A Role of Tyrosine Phosphatase in Acetylcholine Receptor Cluster Dispersal and Formation

Innervation of the skeletal muscle involves local signaling, leading to acetylcholine receptor (AChR) clustering, and global signaling, manifested by the dispersal of preexisting AChR clusters (hot spots). Receptor tyrosine kinase (RTK) activation has been shown to mediate AChR clustering. In this study, the role of tyrosine phosphatase (PTPase) in the dispersal of hot spots was examined. Hot spot dispersal in cultured Xenopus muscle cells was initiated immediately upon the presentation of growth factor–coated beads that induce both AChR cluster formation and dispersal. Whereas the density of AChRs decreased with time, the fine structure of the hot spot remained relatively constant. Although AChR, rapsyn, and phosphotyrosine disappeared, a large part of the original hot spot–associated cytoskeleton remained. This suggests that the dispersal involves the removal of a key linkage between the receptor and its cytoskeletal infrastructure. The rate of hot spot dispersal is inversely related to its distance from the site of synaptic stimulation, implicating the diffusible nature of the signal. PTPase inhibitors, such as pervanadate or phenylarsine oxide, inhibited hot spot dispersal. In addition, they also affected the formation of new clusters in such a way that AChR microclusters extended beyond the boundary set by the clustering stimuli. Furthermore, by introducing a constitutively active PTPase into cultured muscle cells, hot spots were dispersed in a stimulus- independent fashion. This effect of exogenous PTPase was also blocked by pervanadate. These results implicate a role of PTPase in AChR cluster dispersal and formation. In addition to RTK activation, synaptic stimulation may also activate PTPase which acts globally to destabilize preexisting AChR hot spots and locally to facilitate AChR clustering in a spatially discrete manner by countering the action of RTKs.     

ERK1/2 Activities Are Dispensable for Oocyte Growth but Are Required for Meiotic Maturation and Pronuclear Formation in Mouse

Previous studies revealed that extracellular regulated kinase-1 and-2(ERK1/2) cascade plays pivotal roles in regulating oocyte meiotic cell cycle progression. However, most knowledge about the in vivo function of ERK1/2 in mammalian oocytes was indirectly obtained from analyzing the phenotypes of Mos knockout mice. In this study, we knocked out Erk1 and Erk2 in mouse oocytes as early as the primordial follicle stage using the well-characterized Gdf9-Cre mouse model, and for the first time directly investigated the in vivo function of ERK1/2 in mouse oocytes. In this novel mouse model, we observed that ERK1/2 activities in oocyte are dispensable for primordial follicle maintenance,activation and follicle growth. Different from the Mos null oocytes, the ERK1/2-deleted oocytes had well-assembled spindles at metaphase Ⅰ(MⅠ), extruded polar body-1(PB1) with normal sizes, and did not undergo a full parthenogenetic activation characterized for pronuclear formation. However, the ovulated ERK1/2-deleted oocytes had poorly-assembled metaphase Ⅱ(MⅡ) spindles, spontaneously released polar body-2(PB2), and were arrested at another metaphase called metaphase Ⅲ(MⅢ). In addition, ERK1/2 deletion prevented male pronuclear formation after fertilization, and caused female infertility. In conclusion, these results indicate that ERK1/2 activities are required for not only MⅡ-arrest maintenance, but also efficient pronuclear formation in mouse oocytes.     

The Nicotinic Acetylcholine Receptor Dα7 Is Required for an Escape Behavior inDrosophila

Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dα7 nicotinic acetylcholine receptor (nAChR) ofDrosophila shows that it is required for the giant fiber-mediated escape behavior. The Dα7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, we found thatgfA¹, a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele ofDα7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dα7 nAChR in giant fiber-mediated escape inDrosophila.     

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm²) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm², respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm²). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm² at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.     

Rice stripe1-2 and stripe1-3 Mutants Encoding the Small Subunit of Ribonucleotide Reductase Are Temperature Sensitive and Are Required for Chlorophyll Biosynthesis

We induced mutants, stripe1-2 (st1-2) and stripe1-3 (st1-3), from rice (Oryza sativa L.) Indica 9311 using Ethyl methanesulfonate (EMS). Both st1-2 and st1-3 mutants encoded the small subunit of ribonucleotide reductase 1 (RNRS1), differed in the location of the mutated base, and displayed white-stripe from the L2 stage through maturity. The mutants were sensitive to temperature, and their chlorophyll content increased with the increase in temperature; however, they did not revert to normal green leaf phenotype under field conditions. The mutant st1-2 showed loosely arranged thylakoid lamellar structure as compared with wild-type (WT) plants. Contrastingly, st1-3 displayed normal thylakoid lamellar structure, good agronomic traits, and higher yield than st1-2 but lower yield than WT. Three-dimensional structure prediction for RNRS1 indicated that the mutation in Val-171 residue in st1-2 influenced the connection of RNRS1 to iron, causing abnormal development of chloroplasts. Real-time PCR analysis showed that the expression levels associated with chlorophyll biosynthetic pathway and photosynthesis were affected in st1-2 and st1-3 at different temperatures and different developmental stages.     

Lateral Opening and Exit Pore Formation Are Required for BamA Function

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Phs1 and the Synthesis of Very Long Chain Fatty Acids Are Required for Ballistospore Formation

The production and dissemination of spores by members of the fungal kingdom is a major reason for the success of this eukaryotic lineage in colonizing most terrestrial ecosystems. Ballistospores are a type of spore produced by basidiomycete fungi, such as the mushrooms and plant pathogenic rusts. These spores are forcefully discharged through a unique liquid-drop fusion mechanism, enabling the aerosolization of these particles that can contribute to plant disease and human allergies. The genes responsible for this process are unknown due to technical challenges in studying many of the fungi that produce ballistospores. Here, we applied newly-developed techniques in a forward genetic screen to identify genes required for ballistospore formation or function in a tractable red yeast, a species of Sporobolomyces. One strain bearing a mutation in the PHS1 gene was identified as a mirror mutant. PHS1 encodes 3-hydroxyacyl-CoA dehydratase required for the third step in very long chain fatty acid biosynthesis. The Sporobolomyces PHS1 gene complements the essential functions of a S. cerevisiae phs1 mutant. The Sporobolomyces phs1 mutant strain has less dehydratase activity and a reduction in very long chain fatty acids compared to wild type. The mutant strain also exhibits sensitivity to cell wall stress agents and loss of shooting due to a delay in ballistospore formation, indicating that the role of Phs1 in spore dissemination may be primarily in cellular integrity.     

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips

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14-3-3 Proteins Are Required for Maintenance of Raf-1 Phosphorylation and Kinase Activity

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.     

The Nicotinic Acetylcholine Receptor Dα7 Is Required for an Escape Behavior inDrosophila

Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dα7 nicotinic acetylcholine receptor (nAChR) ofDrosophila shows that it is required for the giant fiber-mediated escape behavior. The Dα7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, we found thatgfA¹, a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele ofDα7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dα7 nAChR in giant fiber-mediated escape inDrosophila.     

Low Levels of GSTA1 Expression Are Required for Caco-2 Cell Proliferation

The colonic epithelium continuously regenerates with transitions through various cellular phases including proliferation, differentiation and cell death via apoptosis. Human colonic adenocarcinoma (Caco-2) cells in culture undergo spontaneous differentiation into mature enterocytes in association with progressive increases in expression of glutathione S-transferase alpha-1 (GSTA1). We hypothesize that GSTA1 plays a functional role in controlling proliferation, differentiation and apoptosis in Caco-2 cells. We demonstrate increased GSTA1 levels associated with decreased proliferation and increased expression of differentiation markers alkaline phosphatase, villin, dipeptidyl peptidase-4 and E-cadherin in postconfluent Caco-2 cells. Results of MTS assays, BrdU incorporation and flow cytometry indicate that forced expression of GSTA1 significantly reduces cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increases cells in S-phase and associated cell proliferation. Sodium butyrate (NaB) at a concentration of 1 mM reduces Caco-2 cell proliferation, increases differentiation and increases GSTA1 activity 4-fold by 72 hours. In contrast, 10 mM NaB causes significant toxicity in preconfluent cells via apoptosis through caspase-3 activation with reduced GSTA1 activity. However, GSTA1 down-regulation by siRNA does not alter NaB-induced differentiation or apoptosis in Caco-2 cells. While 10 mM NaB causes GSTA1-JNK complex dissociation, phosphorylation of JNK is not altered. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis.     

The Angiotensin II Type 1 Receptor C-Terminal Lys Residues Interact with Tubulin and Modulate Receptor Export Trafficking

The physiological and pathological functions of angiotensin II are largely mediated through activating the cell surface angiotensin II type 1 receptor (AT1R). However, the molecular mechanisms underlying the transport of newly synthesized AT1R from the endoplasmic reticulum (ER) to the cell surface remain poorly defined. Here we demonstrated that the C-terminus (CT) of AT1R directly and strongly bound to tubulin and the binding domains were mapped to two consecutive Lys residues at positions 310 and 311 in the CT membrane-proximal region of AT1R and the acidic CT of tubulin, suggestive of essentially ionic interactions between AT1R and tubulin. Furthermore, mutation to disrupt tubulin binding dramatically inhibited the cell surface expression of AT1R, arrested AT1R in the ER, and attenuated AT1R-mediated signaling measured as ERK1/2 activation. These data demonstrate for the first time that specific Lys residues in the CT juxtamembrane region regulate the processing of AT1R through interacting with tubulin. These data also suggest an important role of the microtubule network in the cell surface transport of AT1R.     

Mixtures of Complete and pif1- and pif2-Deficient Genotypes Are Required for Increased Potency of an Insect Nucleopolyhedrovirus

The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-BΔ16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-BΔpifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-BΔ16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-BΔ16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-BΔpifs were almost identical to the results observed with SfNIC-BΔ16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-BΔ16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of ∼85% SfNIC-B plus ∼15% SfNIC-BΔ16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNIC-BΔpifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1- and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.When hosts are infected by multiple genotypes of a pathogen, competition between genotypes of low relatedness may favor rapid exploitation of host resources, resulting in an increase in the virulence of the infection, reflected in the degree of damage inflicted on the host (12). Because individual self-interest prevails under such conditions, a rapidly replicating genotype will quickly use up host resources for the production of progeny particles, thereby penalizing a more prudent coinfecting genotype, an interaction known as the “tragedy of the commons” (33, 44, 49). In contrast, when relatedness between genotypes is high, cooperative exploitation of host resources is favored because the rate of exploitation of host resources is often determined by the production of intracellular products by the infecting group, an interaction known as “collective action” (2). Each of these models entail different temptations to cheat or defect from the common goal, by excessive greed in the acquisition of public goods (intracellular products) in the case of the tragedy model, and by overly frugal contribution to the pool of public goods in the case of the collective action model (3). In the case of viruses, such game theory approaches to social dilemmas have provided unique insights into the role of cooperation and defection in the evolution of virulence (2, 11, 24, 44), pathogenesis (45), disease management, and the development of potential therapeutic agents (7, 15, 26).Coinfection by multiple genotypes is a common characteristic of many host-parasite systems, especially insect viruses (5, 6, 14, 19-21). When multiple virus particles infect individual host cells, deletion mutants can arise that have lost genes that are essential for transmission or replication (35). These defective particles survive by sequestering the gene products of complete genotypes in coinfected cells. Recently, we demonstrated that deletion genotypes were prevalent in a genotypically diverse population of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) originally isolated in Nicaragua (SfNIC). The most abundant deletion genotype, named SfNIC-C, comprised a 16.4-kb deletion compared to the complete genotype, named SfNIC-B (40). This deletion included two genes, pif1 and pif2, that encode peroral infection factors that are essential for per os infection (9, 25, 32, 36), which is the usual route of transmission of these viruses. SfNIC-C survives by complementation with PIF-encoding genotypes in the population, a process that requires that complete and deletion genotypes replicate simultaneously in the same cells. The persistence of deletion genotypes at a high frequency in the population signifies that multiple infection of cells is likely to be a common event. However, pif1- and pif2-defective genotypes can replicate autonomously in cell culture or in larvae, if injected. Remarkably, the progeny viruses from insects that had been simultaneously inoculated with a mixture of complete and deletion genotypes, in a ratio similar to that found in nature (∼3:1), were ∼2.5 times more pathogenic, as indicated by concentration-mortality metrics, than the complete genotype alone (28, 38, 39). Whether the increased pathogenicity of mixed genotype inocula was specifically due to the deletion has remained unclear and identifying the gene(s) involved represents a key to our understanding of baculovirus infection strategies.The structure of NPV is complex (46). The multiple NPVs consist of single genomes of double-stranded DNA inside individual nucleocapsids. Groups of approximately one to seven nucleocapsids are then wrapped by an envelope to form occlusion-derived virions (ODVs) that are in turn occluded by a protein matrix to form occlusion bodies (OBs) with approximately 20 to 80 virions within each OB. When consumed by a susceptible insect larva, the OBs dissolve in the alkaline insect midgut releasing numerous virions. The ODV membrane fuses with the membranes of midgut epithelial cells, a process that requires the presence of PIFs in the virion membrane (25, 31). After membrane fusion, the package of nucleocapsids is delivered into the host cell and nucleocapsids migrate to the nucleus, where they uncoat and commence replication. Initially, progeny nucleocapsids migrate from the nucleus and bud from the cell as individual virions, each containing a single genome that spread within the host to initiate secondary infections. It is estimated that each cell of the insect is infected by ∼4 budded virions (4, 38). Later, nucleocapsids are retained in the nucleus, wrapped into ODVs and occluded into OBs. After death, the tegument of the insect breaks down and OBs are released onto plant surfaces for transmission to other susceptible insects. It is clear that both the physical structure and the replication cycle of these viruses foster a high prevalence of infection by multiple genotypes.In the present study we examine the genetic basis for increased pathogenicity and test the hypothesis that coinfection of cells by complete and deleted genotypes results in OBs with increased potency compared to those of the complete genotype alone. Here, we use the term potency to mean the quantity of OBs required to produce a certain prevalence of host mortality, such as the 50% lethal concentration, compared to a standard reference, which in this case are OBs of the complete genotype B. As such, potency is a comparative measure of virus insecticidal activity. We demonstrate that the deletion of the pif1 and pif2 region is necessary and sufficient to explain this increased potency. Finally, we examine the dynamics of mixed infection in serial passage in the natural host and demonstrate that previous findings, in which the ratios of genotypes in experimental mixtures converged to a common equilibrium, were determined by the influence that pif1- and pif2-deficient genotypes exert on OB potency, thereby indirectly influencing the probability of virus transmission.