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Chen L.-Q.,Carnegie Institution for Science
New Phytologist | Year: 2014

Many intercellular solute transport processes require an apoplasmic step, that is, efflux from one cell and subsequent uptake by an adjacent cell. Cellular uptake transporters have been identified for many solutes, including sucrose; however, efflux transporters have remained elusive for a long time. Cellular efflux of sugars plays essential roles in many processes, such as sugar efflux as the first step in phloem loading, sugar efflux for nectar secretion, and sugar efflux for supplying symbionts such as mycorrhiza, and maternal efflux for filial tissue development. Furthermore, sugar efflux systems can be hijacked by pathogens for access to nutrition from hosts. Mutations that block recruitment of the efflux mechanism by the pathogen thus cause pathogen resistance. Until recently, little was known regarding the underlying mechanism of sugar efflux. The identification of sugar efflux carriers, SWEETs (Sugars Will Eventually be Exported Transporters), has shed light on cellular sugar efflux. SWEETs appear to function as uniporters, facilitating diffusion of sugars across cell membranes. Indeed, SWEETs probably mediate sucrose efflux from putative phloem parenchyma into the phloem apoplasm, a key step proceeding phloem loading. Engineering of SWEET mutants using transcriptional activator-like effector nuclease (TALEN)-based genomic editing allowed the engineering of pathogen resistance. The widespread expression of the SWEET family promises to provide insights into many other cellular efflux mechanisms. © 2013 New Phytologist Trust. Source


Hou B.H.,Carnegie Institution for Science
Nature protocols | Year: 2011

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. Genetically encoded Förster resonance energy transfer (FRET) sensors can be used for real-time in vivo detection of metabolites. FRET sensor proteins, for example, for glucose, can be targeted genetically to any cellular compartment, or even to subdomains (e.g., a membrane surface), by adding signal sequences or fusing the sensors to specific proteins. 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. The whole procedure typically takes ∼4 d including seeding and transfection of mammalian cells; the FRET-based analysis of transfected cells takes ∼5 h. Source


Berry J.A.,Carnegie Institution for Science
Annual Review of Plant Biology | Year: 2012

An overriding interest in photosynthesis has propelled my wanderings from chemist to biochemist to plant physiologist and on to global topics. Equations and models have been organizing principles along the way. This fascination started as a reaction to difficulties with written communication, but it has proven to be quite useful in moving across different levels of organization. I conclude with some discussion of the importance of Earth system models for understanding and predicting how human activities may influence the climate, environment, and biota in the future, and some ideas about how disciplinary science might make larger contributions to this interdisciplinary problem. (Note: Selected references are available from the Carnegie Institution Web site at http://dge.stanford.edu/publications/berry/AnnRev2012). © 2012 by Annual Reviews. All rights reserved. Source


Bermejo C.,Carnegie Institution for Science
Nature protocols | Year: 2011

Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded Förster resonance energy transfer (FRET) sensors. 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. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. 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 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. 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 <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors. Source


Kormendy J.,University of Texas at Austin | Ho L.C.,Carnegie Institution for Science
Annual Review of Astronomy and Astrophysics | Year: 2013

Supermassive black holes (BHs) have been found in 85 galaxies by dynamical modeling of spatially resolved kinematics. The Hubble Space Telescope revolutionized BH research by advancing the subject from its proof-of-concept phase into quantitative studies of BH demographics. Most influential was the discovery of a tight correlation between BH mass and the velocity dispersion σ of the bulge component of the host galaxy. Together with similar correlations with bulge luminosity and mass, this led to the widespread belief that BHs and bulges coevolve by regulating each other's growth. Conclusions based on one set of correlations from in brightest cluster ellipticals to in the smallest galaxies dominated BH work for more than a decade. New results are now replacing this simple story with a richer and more plausible picture in which BHs correlate differently with different galaxy components. A reasonable aim is to use this progress to refine our understanding of BH-galaxy coevolution. BHs with masses of 105-106Mȯ are found in many bulgeless galaxies. Therefore, classical (elliptical-galaxy-like) bulges are not necessary for BH formation. On the other hand, although they live in galaxy disks, BHs do not correlate with galaxy disks. Also, any correlations with the properties of disk-grown pseudobulges and dark matter halos are weak enough to imply no close coevolution. The above and other correlations of host-galaxy parameters with each other and with suggest that there are four regimes of BH feedback. (1) Local, secular, episodic, and stochastic feeding of small BHs in largely bulgeless galaxies involves too little energy to result in coevolution. (2) Global feeding in major, wet galaxy mergers rapidly grows giant BHs in short-duration, quasar-like events whose energy feedback does affect galaxy evolution. The resulting hosts are classical bulges and coreless-rotating-disky ellipticals. (3) After these AGN phases and at the highest galaxy masses, maintenance-mode BH feedback into X-ray-emitting gas has the primarily negative effect of helping to keep baryons locked up in hot gas and thereby keeping galaxy formation from going to completion. This happens in giant, core-nonrotating-boxy ellipticals. Their properties, including their tight correlations between and core parameters, support the conclusion that core ellipticals form by dissipationless major mergers. They inherit coevolution effects from smaller progenitor galaxies. Also, (4) independent of any feedback physics, in BH growth modes 2 and 3, the averaging that results from successive mergers plays a major role in decreasing the scatter in correlations from the large values observed in bulgeless and pseudobulge galaxies to the small values observed in giant elliptical galaxies.Copyright ©2013 by Annual Reviews. All rights reserved. Source

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