The Xanthomonas campestris pv. vesicatoria citH gene is expressed early in the infection process of tomato and is positively regulated by the TctDE two-component regulatory system

Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease of tomato and pepper. Previously, we have reported the adaptation of a recombinase- or resolvase-based in vivo expression technology (RIVET) approach to identify Xcv genes that are specifically induced during its interaction with tomato. Analysis of some of these genes revealed that a citH (citrate transporter) homologous gene contributes to Xcv virulence on tomato. Here, we demonstrate that the citH product indeed facilitates citrate uptake by showing the following: citH is specifically needed for Xcv growth in citrate, but not in other carbon sources; the citH promoter is specifically induced by citrate; and the concentration of citrate from tomato leaf apoplast is considerably reduced following growth of the wild-type and a citH-complemented strain, but not the citH mutant. We also show that, in the Xcv-tomato interaction, the promoter activity of the citH gene is induced as early as 2.5h after Xcv is syringe infiltrated into tomato leaves, and continues to be active for at least 96h after inoculation. We identified an operon containing a two-component regulatory system homologous to tctD/tctE influencing citH expression in Xcv, as well as its heterologous expression in Escherichia coli. The expression of hrp genes does not seem to be affected in the citH mutant, and this mutant cannot be complemented for growth in planta when co-inoculated with the wild-type strain, indicating that citrate uptake in the apoplast is important for the virulence of Xcv.     

Muscle-bound primordial stem cells give rise to myofiber-associated myogenic and non-myogenic progenitors

Myofiber cultures give rise to myogenic as well as to non-myogenic cells. Whether these myofiber-associated non-myogenic cells develop from resident stem cells that possess mesenchymal plasticity or from other stem cells such as mesenchymal stem cells (MSCs) remain unsolved. To address this question, we applied a method for reconstructing cell lineage trees from somatic mutations to MSCs and myogenic and non-myogenic cells from individual myofibers that were cultured at clonal density.Our analyses show that (i) in addition to myogenic progenitors, myofibers also harbor non-myogenic progenitors of a distinct, yet close, lineage; (ii) myofiber-associated non-myogenic and myogenic cells share the same muscle-bound primordial stem cells of a lineage distinct from bone marrow MSCs; (iii) these muscle-bound primordial stem-cells first part to individual muscles and then differentiate into myogenic and non-myogenic stem cells.     

Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

Voltage-gated sodium channels (Na_v) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Na_v α subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Na_vs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Na_vs.     

Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering

Molecular switches such as small GTPases of the Ras family cycle between inactive GDP-bound and active GTP-bound states. Their essential role in controlling development and cell homeostasis requires mechanisms which determine amplitude and timing of activation. This is achieved in part by the action of guanine nucleotide exchange factors, which function as highly controlled enzymes whose activity relies on spatial segregation and intra- and intermolecular regulation. Here, we describe two experimental methodologies that permit the identification and characterization of GTPase binding sites on activators by assaying complex formation within a broad range of affinities. In the first assay system, proteins presented on the surface of filamentous phage are used to probe affinity determinants of protein-protein interactions. In this application, a protein-displayed phage library is generated by random mutagenesis and a plate-based selection is performed to identify mutations that confer higher binding affinity to an immobilized target. The second method uses light scattering as a tool for measuring the molecular weight, stoichiometry, and polydispersity of protein complexes in solution. In this application, conventional gel filtration chromatography provides initial fractionation, and in-line light scattering measurements allow accurate determination of molar masses of the eluent. This technique also provides information about conformational homogeneity which can be used as a quality     

Regulation of sprouty stability by Mnk1-dependent phosphorylation

Sprouty (Spry) proteins are negative feedback modulators of receptor tyrosine kinase pathways in Drosophila melanogaster and mammals. Mammalian Spry proteins have been shown to undergo tyrosine and serine phosphorylation in response to growth factor stimulation. While several studies have addressed the function of tyrosine phosphorylation of Spry, little is known about the significance of Spry serine phosphorylation. Here we identify mitogen-activated protein kinase-interacting kinase 1 (Mnk1) as the kinase that phosphorylates human Spry2 (hSpry2) on serines 112 and 121. Mutation of these serine residues to alanine or inhibition of Mnk1 activity increases the rate of ligand-induced degradation of hSpry2. Conversely, enhancement of serine phosphorylation achieved through either the inhibition of cellular phosphatases or the expression of active Mnk1 results in the stabilization of hSpry2. Previous studies have shown that growth factor stimulation induces the proteolytic degradation of hSpry2 by stimulating tyrosine phosphorylation on hSpry2, which in turn promotes c-Cbl binding and polyubiquitination. A mutant of hSpry2 that is deficient in serine phosphorylation displays enhanced tyrosine phosphorylation and c-Cbl binding, indicating that serine phosphorylation stabilizes hSpry2 by exerting an antagonistic effect on tyrosine phosphorylation. Moreover, loss of serine phosphorylation and the resulting enhanced degradation of hSpry2 impair its capacity to antagonize fibroblast growth factor-induced extracellular signal-regulated kinase activation. Our results imply that Mnk1-mediated serine phosphorylation of hSpry2 constitutes a regulatory mechanism to extend the temporal range of Spry activity.     

Adipogenesis and lipid production in adipocytes subjected to sustained tensile deformations and elevated glucose concentration: a living cell-scale model system of diabesity

Adipocyte fate commitment is characterized by morphological changes of fibroblastic pre-adipocyte cells, and specifically by accumulation of lipid droplets (LDs) as part of the adipogenesis metabolism. Formation of LDs indicates the production of triglycerides from glucose through an insulin-regulated glucose internalization process. In obesity, adipocytes typically become insulin resistant, and glucose transport into the cells is impaired, resulting in type 2 diabetes. In the present study, we monitored the adipogenesis in 3T3-L1 cultured cells exposed to high (450 mg/dL hyperglycemia) and low (100 mg/dL physiological) glucose concentrations, in a novel cell culture model system of diabesity. In addition to glucose conditions, cells were concurrently exposed to different substrate tensile strains (12% and control) based on our prior work which revealed that adipogenesis is accelerated in cultures subjected to static, chronic substrate tensile deformations. Phase-contrast images were taken throughout the adipogenesis process (3 weeks) and were analyzed by an image processing algorithm which quantitatively monitors cell differentiation and lipid accumulation (number of LDs per cell and their radius as well as cell size and shape). The results indicated that high glucose concentrations and substrate tensile strains delivered to adipocytes accelerated lipid production by 1.7- and 1.4-fold, respectively. In addition, significant changes in average cell projected area and in other morphological attributes were observed during the differentiation process. The importance of this study is in characterizing the adipogenesis parameters as potential read-outs that can predict the occurrence of insulin resistance in the development of diabesity.     

Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion

Background

Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50–90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP_1,2) on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP_1,2 is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP_1,2 trimeric, viral surface spike. After entry into host endosomes, GP_1,2 is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown.

Principal Findings

We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS) of glycoproteins from two different ebolaviruses that although enzymatic priming of GP_1,2 is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP_1,2. Further, ELISA binding data of primed GP_1,2 to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion.

Significance

The results reveal that the ebolavirus GP_1,2 ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP_1,2 and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.     

Isolation of a dehydrin cDNA from orange and grapefruit citrus fruit that is specifically induced by the combination of heat followed by chilling temperatures

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to environmental stresses such as water stress, salinity and freezing or which occur during the late stages of embryogenesis. Previously, it was reported that citrus contains a small gene family encoding a unique class of dehydrins that differs from most other plant dehydrins in various respects, such as having an unusual K-segment similar to that of gymnosperms. In the present study, we identified by cDNA differential display analysis a 'Navel' orange 202-bp polymerase chain reaction (PCR) fragment, which encoded the typical plant angiosperm-type K-segment consensus sequence, and of which the expression was down-regulated by exposure to low oxygen levels. The full-length cDNA sequence of the orange DHN, designated csDHN (for Citrus sinensis DHN), was further isolated by 5'-and 3'-RACE; it had a total length of 933 bp and encoded a predicted polypeptide of 235 amino acids. In addition, the same 202-bp 'Navel' dehydrin PCR fragment was used to screen a 'Star Ruby' grapefruit flavedo cDNA library, and its full-length grapefruit homologue, designated cpDHN (for C. paradisi DHN) was isolated and found to have a total length of 1024 bp and to encode a predicted polypeptide of 234 amino acids. The defined orange and grapefruit DHN proteins were completely identical in the 196 amino acids of their N-terminus but differed in their C-terminus region. Overall, the csDHN and cpDHN proteins share 84% identity and contain the conserved dehydrin serine cluster (S-segment) and a putative nuclear localization signal, but csDHN has one conserved dehydrin K-segment consensus sequence, whereas cpDHN contains two dehydrin K-segments. Both csDHN and cpDHN represent single copy genes, in 'Navel' orange and 'Star Ruby' grapefruit genomes, respectively. We found that the cpDHN gene was consistently expressed in the fruit peel tissue at harvest, but that its message levels dramatically decreased during storage at either ambient or low temperatures. However, a pre-storage hot water treatment, given to enhance fruit-chilling tolerance, increased cpDHN mRNA levels during the first 3 weeks of cold storage at 2 degrees C, and enabled the message levels to be retained for up to a further 8 weeks of cold storage at 2 degrees C. The hot water treatment by itself had no inductive effect on cpDHN gene expression when the fruits were held at non-chilling temperatures. Other stresses applied to the fruit, such as wounding, UV irradiation, water stress, low oxygen and exposure to the stress hormone ethylene decreased DHN mRNA levels, whereas abscisic acid had no effect at all.