The death receptor CD95 activates the cofilin pathway to stimulate tumour cell invasion

The death receptor CD95 promotes apoptosis through well-defined signalling pathways. In colorectal cancer cells, CD95 primarily stimulates migration and invasion through pathways that are incompletely understood. Here, we identify a new CD95-activated tyrosine kinase pathway that is essential for CD95-stimulated tumour cell invasion. We show that CD95 promotes Tyr 783 phosphorylation of phospholipase C-γ1 through the platelet-derived growth factor receptor-β, resulting in ligand-stimulated phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis. PIP(2) hydrolysis liberates the actin-severing protein cofilin from the plasma membrane to initiate cortical actin remodelling. Cofilin activation is required for CD95-stimulated formation of membrane protrusions and increased tumour cell invasion.     

Characterization of a FGF19 variant with altered receptor specificity revealed a central role for FGFR1c in the regulation of glucose metabolism

Diabetes and associated metabolic conditions have reached pandemic proportions worldwide, and there is a clear unmet medical need for new therapies that are both effective and safe. FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Both have potent effects on normalizing glucose, lipid, and energy homeostasis, and therefore, represent attractive potential next generation therapies for combating the growing epidemics of type 2 diabetes and obesity. The mechanism responsible for these impressive metabolic effects remains unknown. While both FGF19 and FGF21 can activate FGFRs 1c, 2c, and 3c in the presence of co-receptor βKlotho in vitro, which receptor is responsible for the metabolic activities observed in vivo remains unknown. Here we have generated a variant of FGF19, FGF19-7, that has altered receptor specificity with a strong bias toward FGFR1c. We show that FGF19-7 is equally efficacious as wild type FGF19 in regulating glucose, lipid, and energy metabolism in both diet-induced obesity and leptin-deficient mouse models. These results are the first direct demonstration of the central role of the βKlotho/FGFR1c receptor complex in glucose and lipid regulation, and also strongly suggest that activation of this receptor complex alone might be sufficient to achieve all the metabolic functions of endocrine FGF molecules.     

Impaired Auxin Biosynthesis in the defective endosperm18 Mutant Is Due to Mutational Loss of Expression in the ZmYuc1 Gene Encoding Endosperm-Specific YUCCA1 Protein in Maize

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.The phytohormone auxin, as a signaling molecule, controls and coordinates numerous aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most predominant in planta auxin and regulates diverse processes, including cell division, cell elongation, formation and maintenance of meristems, vascular tissue differentiation, phototropism, flowering, and endosperm and embryo growth in developing seeds (Davies, 2010). Despite its critical roles, basic components of IAA biosynthesis are poorly understood, compared with transport and signaling aspects. However, the use of appropriate genetic screens in Arabidopsis (Arabidopsis thaliana) and the use of sensitive analytical tools in the identification of metabolic intermediates have led to significant advancements toward a better understanding of biosynthesis. Currently, there are four proposed Trp-dependent pathways of de novo IAA biosynthesis in plants (Woodward and Bartel, 2005; Pollmann et al., 2009; Normanly, 2010); of these, indole-3-pyruvic acid (IPA) was recently suggested to predominate in Arabidopsis (Mashiguchi et al., 2011; Won et al., 2011; Stepanova et al., 2011) and in pea (Pisum sativum) seeds (Tivendale et al., 2012).The first step of the IPA pathway involves the conversion of Trp to IPA by Trp aminotransferases, first demonstrated in Arabidopsis by Stepanova et al. (2008) and Tao et al. (2008). The mutants of Arabidopsis Trp-aminotransferase (taa1) are defective in shade avoidance syndrome due to reduced levels of IAA. In maize (Zea mays), orthologs of the TAA1 gene include an endosperm-specific gene, ZmTar1 (for TA-Related1; Chourey et al., 2010) and Vanishing tassel2 (Vt2), which encode grass-specific Trp aminotransferases (Phillips et al., 2011). The vt2 mutant is marked by severe developmental abnormality, attributed to approximately 60% reduced IAA levels in the mutant seedlings. These results are significant in showing the functionality of the TAR enzyme and the IPA pathway in IAA biosynthesis in maize. Recently, it was suggested that the IPA pathway also involves the YUCCA (YUC) genes, which encode flavin monooxygenases that are now believed to catalyze the conversion of IPA to IAA (Phillips et al., 2011; Mashiguchi et al., 2011; Stepanova et al., 2011; Won et al., 2011; Kriechbaumer et al., 2012). This is based in part on evidence that the Arabidopsis YUC2 protein, expressed in Escherichia coli, converted IPA to IAA in vitro (Mashiguchi et al., 2011). In Arabidopsis, three Yuc genes, Yuc-1, -4, and -10, are expressed in an overlapping fashion in developing seeds and are considered essential in embryogenesis (Cheng et al., 2007); however, single or double mutant yuc1 yuc4 do not show detectable defects in embryogenesis or seed phenotype.Orthologs of the AtYuc genes are now described in several plant groups, including maize (Gallavotti et al., 2008; LeClere et al., 2010). The first Yuc-like gene in maize was isolated through positional cloning of the sparse inflorescence1 (spi1) locus; spi1 mutants showed auxin-deficient-related characteristics in the male inflorescence (Gallavotti et al., 2008). The second gene, ZmYuc1, is highly endosperm specific and its temporal expression pattern coincided with IAA biosynthesis at various stages of seed development (LeClere et al., 2010). In pea, two highly similar PsYuc-like genes, PsYuc1 and PsYuc2, showed seed- and root-specific expression, respectively (Tivendale et al., 2010). Metabolic studies in pea, however, showed that only the roots but not seeds can metabolize Trp to IAA through the proposed TAM pathway (Quittenden et al., 2009; Tivendale et al., 2010).In contrast with many studies on auxin-related mutants that affect vegetative parts of the plant, very limited data are available on auxin mutants affecting seed development, even though seeds accumulate higher levels of IAA than any other tissue of the plant. In maize, endosperm synthesizes nearly 100- to 500-fold higher levels of IAA relative to vegetative tissues (Jensen and Bandurski, 1994; LeClere et al., 2008; Phillips et al., 2011). The significance of the large abundance of IAA in developing endosperm remains to be understood, except that it may be used during the very early stages of seed germination because >90% of the total IAA is in biologically inactive conjugated storage form (Jensen and Bandurski, 1994; LeClere et al., 2008). Such a role in germination is consistent with the fact that there are very few viable seed mutants reported in maize that are linked to IAA deficiency, although single-locus recessive mutants (defective kernels [dek]) with various abnormalities in either embryo or endosperm development and with low IAA levels (measured by ELISA) were reported by Lur and Setter (1993). It is significant in this regard that a viable defective endosperm-B18 (hereafter, de18) was identified as associated with IAA deficiency (Torti et al., 1986). Although not quantified by mass spectrometry, de18 endosperms contained total IAA levels (including conjugates) in the range of 6% to 0.3% of the wild type B37 (hereafter, De18) values, during 12 to 40 d after pollination (DAP). At the early stages, the mutant seed phenotype is <50% of the wild type in seed weight, and throughout seed development, mutant seeds are reduced in kernel size and accumulate less dry matter. Furthermore, application of the synthetic auxin, naphthalene acetic acid, to developing seeds largely rescued the de18 mutant phenotype, indicating impairment in IAA biosynthesis or metabolism as the cause of the phenotypic changes (Torti et al., 1986). Recent cellular-level studies also indicated the IAA deficiency of the de18 endosperm; high levels of immunosignal for IAA were detected in the basal endosperm transfer layer (BETL), aleurone, embryo surrounding region domains, and maternal chalazal tissue in De18 but not in the mutant (Forestan et al., 2010). Overall, the maize de18 and the pea tar2 (Tivendale et al., 2012) mutants are thus far the only seed-specific viable mutants linked to auxin deficiency. The objective of this study is to further extend our knowledge on IAA deficit in the de18 kernels, to specifically analyze temporal expression of two major IAA biosynthetic genes and to elucidate the possible molecular basis of the mutant. Our collective data, based on the cloning and sequencing of ZmYuc1 and on mapping studies, indicate that ZmYuc1 and De18 are tightly associated and that the aberrant YUC1 protein in de18 is the causal basis of IAA deficiency and the small seed phenotype in that mutant.     

Balancing sex chromosome expression and satisfying the sexes

Equalizing sex chromosome expression between the sexes when they have largely differing gene content appears to be necessary, and across species, is accomplished in a variety of ways. Even in birds, where the process is less than complete, a mechanism to reduce the difference in gene dose between the sexes exists. In early development, while the dosage difference is unregulated and still in flux, it is frequently exploited by sex determination mechanisms. The Drosophila female sex determination process is one clear example, determining the sexes based on X chromosome dose. Recent data show that in Drosophila, the female sex not only reads this gene balance difference, but at the same time usurps the moment. Taking advantage of the transient default state of male dosage compensation, the sex determination master-switch Sex-lethal which resides on the X, has its expression levels enhanced before it works to correct the gene imbalance. Intriguingly, key developmental genes which could create developmental havoc if their levels were unbalanced show more exquisite regulation, suggesting nature distinguishes them and ensures their expression is kept in the desirable range.     

A Rapid Genotyping Assay for Segregating Human Olfactory Receptor Pseudogenes

Variation in odor perception between individuals is initiated by binding of “odorant” molecules to olfactory receptors (ORs) located in the nasal cavity. To determine the mechanism for variation in odor perception, identification of specific ligands for a large number of ORs is required. However, it has been difficult to identify specific ligands, and ligands have been identified for only 2–3% of the hundreds of mammalian ORs. One way to increase the number of identified ligands is to take advantage of >60 human OR genes that are segregating as a result of a single nucleotide polymorphism, between a functional intact allele and a nonfunctional pseudogene allele. Potential ligands for these ORs can be identified by correlating odor perception of an individual with their genotype [intact/intact (I/I) vs. pseudogene/pseudogene (P/P)] for an OR gene. For this type of study, genotypes must be determined for a large number of individuals. We have developed a PCR-based assay to distinguish between the intact and pseudogene alleles of 49 segregating human OR genes and to determine an individual''s genotype for these genes. To facilitate rapid determination of genotypes for a large number of individuals, the assay uses a small number of simple steps and equipment commonly found in most molecular biology and biochemistry laboratories. Although this assay was developed to distinguish between polymorphisms in OR genes, it can easily be adapted for use in distinguishing single nucleotide polymorphisms in any gene or chromosomal locus.     

Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase

Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680-709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor V(Delta680-709)) and a mutant molecule with the (695)DYDY (698) --> AAAA substitutions (factor V(4A)). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor Va (Wt)). Using an assay employing purified reagents, we found that prothrombinase assembled with factor Va(Delta680-709) displayed an approximately 39% increase in k cat, while prothrombinase assembled with factor Va(4A) exhibited an approximately 20% increase in k cat for the activation of prothrombin as compared to prothrombinase assembled with factor Va(Wt). Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg(271) and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase.