mating-based split-ubiquitin assay. These data indicate that the binding affinity of AMT1 subunits is stabilized by cysteines in the N-terminus and suggest a role for disulphide bridge formation via apoplastic N-terminal cysteine residues.

Precise and dynamic measurement of intracellular metabolite levels has been hampered by difficulties in differentiating between adsorbed and imported fractions and the subcellular distribution between cytosol, endomembrane compartments and mitochondria. Moreover, by using the FRET sensors in a microfluidic platform, we were able to monitor in vivo changes of intracellular free glucose in individual yeast cells.

Sugar efflux transporters are essential for the maintenance of animal blood glucose levels, plant nectar production, and plant seed and pollen development. Despite broad biological importance, the identity of sugar efflux transporters has remained elusive. Using optical glucose sensors, we identified a new class of sugar transporters, named SWEETs, and show that at least six out of seventeen Arabidopsis, two out of over twenty rice and two out of seven homologues in Caenorhabditis elegans, and the single copy human protein, mediate glucose transport.

Bacterial symbionts and fungal and bacterial pathogens induce the expression of different SWEET genes, indicating that the sugar efflux function of SWEET transporters is probably targeted by pathogens and symbionts for nutritional gain. The metazoan homologues may be involved in sugar efflux from intestinal, liver, epididymis and mammary cells. Although biochemical studies suggested a high permeability of the endoplasmic reticulum ER membrane for small molecules, proteomics identified few specialized ER transporters.

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HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEKT cells to take up glucose across the plasma membrane correlated with low ER transport. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport.

Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability. AMT1;1 exists in active and inactive states, probably controlled by the spatial positioning of the C-terminus. Ammonium triggers the phosphorylation of a conserved threonine residue T in the C-terminus of AMT1;1 in a time- and concentration-dependent manner.

The T phosphorylation level correlates with a decrease of root ammonium uptake. We propose that ammonium-induced phosphorylation modulates ammonium uptake as a general mechanism to protect against ammonium toxicity. Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 GDU1 has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap.

In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gduD. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gduD compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system.

Each of the seven Arabidopsis Arabidopsis thaliana GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism.

Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis. The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels.

The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr T in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T leads to inactivation of the trimeric complex. This study shows that phosphorylation of T is triggered by ammonium in a time- and concentration-dependent manner.

Neither Gln nor l-methionine sulfoximine-induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots.

Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor i. The Arabidopsis AMT1 proteins mediate uptake of the ionic form of ammonium. AMT transport activity is controlled allosterically via a highly conserved cytosolic C terminus that interacts with neighboring subunits in a trimer.

The C terminus is thus capable of modulating the conductivity of the pore.

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To gain insight into the underlying mechanism, pore mutants suppressing the inhibitory effect of mutations in the C-terminal trans-activation domain were characterized. To explore whether the transport mechanism was altered, the AMT1;1-Q57H mutant was expressed in Xenopus oocytes and analyzed electrophysiologically. AMT1;1-Q57H was characterized by increased ammonium-induced and reduced methylammonium-induced currents. AMT1;1-Q57H possesses a x lower affinity for ammonium K m and a fold higher V max as compared with the wild type form.

To test whether the trans-regulatory mechanism is conserved in archaeal homologs, AfAmt-2 from Archaeoglobus fulgidus was expressed in yeast. The transport function of AfAmt-2 also depends on trans-activation by the C terminus, and mutations in pore-residues corresponding to Q57H of AMT1;1 suppress nonfunctional AfAmt-2 mutants lacking the activating C terminus. Altogether, our data suggest that bacterial and plant AMTs use a conserved allosteric mechanism to control ammonium flux, potentially using a gating mechanism that limits flux to protect against ammonium toxicity.

Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. HEKT cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels.

Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEKT cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced.

Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity. Hormones typically serve as long distance signaling molecules. To reach their site of action, hormones need to be transported from the sites of synthesis.

Many plant hormones are mobile, thus requiring specific transport systems for the export from their source cells as well as subsequent import into target cells. Hormone transport in general is still poorly understood. Auxin is probably the most intensively studied plant hormone concerning transport in the moment. To advance our understanding of hormone transport we need two principal data sets: information on the properties of the transport systems including substrate specificity and kinetics, and we need to identify candidate genes for the respective transporters.

Physiological transport data can provide an important basis for identifying and characterizing candidate transporters and to define their in vivo role. A recent publication in Plant Physiology highlights how kinetic and specificity studies may help to identify cytokinin transporters. Fluorescent proteins have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. While the most common applications of fluorescent proteins are to image expression, localization, and dynamics of protein chimeras, there is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells.

This tutorial review provides an overview of the progress made in the development of fluorescent protein-based biosensors to date. The recent development of analytic technologies allows fast analysis of metabolism in real time. Fluxomics aims to define the genes involved in regulation of flux through a metabolic or signaling pathway.

Flux through a metabolic or signaling pathway is determined by the activity of its individual components; regulation can occur at many levels, including transcriptional, posttranslational, and allosteric levels. Currently two technologies are used to monitor fluxes. The first is pulse labeling of the organism with a tracer such as C13, followed by mass spectrometric analysis of the partitioning of label into different compounds.

The second approach is based on the use of flux sensors, proteins that respond with a conformational change to ligand binding. Fluorescence resonance energy transfer FRET detects the conformational change and serves as a proxy for ligand concentration.

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Both methods provide high time resolution. In contrast to mass spectrometry assays, FRET nanosensors monitor only a single compound, but the advantage of FRET nanosensors is that they yield data with cellular and subcellular resolution. In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function.

We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 MCC. However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins.

Although soil contains only traces of soluble carbohydrates, plant roots take up glucose and sucrose efficiently when supplied in artificial media. Soluble carbohydrates and other small metabolites found in soil are in part products from exudation from plant roots. The molecular nature of the transporters for uptake and exudation is unknown. Here, fluorescence resonance energy transfer FRET glucose and sucrose sensors were used to characterize accumulation and elimination of glucose and sucrose in Arabidopsis roots tips. Using an improved image acquisition set-up, FRET responses to perfusion with carbohydrates were detectable in roots within less than 10 sec and over a wide concentration range.

The rate of elimination was unaffected by pre-incubation with high concentrations of glucose, suggesting that elimination is not due to accumulation in a short-term buffer such as the vacuole. Glucose and sucrose accumulation was insensitive to protonophores, was comparable in media differing in potassium levels, and was similar at pH 5. The root expression maps may help to pinpoint candidate genes for uptake and release of carbohydrates from roots. Cytokinins are distributed through the vascular system and trigger responses of target cells via receptor-mediated signal transduction.

Perception and transduction of the signal can occur at the plasma membrane or in the cytosol. The signal is terminated by the action of extra- or intracellular cytokinin oxidases. While radiotracer studies have been used to study transport and metabolism of cytokinins in plants, little is known about the kinetic properties of cytokinin transport. To provide a reference dataset, radiolabeled trans-zeatin tZ was used for uptake studies in Arabidopsis Arabidopsis thaliana cell culture. Uptake kinetics of tZ are multiphasic, indicating the presence of both low- and high-affinity transport systems.

Other physiological cytokinins, such as isopentenyl adenine and benzylaminopurine, are effective competitors of tZ uptake, whereas allantoin has no inhibitory effect. Adenine competes for zeatin uptake, indicating that the degradation product of cytokinin oxidases is transported by the same systems. Comparison of adenine and tZ uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties, as well as substrate specificity determined in cell cultures, are compatible with the hypothesis that members of the plant-specific purine permease family play a role in adenine transport for scavenging extracellular adenine and may, in addition, be involved in low-affinity cytokinin uptake.

In solanaceous plants such as tomato and tobacco, the sucrose transporter SUT1 is crucial for phloem loading. Using GUS as a reporter, the promoter and other regulatory cis elements required for the tomato LeSUT1 expression were analyzed by heterologous expression of translational chimeric constructs in tobacco. In GUS-positive transformants, expression levels were low and limited to leaf phloem. Increasing or decreasing the length of LeSUT1 promoter did not lead to a significant increase in positive transformants or higher expression levels.

LeSUT1 expression was detected in ab- and adaxial phloem companion cells, trichomes and guard cells. Results showed remarkable functions for the three introns for SUT1 expression in trichomes, guard cells and phloem cells. Intron 3 is responsible for expression in trichomes, whereas intron 2 is necessary for expression in companion cells and guard cells.

The combination of all introns is required for the full expression pattern in phloem, guard cells and trichomes. Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays.

Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. An Escherichia coli expression vector compatible with phage lambda recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose.

In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates.

The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation.

However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a fold higher ratio change and dynamic range 0.

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GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens.

The neurotransmitter glutamate is the mediator of excitatory neurotransmission in the brain. We have developed a novel technique to visualize glutamate release in brain slices using three purified fluorescence Forster energy resonance transfer FRET -based glutamate sensor proteins.

Using a simple loading protocol, the FRET sensor proteins diffuse deeply into the extracellular space and remain functional for many tens of minutes. This allows imaging of glutamate release in brain slices with simultaneous electrophysiological recordings and provides temporal and spatial resolution not previously possible. Using this glutamate FRET sensor loading and imaging protocol, we show that changes in network excitability and glutamate re-uptake alter evoked glutamate transients and produce correlated changes in evoked-cortical field potentials.

Given the sophisticated advantages of brain slices for electrophysiological and imaging protocols, the ability to perform real-time imaging of glutamate in slices should lead to key insights in brain function relevant to plasticity, development and pathology. This technique also provides a unique assay of network activity that compliments alternative techniques such as voltage-sensitive dyes and multi-electrode arrays.

Homotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies.

Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics.

In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen ; this issue, pp.

In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces. Phosphate sensors were developed by embedding fluorescent reporter proteins FLIPPi in polyacrylamide nanoparticles with diameters from 40 to nm. The experimental results prove that the cross-linked polyacrylamide matrix could protect FLIPPi from degradation by soluble proteases to some extent. This nanoparticle embedding method provides a novel promising tool for in vivo metabolite studies.

It also demonstrates a universal method for embedding different fragile bioactive elements, such as antibodies, genes, enzymes, and other functional proteins, in nanoparticles for, for example, sensing, biological catalysis, and gene delivery. Little is known about regulatory networks that control metabolic flux in plant cells. Detailed understanding of regulation is crucial for synthetic biology.

The difficulty of measuring metabolites with cellular and subcellular precision is a major roadblock. New tools have been developed for monitoring extracellular, cytosolic, organellar and vacuolar ion and metabolite concentrations with a time resolution of milliseconds to hours. Genetically encoded sensors allow quantitative measurement of steady-state concentrations of ions, signaling molecules and metabolites and their respective changes over time. Fluorescence resonance energy transfer FRET sensors exploit conformational changes in polypeptides as a proxy for analyte concentrations.

Subtle effects of analyte binding on the conformation of the recognition element are translated into a FRET change between two fused green fluorescent protein GFP variants, enabling simple monitoring of analyte concentrations using fluorimetry or fluorescence microscopy. Fluorimetry provides information averaged over cell populations, while microscopy detects differences between cells or populations of cells.

The genetically encoded sensors can be targeted to subcellular compartments or the cell surface. Confocal microscopy ultimately permits observation of gradients or local differences within a compartment. The FRET assays can be adapted to high-throughput analysis to screen mutant populations in order to systematically identify signaling networks that control individual steps in metabolic flux.

Mammalian cells rely on cellular uptake of the essential amino acid tryptophan. Tryptophan sequestration by up-regulation of the key enzyme for tryptophan degradation, indoleamine 2,3-dioxygenase IDO , e. Additionally, the excreted tryptophan catabolites kynurenines induce apoptosis of lymphocytes. Whereas tryptophan transport systems have been identified, the molecular nature of kynurenine export remains unknown.

To measure cytosolic tryptophan steady-state levels and flux in real time, we developed genetically encoded fluorescence resonance energy transfer nanosensors FLIPW. The strict coupling protects cells that overproduce IDO from kynurenine accumulation. Consequently, this mechanism may contribute to immunosuppression involved in autoimmunity and tumor immune escape. Sucrose is the main product of photosynthesis and the most common transport form of carbon in plants.

In addition, sucrose is a compound that serves as a signal affecting metabolic flux and development. Here we provide first results of externally induced phosphorylation changes of plasma membrane proteins in Arabidopsis. In an unbiased approach, seedlings were grown in liquid medium with sucrose and then depleted of carbon before sucrose was resupplied.

Plasma membranes were purified, and phosphopeptides were enriched and subsequently analyzed quantitatively by mass spectrometry. In total, 67 phosphopeptides were identified, most of which were quantified over five time points of sucrose resupply.

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This corresponded to a significant increase of proton pumping activity of plasma membrane vesicles from sucrose-supplied seedlings. This phosphorylation site was shown to be crucial for ATPase activity and overrode regulation via the well known C-terminal phosphorylation site. Novel phosphorylation sites were identified for both receptor kinases and cytosolic kinases that showed rapid increases in relative intensities after short times of sucrose treatment.

Seven response classes were identified including non-responsive, rapid increase within 3 min , slow increase, and rapid decrease. Relative quantification of phosphorylation changes by phosphoproteomics provides a means for identification of fast responses to external stimuli in plants as a basis for further functional characterization.

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  4. The recent development of analytic high-throughput technologies enables us to take a bird's view of how metabolism is regulated in real time. We have known for a long time that metabolism is highly regulated at all levels, including transcriptional, posttranslational and allosteric controls. Flux through a metabolic or signaling pathway is determined by the activity of its individual components.

    Fluxomics aims to define the genes involved in regulation by following the flux. Two technologies are used to monitor fluxes. Pulse labeling of the organism or cell with a tracer, such as 13C, followed by mass spectrometric analysis of the partitioning of label into different compounds provides an efficient tool to study flux and to compare the effect of mutations on flux.

    In contrast to the mass spectrometry assays, FRET nanosensors monitor only a single compound. The major advantages of FRET nanosensors are that they yield data with cellular and subcellular resolution and the method is minimally invasive. Polytopic membrane proteins are essential for cellular uptake and release of nutrients. To prevent toxic accumulation, rapid shut-off mechanisms are required.

    Here we show that the soluble cytosolic carboxy terminus of an oligomeric ammonium transporter from Arabidopsis thaliana serves as an allosteric regulator essential for function; mutations in the C-terminal domain, conserved between bacteria, fungi and plants, led to loss of transport activity. When co-expressed with intact transporters, mutants inactivated functional subunits, but left their stability unaffected.

    Co-expression of two inactive transporters, one with a defective pore, the other with an ablated C terminus, reconstituted activity. The crystal structure of an Archaeoglobus fulgidus ammonium transporter AMT suggests that the C terminus interacts physically with cytosolic loops of the neighbouring subunit. Phosphorylation of conserved sites in the C terminus are proposed as the cognate control mechanism.

    Conformational coupling between monomers provides a mechanism for tight regulation, for increasing the dynamic range of sensing and memorizing prior events, and may be a general mechanism for transporter regulation. Today's major excitement in biology centers on signaling: How can a cell or organism measure the myriad of environmental cues, integrate it, and acclimate to the new conditions? Hormonal signals and second messengers are in the focus of most of these studies, e.

    But if we look to other organisms such as E. To investigate whether AMTs are regulated at the posttranscriptional level, a gene construct consisting of the cauliflower mosaic virus 35S promoter driving the Arabidopsis Arabidopsis thaliana AMT1;1 gene was introduced into tobacco Nicotiana tabacum. When ammonium was supplied as the major nitrogen N form but at limiting amounts to soil-grown plants, transgenic lines overexpressing AtAMT1;1 did not show enhanced growth or N acquisition relative to wild-type plants. Surprisingly, steady-state transcript levels of AtAMT1;1 accumulated to higher levels in N-deficient roots and shoots of transgenic tobacco plants in spite of expression being controlled by the constitutive 35S promoter.

    Moreover, steady-state transcript levels were decreased after addition of ammonium or nitrate in N-deficient roots, suggesting a role for N availability in regulating AtAMT1;1 transcript abundance. Evidence for a regulatory role of the 3'-untranslated region of AtAMT1;1 alone in N-dependent transcript accumulation was not found. These results show that the accumulation of AtAMT1;1 transcripts is regulated in a N- and organ-dependent manner and suggest mRNA turnover as an additional mechanism for the regulation of AtAMT1;1 in response to the N nutritional status of plants.

    The recovery of free purine and pyrimidine bases and their degradation products represent alternative pathways in plant cells either to synthesize nucleotides salvage pathways by low energy consumption or to reuse organic nitrogen. Such recycling of metabolites often requires their uptake into the cell by specialized transport systems residing in the plasma membrane.

    In plants, it has been suggested that several protein families are involved in this process, but only a few transporters have so far been characterized. In this work, gene expression, substrate specificities, and transport mechanisms of members of the Ureide Permease family in Arabidopsis AtUPS were analyzed and compared. Promoter-GUS studies indicated that the members of the family have distinct and partially overlapping expression patterns with regard to developmental stages or tissue specific localization. In addition, two alternative splice variants of AtUPS5, a novel member of the transporter family, were identified and investigated.

    The abundance of both alternative mRNAs varied in different organs, while the relative amounts were comparable. In contrast, AtUPS5s shorter isoform lacks two transmembrane domains as structural consequence of the additional splice event. AtUPS5s, however, could not be shown to mediate uptake of these compounds into yeast cells and might therefore be defective or have a different function.

    FLIPPi sensors are suitable for real-time monitoring of P i metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P i during cell migration. Glucose is the main sugar transport form in animals, whereas plants use sucrose to supply non-photosynthetic organs with carbon skeletons and energy. Many aspects of sucrose transport, metabolism, and signaling are not well understood, including the route of sucrose efflux from leaf mesophyll cells and transport across vacuolar membranes.

    Tools that can detect sucrose with high spatial and temporal resolution in intact organs may help elucidate the players involved. Here, FRET sensors were generated by fusing putative sucrose-binding proteins to green fluorescent protein variants. Plant-associated bacteria such as Rhizobium and Agrobacterium can use sucrose as a nutrient source; sugar-binding proteins were, thus, used as scaffolds for developing sucrose nanosensors.

    Among a set of putative sucrose-binding protein genes cloned in between eCFP and eYFP and tested for sugar-dependent FRET changes, an Agrobacterium sugar-binding protein bound sucrose with 4 mum affinity. This FLIPsuc-4mu protein also recognized other sugars including maltose, trehalose, and turanose and, with lower efficiency, glucose and palatinose.

    Homology modeling enabled the prediction of binding pocket mutations to modulate the relative affinity of FLIPsuc-4mu for sucrose, maltose, and glucose. Mutant nanosensors showed up to and fold increases in specificity for sucrose over maltose and glucose, respectively, and the sucrose binding affinity was simultaneously decreased to allow detection in the physiological range. In addition, the signal-to-noise ratio of the sucrose nanosensor was improved by linker engineering. This novel reagent complements FLIPs for glucose, maltose, ribose, glutamate, and phosphate and will be used for analysis of sucrose-derived carbon flux in bacterial, fungal, plant, and animal cells.

    Genetically encoded glucose nanosensors have been used to measure steady state glucose levels in mammalian cytosol, nuclei, and endoplasmic reticulum. Unfortunately, the same nanosensors in Arabidopsis thaliana transformants manifested transgene silencing and undetectable fluorescence resonance energy transfer changes. Expressing nanosensors in sgs3 and rdr6 transgene silencing mutants eliminated silencing and resulted in high fluorescence levels.

    To measure glucose changes over a wide range nanomolar to millimolar , nanosensors with higher signal-to-noise ratios were expressed in these mutants. Perfusion of leaf epidermis with glucose led to concentration-dependent ratio changes for nanosensors with in vitro K d values of microM FLIPglu microDelta13 and 3. In intact roots, FLIPglu These results demonstrate that cytosolic steady state glucose levels depend on external supply in both leaves and roots, but under the conditions tested they are lower in root versus epidermal and guard cells.

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    Without photosynthesis and external supply, cytosolic glucose can decrease to View details for DOI Hebeloma cylindrosporum is a model fungus for mycorrhizal studies because of its fast growth rate, simple nutritional requirements, and completion of its life cycle in vitro, and because it is amenable to transformation.

    To advance cell biological research during establishment of symbiosis, a tool that would enable the direct visualisation of fusion proteins in the different symbiotic tissues [namely, the expression of reporter genes such as Green Fluorescent Protein GFP ] was still a missing tool. In the present study, H. EGFP expression was successfully detected in transformants. The fluorescence was uniformly distributed in the hyphae, while no significant background signal was detected in control hyphae.

    The suitability of EGFP for reporter gene studies in Hebeloma cylindrosporum was demonstrated opening up new perspectives in the Hebeloma genetics. Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease AAP subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs.

    Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue.

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    These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.

    Vitamin B6 represents a highly important group of compounds ubiquitous in all living organisms. By means of a genetic approach, we have identified a novel mutant, rsr for reduced sugar response , with aberrant root and leaf growth that requires supplementation of vitamin B6 for normal development. Consequently, metabolism is broadly altered, mainly affecting amino acid, raffinose, and shikimate contents and trichloroacetic acid cycle constituents. Yeast two-hybrid and pull-down analyses showed that Arabidopsis thaliana PDX1 proteins can form oligomers.

    Interestingly, the mutant form of PDX1 has severely reduced capability to oligomerize, potentially suggesting that oligomerization is important for function. In summary, our results demonstrate the critical function of the PDX1 protein family for metabolism, whole-plant development, and vitamin B6 biosynthesis in higher plants. Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents.

    However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 AtUPS1 , previously cloned by functional complementation in yeast.

    Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane-bound proteins.

    Constraints on plant growth imposed by low availability of nitrogen are a characteristic feature of ecosystems dominated by ectomycorrhizal plants. Ectomycorrhizal fungi play a key role in the N nutrition of plants, allowing their host plants to access decomposition products of dead plant and animal materials. Ectomycorrhizal plants are thus able to compensate for the low availability of inorganic N in forest ecosystems. The capacity to take up peptides, as well as the transport mechanisms involved, were analysed in the ectomycorrhizal fungus Hebeloma cylindrosporum.

    The present study demonstrated that H. Uptake capacities and expression regulation of both genes were analysed, indicating that HcPTR2A was involved in the high-efficiency peptide uptake under conditions of limited N availability, whereas HcPTR2B was expressed constitutively. Fluorescent labels have revolutionized cell biology. Signaling intermediates and metabolites can be measured in real time with subcellular spatial resolution. Most of these sensors are based on fluorescent proteins, and many report fluorescence resonance energy transfer.

    Because the biosensors are genetically encoded, a toolbox for addressing cell biological questions at the systems level is now available. Fluorescent biosensors are able to determine the localization of proteins and their dynamics, to reveal the cellular and subcellular localization of the respective interactions and activities, and to provide complementary data on the steady state levels of ions, metabolites, and signaling intermediates with high temporal and spatial resolution.

    They represent the basis for cell-based high-throughput assays that are necessary for a systems perspective on plant cell function. Glucose release from hepatocytes is important for maintenance of blood glucose levels. Glucosephosphate phosphatase, catalyzing the final metabolic step of gluconeogenesis, faces the endoplasmic reticulum ER lumen.

    Thus, glucose produced in the ER has to be either exported from the ER into the cytosol before release into circulation or exported directly by a vesicular pathway. To measure ER transport of glucose, fluorescence resonance energy transfer-based nanosensors were targeted to the cytosol or the ER lumen of HepG2 cells. Glucose levels and kinetics inside the ER were indistinguishable from cytosolic levels, suggesting rapid bidirectional glucose transport across the ER membrane.

    A dynamic model incorporating rapid bidirectional ER transport yields a very good fit with the observed kinetics. Plasma membrane and ER membrane glucose transport differed regarding sensitivity to cytochalasin B and showed different relative kinetics for galactose uptake and release, suggesting catalysis by distinct activities at the two membranes. The presence of a high-capacity glucose transport system on the ER membrane is consistent with the hypothesis that glucose export from hepatocytes occurs via the cytosol by a yet-to-be-identified set of proteins.

    A family of genetically-encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins PBPs linearly fused to protein fluorophores. The ligand-induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer FRET -compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand-measurement and visualization.

    Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter-fluorophore separation contribute to FRET transfer efficiency and the resulting ligand-dependent spectral changes.

    Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter kappa2 to FRET efficiency.