Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.     

In Situ Speciation and Distribution of Toxic Selenium in Hydrated Roots of Cowpea

The speciation and spatial distribution of selenium (Se) in hydrated plant tissues is not well understood. Using synchrotron-based x-ray absorption spectroscopy and x-ray fluorescence microscopy (two-dimensional scanning [and associated mathematical model] and computed tomography), the speciation and distribution of toxic Se were examined within hydrated roots of cowpea (Vigna unguiculata) exposed to either 20 µm selenite or selenate. Based upon bulk solution concentrations, selenate was 9-fold more toxic to the roots than selenite, most likely due to increased accumulation of organoselenium (e.g. selenomethionine) in selenate-treated roots. Specifically, uptake of selenate (probably by sulfate transporters) occurred at a much higher rate than for selenite (apparently by both passive diffusion and phosphate transporters), with bulk root tissue Se concentrations approximately 18-fold higher in the selenate treatment. Although the proportion of Se converted to organic forms was higher for selenite (100%) than for selenate (26%), the absolute concentration of organoselenium was actually approximately 5-fold higher for selenate-treated roots. In addition, the longitudinal and radial distribution of Se in roots differed markedly: the highest tissue concentrations were in the endodermis and cortex approximately 4 mm or more behind the apex when exposed to selenate but in the meristem (approximately 1 mm from the apex) when exposed to selenite. The examination of the distribution and speciation of Se in hydrated roots provides valuable data in understanding Se uptake, transport, and toxicity.Selenium (Se) is an essential micronutrient for humans and other animals (Rayman, 2008). At elevated concentrations, however, it is toxic, and the concentration range from deficiency to lethality is unusually narrow (Terry et al., 2000). Plants represent a direct entrance to the wider food chain as the main sources of dietary Se (Rayman, 2008). The uptake and accumulation of Se by plants is an important process in controlling the health risks resulting from Se deficiency or toxicity. Se toxicity to plants has been observed in arid and semiarid soils derived from seleniferous rocks and shales, although anthropogenic contamination is also of concern (Terry et al., 2000). Therefore, it is important that the mechanisms of Se uptake, transformation, and toxicity in plants are understood in order to reduce health risks.Selenite (Se[IV]) and selenate (Se[VI]) are the two dominant inorganic species in soils depending upon the redox potential and pH (Elrashidi et al., 1987). The mechanism of Se[VI] uptake is well known: it is taken up by plant roots via the high-affinity sulfate transporters (Terry et al., 2000) due to the similarity between Se[VI] and sulfate. By contrast, little is known about the uptake mechanism involved in Se[IV] in plant roots. Some studies suggested that Se[IV] is taken up via passive diffusion (Shrift and Ulrich, 1969; Arvy, 1993). Recently, Zhao et al. (2010) reported that the uptake of Se[IV] is mediated by the silicon (Si) influx transporter Lsi1 (OsNIP2;1) in rice (Oryza sativa). Furthermore, Se[IV] uptake was found to occur via both passive diffusion and phosphate transporters in the marine coccolithophore Emiliania huxleyi (Araie et al., 2011). Apart from the difference in their mechanisms of uptake, they also differ in their mobility within plants (Li et al., 2008). Se[VI] is relatively easily translocated from roots to shoots, whereas Se[IV] tends to accumulate within the roots (Arvy, 1993). Despite this important progress, much less is known about the sites of uptake of Se[IV]/Se[VI] and their possible chemical transformations in hydrated plant roots. This information regarding the in situ distribution and chemical forms of Se would be helpful in elucidating the mechanism(s) responsible for Se uptake, transformation, and toxicity in plants.Recent advances in synchrotron-based techniques allow in situ measurement of the distribution of metal(loid)s in hydrated fresh plant tissues (Kopittke et al., 2011, 2012; Lombi et al., 2011a). In particular, the prototype Maia detector system, jointly developed by the Australian Synchrotron, the Commonwealth Scientific and Industrial Research Organization, and the Brookhaven National Laboratory, represents a new-generation x-ray fluorescence detector and real-time processing approach that provides unprecedented capabilities in in situ element imaging and measurement (Lombi et al., 2011b). The Maia uses an annular array of 384 silicon-diode detectors positioned in a backscatter geometry to subtend a large solid angle (approximately 1.3 steradian) and to achieve high count-rate capacity (Kirkham et al., 2010). Data acquisition times are approximately 10 to 100 times faster in the Maia than for other detectors, thereby allowing analysis of highly hydrated biological specimens (e.g. roots) without observable damage (Lombi et al., 2011a). This has allowed us to overcome the analytical challenges of examining the two-dimensional and virtual three-dimensional distribution of low-concentration metal(loid)s in hydrated and fresh plant tissues (Kopittke et al., 2011, 2012; Lombi et al., 2011a, 2011c).In this study, we investigated the speciation and quantified the longitudinal and radial distribution of Se in hydrated roots of cowpea (Vigna unguiculata) exposed to either Se[IV] or Se[VI]. Cowpea is a model species of rhizotoxicity and is also one of the most important food legume crops in the semiarid tropics, where Se toxicity is often a concern. The chemical forms of Se in cowpea roots were first examined using x-ray absorption spectroscopy (XAS). Second, with x-ray fluorescence microscopy (µ-XRF), we used two-dimensional imaging (coupled with an associated mathematical model to calculate concentrations of Se within various tissues of the root cylinder) to determine the spatially resolved distribution of Se within root tissues. Additionally, sequential tomography was used to provide virtual three-dimensional reconstructions of Se distribution in roots, enabling comparison of computed tomography with the mathematical model.     

Rspo1/Wnt signaling promotes angiogenesis via Vegfc/Vegfr3

Here, we show that a novel Rspo1-Wnt-Vegfc-Vegfr3 signaling pathway plays an essential role in developmental angiogenesis. A mutation in R-spondin1 (rspo1), a Wnt signaling regulator, was uncovered during a forward-genetic screen for angiogenesis-deficient mutants in the zebrafish. Embryos lacking rspo1 or the proposed rspo1 receptor kremen form primary vessels by vasculogenesis, but are defective in subsequent angiogenesis. Endothelial cell-autonomous inhibition of canonical Wnt signaling also blocks angiogenesis in vivo. The pro-angiogenic effects of Rspo1/Wnt signaling are mediated by Vegfc/Vegfr3(Flt4) signaling. Vegfc expression is dependent on Rspo1 and Wnt, and Vegfc and Vegfr3 are necessary to promote angiogenesis downstream from Rspo1-Wnt. As all of these molecules are expressed by the endothelium during sprouting stages, these results suggest that Rspo1-Wnt-VegfC-Vegfr3 signaling plays a crucial role as an endothelial-autonomous permissive cue for developmental angiogenesis.     

In situ distribution and speciation of toxic copper, nickel, and zinc in hydrated roots of cowpea

The phytotoxicity of trace metals is of global concern due to contamination of the landscape by human activities. Using synchrotron-based x-ray fluorescence microscopy and x-ray absorption spectroscopy, the distribution and speciation of copper (Cu), nickel (Ni), and zinc (Zn) was examined in situ using hydrated roots of cowpea (Vigna unguiculata) exposed to 1.5 μm Cu, 5 μm Ni, or 40 μm Zn for 1 to 24 h. After 24 h of exposure, most Cu was bound to polygalacturonic acid of the rhizodermis and outer cortex, suggesting that binding of Cu to walls of cells in the rhizodermis possibly contributes to the toxic effects of Cu. When exposed to Zn, cortical concentrations remained comparatively low with much of the Zn accumulating in the meristematic region and moving into the stele; approximately 60% to 85% of the total Zn stored as Zn phytate within 3 h of exposure. While Ni concentrations were high in both the cortex and meristem, concentrations in the stele were comparatively low. To our knowledge, this is the first report of the in situ distribution and speciation of Cu, Ni, and Zn in hydrated (and fresh) plant tissues, providing valuable information on the potential mechanisms by which they are toxic.     

Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm

Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmia-esophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2(EGFP), appear normal. However, further reductions in Sox2, using Sox2(LP) and Sox2(COND) hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2(EGFP/COND) embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63(+) basal cells. By contrast, the abnormal foregut of Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2(EGFP/COND) embryos in which Sox2 levels are approximately 18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63(+) cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach.     

Out from Underfoot: Beasts Before Us Brings Mammalian Ancestry to the Forefront

Journal of Mammalian Evolution -     

Differential cholesterol binding by class II fusion proteins determines membrane fusion properties

The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.     

A hybrid approach for the automated finishing of bacterial genomes

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.