Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of ...inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3-5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single mammalian cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•SemiSWEETs and SWEETs are sugar transporters present in all kingdoms of life.•SWEETs serve critical roles in sugar allocation in plants.•Physiological roles in plants predominantly relate to ...cellular efflux.•Bacterial SemiSWEETs, among the smallest known transporters, are built from a polypeptide comprising a triple helix bundle.•The minimal functional unit of the SemiSWEETs is a dimer.•SemiSWEETs have been crystallized in three states: outward open, occluded, and inward open.•The eukaryotic SWEETs comprise a duplicated triple helix bundle connected via an inversion linker helix.
SemiSWEETs and SWEETs have emerged as unique sugar transporters. First discovered in plants with the help of fluorescent biosensors, homologs exist in all kingdoms of life. Bacterial and plant homologs transport hexoses and sucrose, whereas animal SWEETs transport glucose. Prokaryotic SemiSWEETs are small and comprise a parallel homodimer of an approximately 100 amino acid-long triple helix bundle (THB). Duplicated THBs are fused to create eukaryotic SWEETs in a parallel orientation via an inversion linker helix, producing a similar configuration to that of SemiSWEET dimers. Structures of four SemiSWEETs have been resolved in three states: open outside, occluded, and open inside, indicating alternating access. As we discuss here, these atomic structures provide a basis for exploring the evolution of structure–function relations in this new class of transporters.
Despite the relevance of seed-filling mechanisms for crop yield, we still have only a rudimentary understanding of the transport processes that supply the caryopsis with sugars. We hypothesized that ...SWEET sucrose transporters may play important roles in nutrient import pathways in the rice caryopsis.
We used a combination of mRNA quantification, histochemical analyses, translational promoter–reporter fusions and analysis of knockout mutants created by genomic editing to evaluate the contribution of SWEET transporters to seed filling.
In rice caryopses, SWEET11 and 15 had the highest mRNA levels and proteins localized to four key sites: all regions of the nucellus at early stages; the nucellar projection close to the dorsal vein; the nucellar epidermis that surrounds the endosperm; and the aleurone. ossweet11;15 double knockout lines accumulated starch in the pericarp, whereas caryopses did not contain a functional endosperm.
Jointly, SWEET11 and 15 show all the hallmarks of being necessary for seed filling with sucrose efflux functions at the nucellar projection and a role in transfer across the nucellar epidermis/aleurone interface, delineating two major steps for apoplasmic seed filling, observations that are discussed in relation to observations made in rice and barley regarding the relative prevalence of these two potential import routes.
Transport of Sugars Chen, Li-Qing; Cheung, Lily S; Feng, Liang ...
Annual review of biochemistry,
06/2015, Letnik:
84, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Soluble sugars serve five main purposes in multicellular organisms: as sources of carbon skeletons, osmolytes, signals, and transient energy storage and as transport molecules. Most sugars are ...derived from photosynthetic organisms, particularly plants. In multicellular organisms, some cells specialize in providing sugars to other cells (e.g., intestinal and liver cells in animals, photosynthetic cells in plants), whereas others depend completely on an external supply (e.g., brain cells, roots and seeds). This cellular exchange of sugars requires transport proteins to mediate uptake or release from cells or subcellular compartments. Thus, not surprisingly, sugar transport is critical for plants, animals, and humans. At present, three classes of eukaryotic sugar transporters have been characterized, namely the glucose transporters (GLUTs), sodium-glucose symporters (SGLTs), and SWEETs. This review presents the history and state of the art of sugar transporter research, covering genetics, biochemistry, and physiology-from their identification and characterization to their structure, function, and physiology. In humans, understanding sugar transport has therapeutic importance (e.g., addressing diabetes or limiting access of cancer cells to sugars), and in plants, these transporters are critical for crop yield and pathogen susceptibility.
The EU and New Zealand are the only legislations where genome-edited plants are considered and regulated as GMOs while many other countries move to exempt genome-edited crops.
Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major ...transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.
Molecular activities are highly dynamic and can occur locally in subcellular domains or compartments. Neighboring cells in the same tissue can exist in different states. Therefore, quantitative ...information on the cellular and subcellular dynamics of ions, signaling molecules, and metabolites is critical for functional understanding of organisms. Mass spectrometry is generally used for monitoring ions and metabolites; however, its temporal and spatial resolution are limited. Fluorescent proteins have revolutionized many areas of biology-e.g., fluorescent proteins can report on gene expression or protein localization in real time-yet promoter-based reporters are often slow to report physiologically relevant changes such as calcium oscillations. Therefore, novel tools are required that can be deployed in specific cells and targeted to subcellular compartments in order to quantify target molecule dynamics directly. We require tools that can measure enzyme activities, protein dynamics, and biophysical processes (e.g., membrane potential or molecular tension) with subcellular resolution. Today, we have an extensive suite of tools at our disposal to address these challenges, including translocation sensors, fluorescence-intensity sensors, and Förster resonance energy transfer sensors. This review summarizes sensor design principles, provides a database of sensors for more than 70 different analytes/processes, and gives examples of applications in quantitative live cell imaging.
Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 ...sucrose/H+ symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known.
Here we analysed the contribution of SWEETs to phloem loading in maize.
We identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. ZmSWEET13 paralogues (a, b, c) are among the most highly expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants were severely stunted. Photosynthesis of mutants was impaired and leaves accumulated high levels of soluble sugars and starch. RNA-seq revealed profound transcriptional deregulation of genes associated with photosynthesis and carbohydrate metabolism. Genome-wide association study (GWAS) analyses may indicate that variability in ZmSWEET13s correlates with agronomical traits, especifically flowering time and leaf angle.
This work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Z. mays.
Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae (Xoo), secretes one or more of six known transcription-activator-like effectors ...(TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11, SWEET13 and SWEET14, the expression of which is required for disease susceptibility. We used CRISPR-Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14, which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.
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