Yellow Fluorescent Protein Fusion Research Articles

Biosynthesis of polyprenylated xanthones in Hypericum perforatum roots involves 4-prenyltransferase.

Polyprenylated xanthones are natural products with a multitude of biological and pharmacological activities. However, their biosynthetic pathway is not completely understood. In this study, metabolic profiling revealed the presence of 4-prenylated 1,3,5,6-tetrahydroxyxanthone derivatives in St. John's wort (Hypericum perforatum) root extracts. Transcriptomic data mining led to the detection of five variants of xanthone 4-prenyltransferase (HpPT4px) comprising four long variants (HpPT4px-v1 to HpPT4px-v4) and one short variant (HpPT4px-sh). The full-length sequences of all five variants were cloned and heterologously expressed in yeast (Saccharomyces cerevisiae). Microsomes containing HpPT4px-v2, HpPT4px-v4, and HpPT4px-sh catalyzed the addition of a prenyl group at the C-4 position of 1,3,5,6-tetrahydroxyxanthone, 1,3,5-trihydroxyxanthone, and 1,3,7-trihydroxyxanthone, whereas microsomes harboring HpPT4px-v1 and HpPT4px-v3 additionally accepted 1,3,6,7-tetrahydroxyxanthone. HpPT4px-v1 produced in Nicotiana benthamiana displayed the same activity as in yeast, while HpPT4px-sh was inactive. The kinetic parameters of HpPT4px-v1 and HpPT4px-sh chosen as representative variants indicated 1,3,5,6-tetrahydroxyxanthone as the preferred acceptor substrate, rationalizing that HpPT4px catalyzes the first prenylation step in the biosynthesis of polyprenylated xanthones in H. perforatum. Dimethylallyl pyrophosphate was the exclusive prenyl donor. Expression of the HpPT4px transcripts was highest in roots and leaves, raising the question of product translocation. C-terminal yellow fluorescent protein fusion of HpPT4px-v1 localized to the envelope of chloroplasts in N. benthamiana leaves, whereas short, truncated, and masked signal peptides led to the disruption of plastidial localization. These findings pave the way for a better understanding of the prenylation of xanthones in plants and the identification of additional xanthone-specific prenyltransferases.

Characterization of basal forebrain glutamate neurons suggests a role in control of arousal and avoidance behavior.

The basal forebrain (BF) is involved in arousal, attention, and reward processing but the role of individual BF neuronal subtypes is still being uncovered. Glutamatergic neurons are the least well-understood of the three main BF neurotransmitter phenotypes. Here we analyzed the distribution, size, calcium-binding protein content and projections of the major group of BF glutamatergic neurons expressing the vesicular glutamate transporter subtype 2 (vGluT2) and tested the functional effect of activating them. Mice expressing Cre recombinase under the control of the vGluT2 promoter were crossed with a reporter strain expressing the red fluorescent protein, tdTomato, to generate vGluT2-cre-tdTomato mice. Immunohistochemical staining for choline acetyltransferase and a cross with mice expressing green fluorescent protein selectively in GABAergic neurons confirmed that cholinergic, GABAergic and vGluT2+ neurons represent distinct BF subpopulations. Subsets of BF vGluT2+ neurons expressed the calcium-binding proteins calbindin or calretinin, suggesting that multiple subtypes of BF vGluT2+ neurons exist. Anterograde tracing using adeno-associated viral vectors expressing channelrhodopsin2-enhanced yellow fluorescent fusion proteins revealed major projections of BF vGluT2+ neurons to neighboring BF cholinergic and parvalbumin neurons, as well as to extra-BF areas involved in the control of arousal or aversive/rewarding behavior such as the lateral habenula and ventral tegmental area. Optogenetic activation of BF vGluT2+ neurons elicited a striking avoidance of the area where stimulation was given, whereas stimulation of BF parvalbumin or cholinergic neurons did not. Together with previous optogenetic findings suggesting an arousal-promoting role, our findings suggest that BF vGluT2 neurons play a dual role in promoting wakefulness and avoidance behavior.

Indole-3-glycerolphosphate synthase, a branchpoint for the biosynthesis of tryptophan, indole, and benzoxazinoids in maize.

The maize (Zea mays) genome encodes three indole-3-glycerolphosphate synthase enzymes (IGPS1, 2, and 3) catalyzing the conversion of 1-(2-carboxyphenylamino)-l-deoxyribulose-5-phosphate to indole-3-glycerolphosphate. Three further maize enzymes (BX1, benzoxazinoneless 1; TSA, tryptophan synthase alpha subunit; and IGL, indole glycerolphosphate lyase) convert indole-3-glycerolphosphate to indole, which is released as a volatile defense signaling compound and also serves as a precursor for the biosynthesis of tryptophan and defense-related benzoxazinoids. Phylogenetic analyses showed that IGPS2 is similar to enzymes found in both monocots and dicots, whereas maize IGPS1 and IGPS3 are in monocot-specific clades. Fusions of yellow fluorescent protein with maize IGPS enzymes and indole-3-glycerolphosphate lyases were all localized in chloroplasts. In bimolecular fluorescence complementation assays, IGPS1 interacted strongly with BX1 and IGL, IGPS2 interacted primarily with TSA, and IGPS3 interacted equally with all three indole-3-glycerolphosphate lyases. Whereas IGPS1 and IGPS3 expression was induced by insect feeding, IGPS2 expression was not. Transposon insertions in IGPS1 and IGPS3 reduced the abundance of both benzoxazinoids and free indole. Spodoptera exigua (beet armyworm) larvae show improved growth on igps1 mutant maize plants. Together, these results suggest that IGPS1 and IGPS3 function mainly in the biosynthesis of defensive metabolites, whereas IGPS2 may be involved in the biosynthesis of tryptophan. This metabolic channeling is similar to, though less exclusive than, that proposed for the three maize indole-3-glycerolphosphate lyases.

Genetic Variation in Complex Traits in Transgenic α-Synuclein Strains of Caenorhabditis elegans.

Different genetic backgrounds can modify the effect of mutated genes. Human α-synuclein (SNCA) gene encodes α-synuclein, and its oligomeric complexes accumulate with age and mediate the disruption of cellular homeostasis, resulting in the neuronal death that is characteristic of Parkinson’s Disease. Polymorphic variants modulate this complex pathologic mechanism. Previously, we constructed five transgenic introgression lines of a Caenorhabditis elegans model of α-synuclein using genetic backgrounds that are genetically diverse from the canonical wild-type Bristol N2. A gene expression analysis revealed that the α-synuclein transgene differentially affects genome-wide transcription due to background modifiers. To further investigate how complex traits are affected in these transgenic lines, we measured the α-synuclein transgene expression, the overall accumulation of the fusion protein of α-synuclein and yellow fluorescent protein (YFP), the lysosome-related organelles, and the body size. By using quantitative PCR (qPCR), we demonstrated stable and similar expression levels of the α-synuclein transgene in different genetic backgrounds. Strikingly, we observed that the levels of the a-synuclein:YFP fusion protein vary in different genetic backgrounds by using the COPAS™ biosorter. The quantification of the Nile Red staining assay demonstrates that α-synuclein also affects lysosome-related organelles and body size. Our results show that the same α-synuclein introgression in different C. elegans backgrounds can produces differing effects on complex traits due to background modifiers.

Transcriptional and Biochemical Characterization of Cytosolic Pyruvate Kinases in Arabidopsis thaliana.

Glycolysis is a central catabolic pathway in every living organism with an essential role in carbohydrate breakdown and ATP synthesis, thereby providing pyruvate to the tricarboxylic acid cycle (TCA cycle). The cytosolic pyruvate kinase (cPK) represents a key glycolytic enzyme by catalyzing phosphate transfer from phosphoenolpyruvate (PEP) to ADP for the synthesis of ATP. Besides its important functions in cellular energy homeostasis, the activity of cytosolic pyruvate kinase underlies tight regulation, for instance by allosteric effectors, that impact stability of its quaternary structure. We determined five cytosol-localized pyruvate kinases, out of the fourteen putative pyruvate kinase genes encoded by the Arabidopsis thaliana genome, by investigation of phylogeny and localization of yellow fluorescent protein (YFP) fusion proteins. Analysis of promoter β-glucuronidase (GUS) reporter lines revealed an isoform-specific expression pattern for the five enzymes, subject to plant tissue and developmental stage. Investigation of the heterologously expressed and purified cytosolic pyruvate kinases revealed that these enzymes are differentially regulated by metabolites, such as citrate, fructose-1,6-bisphosphate (FBP) and ATP. In addition, measured in vitro enzyme activities suggest that pyruvate kinase subunit complexes consisting of cPK2/3 and cPK4/5 isoforms, respectively, bear regulatory properties. In summary, our study indicates that the five identified cytosolic pyruvate kinase isoforms adjust the carbohydrate flux through the glycolytic pathway in Arabidopsis thaliana, by distinct regulatory qualities, such as individual expression pattern as well as dissimilar responsiveness to allosteric effectors and enzyme subgroup association.

Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux

Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe3+ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP) fusions of Heph and APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, (K/R)EWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.

Sequential regiospecific gem-diprenylation of tetrahydroxyxanthone by prenyltransferases from Hypericum sp.

Polyprenylated acylphloroglucinol derivatives, such as xanthones, are natural plant products with interesting pharmacological properties. They are difficult to synthesize chemically. Biotechnological production is desirable but it requires an understanding of the biosynthetic pathways. cDNAs encoding membrane-bound aromatic prenyltransferase (aPT) enzymes from Hypericum sampsonii seedlings (HsPT8px and HsPTpat) and Hypericum calycinum cell cultures (HcPT8px and HcPTpat) were cloned and expressed in Saccharomyces cerevisiae and Nicotiana benthamiana, respectively. Microsomes and chloroplasts were used for functional analysis. The enzymes catalyzed the prenylation of 1,3,6,7-tetrahydroxyxanthone (1367THX) and/or 1,3,6,7-tetrahydroxy-8-prenylxanthone (8PX) and discriminated nine additionally tested acylphloroglucinol derivatives. The transient expression of the two aPT genes preceded the accumulation of the products in elicitor-treated H.calycinum cell cultures. C-terminal yellow fluorescent protein fusions of the two enzymes were localized to the envelope of chloroplasts in N.benthamiana leaves. Based on the kinetic properties of HsPT8px and HsPTpat, the enzymes catalyze sequential rather than parallel addition of two prenyl groups to the carbon atom 8 of 1367THX, yielding gem-diprenylated patulone under loss of aromaticity of the gem-dialkylated ring. Coexpression in yeast significantly increased product formation. The patulone biosynthetic pathway involves multiple subcellular compartments. The aPTs studied here and related enzymes may be promising tools for plant/microbe metabolic pathway engineering.

Visualization of PAX7 protein dynamics in muscle satellite cells in a YFP knock-in-mouse line

BackgroundSatellite cells are residential muscle stem cells that express a paired box protein, PAX7.ResultsHere, we report a knock-in mouse line expressing a PAX7-enhanced yellow fluorescent protein (YFP) fusion protein that enables visualization of PAX7 protein dynamics in living satellite cells through YFP fluorescence. The YFP fluorescence signals in Pax7-YFP knock-in mice clearly recapitulated the endogenous expression of PAX7 protein in satellite cells. YFP+ satellite cells were efficiently isolated from muscle tissues by fluorescence-activated cell sorting. Homozygous Pax7-YFP knock-in mice (Pax7YFP/YFP) were viable, grew and regenerated muscle normally, and Pax7YFP/YFP mouse-derived satellite cells proliferated, differentiated, and self-renewed as efficiently as those from wild-type (Pax7+/+) mice.ConclusionsTaken together, our Pax7-YFP mouse line is a useful tool to aid the development of stem-cell-based therapies for muscle diseases.

Constitutive expression of CmSKOR, an outward K+ channel gene from melon, in Arabidopsis thaliana involved in saline tolerance

Shaker-like K+ outward rectifying channel (SKOR) is involved in mediating long-distance K+ transport from roots to shoots. In this study, a Shaker-like outward K+ channel gene CmSKOR (GenBank accession number MF447462) was isolated from melon (Cucumis melo L.). Phylogenetic analysis showed that CmSKOR belongs to the SKOR-subfamily in the Shaker-like K+ channel family. Electrophysiological experiments indicated that CmSKOR was a K+-permeable channel with low affinity. Expressed in Xenopus oocytes, CmSKOR displayed classical Shaker-like outwardly rectifying K+ currents. Confocal imaging of a CmSKOR – yellow fluorescent fusion protein (YFP) in transgenic Nicotiana tabacum leaves indicated that CmSKOR was located in the plasma membrane. Transcript analysis showed CmSKOR predominantly expressed in melon roots and with lower abundance in stem and leaves. In addition, both external K+ and NaCl treatment could up-regulate the expression of CmSKOR in melon and enhance the K+ content in shoot. Constitutive overexpressed CmSKOR in Arabidopsis thaliana, the transgenic plants showed changes in root length in MS plates, displayed higher maximum photochemical efficiency of PSII (Fv/Fm), higher fresh and dry weight, and accumulation of K+ in shoot, together with the changes of transcript amount of CmSKOR with NaCl treatments in mixture substrate. In conclusion, it was proposed that CmSKOR may play the role on distributing K+ to the shoot in melon and its constitutive expression in Arabidopsis improved saline tolerance by maintaining K+ homeostasis in the plant.

The Synechocystis Manganese Exporter Mnx Is Essential for Manganese Homeostasis in Cyanobacteria.

The essential micronutrient manganese (Mn) functions as redox-active cofactor in active sites of enzymes and, thus, is involved in various physiological reactions. Moreover, in oxygenic photosynthetic organisms, Mn is of special importance, since it is central to the oxygen-evolving complex in photosystem II. Although Mn is an essential micronutrient, increased amounts are detrimental to the organism; thus, only a small window exists for beneficial concentrations. Accordingly, Mn homeostasis must be carefully maintained. In contrast to the well-studied uptake mechanisms in cyanobacteria, it is largely unknown how Mn is distributed to the different compartments inside the cell. We identified a protein with so far unknown function as a hypothetical Mn transporter in the cyanobacterial model strain Synechocystis sp. PCC 6803 and named this protein Mnx for Mn exporter. The knockout mutant Δmnx showed increased sensitivity toward externally supplied Mn and Mn toxicity symptoms, which could be linked to intracellular Mn accumulation. 54Mn chase experiments demonstrated that the mutant was not able to release Mn from the internal pool. Microscopic analysis of a Mnx::yellow fluorescent protein fusion showed that the protein resides in the thylakoid membrane. Heterologous expression of mnx suppressed the Mn-sensitive phenotype of the Saccharomyces cerevisiae mutant Δpmr1 Our results indicate that Mnx functions as a thylakoid Mn transporter and is a key player in maintaining Mn homeostasis in Synechocystis sp. PCC 6803. We propose that Mn export from the cytoplasm into the thylakoid lumen is crucial to prevent toxic cytoplasmic Mn accumulation and to ensure Mn provision to photosystem II.

Binding characteristics of galectin-3 fusion proteins.

Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant Kd) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1-116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1-62) and galectin-3(Δ1-116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy.

Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed. Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae This tiny, 2-μm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma "gliding motility" is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism.

Functional characterization of Rorippa indica defensin and its efficacy against Lipaphis erysimi.

Rorippa indica, a wild crucifer, has been previously reported as the first identified plant in the germplasm of Brassicaceae known to be tolerant towards the mustard aphid Lipaphis erysimi Kaltenbach. We herein report the full-length cloning, expression, purification and characterization of a novel R. indica defensin (RiD) and its efficacy against L. erysimi. Structural analysis through homology modeling of RiD showed longer α-helix and 3rd β-sheet as compared to Brassica juncea defensin (BjD). Recombinant RiD and BjD was purified for studying its efficacy against L. erysimi. In the artificial diet based insect bioassay, the LC50 value of RiD against L. erysimi was found to be 9.099 ± 0.621 µg/mL which is far lower than that of BjD (43.51 ± 0.526 µg/mL). This indicates the possibility of RiD having different interacting partner and having better efficacy against L. erysimi over BjD. In the transient localization studies, RiD signal peptide directed the RiD: yellow fluorescent protein (YFP) fusion protein to the apoplastic regions which indicates that it might play a very important role in inhibiting nutrient uptake by aphids which follow mainly extracellular route to pierce through the cells. Hence, the present study has a significant implication for the future pest management program of B. juncea through the development of aphid tolerant transgenic plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2144-2) contains supplementary material, which is available to authorized users.

Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models.

BackgroundWhile many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions – e.g. neurodegenerative diseases – often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field.ResultsWe describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms’ tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system’s ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model.ConclusionOur microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0083-6) contains supplementary material, which is available to authorized users.

Dual-Reporter β-Cell-Specific Male Transgenic Rats for the Analysis of β-Cell Functional Mass and Enrichment by Flow Cytometry.

Mouse β-cell-specific reporter lines have played a key role in diabetes research. Although the rat provides several advantages, its use has lagged behind the mouse due to the relative paucity of genetic models. In this report we describe the generation and characterization of transgenic rats expressing a Renilla luciferase (RLuc)-enhanced yellow fluorescent protein (YFP) fusion under control of a 9-kb genomic fragment from the rat ins2 gene (RIP7-RLuc-YFP). Analysis of RLuc luminescence and YFP fluorescence revealed that reporter expression is restricted to β-cells in the adult rat. Physiological characteristics including body weight, fat and lean mass, fasting and fed glucose levels, glucose and insulin tolerance, and β-cell mass were similar between two RIP7-RLuc-YFP lines and wild-type littermates. Glucose-induced insulin secretion in isolated islets was indistinguishable from controls in one of the lines, whereas surprisingly, insulin secretion was defective in the second line. Consequently, subsequent studies were limited to the former line. We asked whether transgene activity was responsive to glucose as shown previously for the ins2 gene. Exposing islets ex vivo to high glucose (16.7 mM) or in vivo infusion of glucose for 24 hours increased luciferase activity in islets, whereas the fraction of YFP-positive β-cells after glucose infusion was unchanged. Finally, we showed that fluorescence-activated cell sorting of YFP-positive islet cells can be used to enrich for β-cells. Overall, this transgenic line will enable for the first time the application of both fluorescence and bioluminescence/luminescence-based approaches for the study of rat β-cells.

Construction of a linker library with widely controllable flexibility for fusion protein design.

Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions.

Ultrastructure and Membrane Traffic During Cell Division in the Marine Pennate Diatom Phaeodactylum tricornutum

The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum.

Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance.

Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data indicate that Aux/IAA family members have protein-specific degradation rates and that ubiquitination of Aux/IAAs can occur on multiple types of amino residues to promote rapid auxin-mediated degradation.

The cytoplasmic localization of the catalytic site of CSLF6 supports a channeling model for the biosynthesis of mixed-linkage glucan.

Mixed-linkage glucan (MLG) is a significant cell wall carbohydrate in grasses and an important carbon source for human consumption and biofuel production. MLG biosynthesis depends on the biochemical activity of membrane spanning glucan synthases encoded by the CSLH and CSLF cellulose synthase-like gene families. CSLF proteins are the best characterized to date but relatively little information is known about their topology with respect to the biosynthetic membranes. In this study, we report on the topology of CSLF6 protein derived from the model grass species Brachypodium distachyon (BdCSLF6) when it is expressed in heterologous systems. Using live cell imaging and immuno-electron microscopy analyses of tobacco epidermal cells expressing BdCSLF6, we demonstrate that a functional yellow fluorescent protein (YFP) fusion of BdCSLF6 is localized to the Golgi apparatus and that the Golgi localization of BdCSLF6 is sufficient for MLG biosynthesis. By implementing protease protection assays of BdCSLF6 expressed in the yeast Pichia pastoris, we also demonstrate that the catalytic domain, the N-terminus and the C- terminus of the protein are exposed in the cytosol. Furthermore, we found that BdCSLF6 is capable of producing MLG not only in tobacco cells but also in Pichia, which generally does not produce MLG. Together, these results support the conclusion that BdCSLF6 can produce both of the linkages present in the (1,3;1,4)-β-d-glucan chain of MLG and that the product is channelled at the Golgi into the secretory pathway for deposition into the cell wall.