The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H⁺-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe⁵, Met¹⁰, and Gln¹⁴ involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.     

Collective cell migration of smooth muscle and endothelial cells: impact of injury versus non-injury stimuli

Background

Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration – some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces.

Results

Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour.

Conclusion

For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

     

N-terminal domain of the V-ATPase a2-subunit displays integral membrane protein properties

V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N_1–402) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N_1–402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINE®12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N_1–402 in NDSB-256 was successfully used in pull-down and BIAcore™ protein–protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N_1–402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called “cytosolic” tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N_1–402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.     

A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates

A taxonomic survey was conducted to determine the microbial diversity held within the Harbor Branch Oceanographic Marine Microbial Culture Collection (HBMMCC). The collection consists of approximately 17,000 microbial isolates, with 11,000 from a depth of greater than 150 ft seawater. A total of 2273 heterotrophic bacterial isolates were inventoried using the DNA fingerprinting technique amplified rDNA restriction analysis on approximately 750-800 base pairs (bp) encompassing hypervariable regions in the 5' portion of the small subunit (SSU) 16S rRNA gene. Restriction fragment length polymorphism patterns obtained from restriction digests with RsaI, HaeIII, and HhaI were used to infer taxonomic similarity. SSU 16S rDNA fragments were sequenced from a total of 356 isolates for more definitive taxonomic analysis. Sequence results show that this subset of the HBMMCC contains 224 different phylotypes from six major bacterial clades (Proteobacteria (Alpha, Beta, Gamma), Cytophaga, Flavobacteria, and Bacteroides (CFB), Gram + high GC content, Gram + low GC content). The 2273 microorganisms surveyed encompass 834 alpha-Proteobacteria (representing 60 different phylotypes), 25 beta-Proteobacteria (3 phylotypes), 767 gamma-Proteobacteria (77 phylotypes), 122 CFB (17 phylotypes), 327 Gram + high GC content (43 phylotypes), and 198 Gram + low GC content isolates (24 phylotypes). Notably, 11 phylotypes were < or =93% similar to the closest sequence match in the GenBank database even after sequencing a larger portion of the 16S rRNA gene (approximately 1400 bp), indicating the likely discovery of novel microbial taxa. Furthermore, previously reported "uncultured" microbes, such as sponge-specific isolates, are part of the HBMMCC. The results of this research will be available online as a searchable taxonomic database (www.hboi.edu/dbmr/dbmr_hbmmd.html).     

Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes

Several insect groups have obligate, vertically transmitted bacterial symbionts that provision hosts with nutrients that are limiting in the diet. Some of these bacteria have been shown to descend from ancient infections. Here we show that the large group of related insects including cicadas, leafhoppers, treehoppers, spittlebugs, and planthoppers host a distinct clade of bacterial symbionts. This newly described symbiont lineage belongs to the phylum Bacteroidetes. Analyses of 16S rRNA genes indicate that the symbiont phylogeny is completely congruent with the phylogeny of insect hosts as currently known. These results support the ancient acquisition of a symbiont by a shared ancestor of these insects, dating the original infection to at least 260 million years ago. As visualized in a species of spittlebug (Cercopoidea) and in a species of sharpshooter (Cicadellinae), the symbionts have extraordinarily large cells with an elongate shape, often more than 30 mum in length; in situ hybridizations verify that these correspond to the phylum Bacteroidetes. "Candidatus Sulcia muelleri" is proposed as the name of the new symbiont.     

Peanut gene expression profiling in developing seeds at different reproduction stages during <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>infection

Background  

Peanut (Arachis hypogaea L.) is an important crop economically and nutritionally, and is one of the most susceptible host crops to colonization of Aspergillus parasiticus and subsequent aflatoxin contamination. Knowledge from molecular genetic studies could help to devise strategies in alleviating this problem; however, few peanut DNA sequences are available in the public database. In order to understand the molecular basis of host resistance to aflatoxin contamination, a large-scale project was conducted to generate expressed sequence tags (ESTs) from developing seeds to identify resistance-related genes involved in defense response against Aspergillus infection and subsequent aflatoxin contamination.     

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study

BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.ConclusionsThis study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.