In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-catenin interactions

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.     

Organization of human interferon gamma-heparin complexes from solution properties and hydrodynamics

Heparan sulfate (HS) recognizes a variety of proteins, one of which is the pleiotropic cytokine IFN-gamma, and as such modulates many biological processes. IFN-gamma is a homodimer with a well-defined core and two flexible C-termini that constitute HS binding domains. We show here using molecular modeling that an extended IFN-gamma structure overlaps a HS fragment of 16 disaccharides (16 nm). Since a 21-24-disaccharide HS fragment was experimentally defined as the minimum size that interacts with IFN-gamma [Lortat-Jacob, H., Turnbull, J. E., and Grimaud, J. A. (1995) Biochem. J. 310 (Part 2), 497-505], this raises the question of the complexe organization. We combine analytical ultracentrifugation, size exclusion chromatography, and hydrodynamic bead modeling to characterize the complexes formed in solution with heparin oligosaccharides. For oligosaccharides of 14 and 20 nm, two types of complexes are formed with one IFN-gamma and one or two heparin molecules. Complexes consisting of two IFN-gamma and one or two heparin molecules are present for a fragment of 25 nm and aggregates for a fragment of 35 nm. The complexes are rather compact and can be formed without major conformational changes of the partners. The complex pattern of interaction is related to the size of the partners and their multiple binding possibilities. These various possibilities suggest networks of interactions at the crowded surface of the cells. Hydrodynamic methods used here proved to be very efficient tools for describing protein-HS complexes that, due to the intrinsic heterogeneity and flexibility of the partners, are otherwise very difficult to analyze.     

Functional effects of intramembranous proline substitutions in the staphylococcal multidrug transporter QacA

The QacA multidrug transporter is encoded on Staphylococcus aureus multidrug resistance plasmids and confers broad-range antimicrobial resistance to more than 30 monovalent and bivalent lipophilic, cationic compounds from at least 12 different chemical classes. QacA contains 10 proline residues predicted to be within transmembrane regions, several of which are conserved in related export proteins. Proline residues are classically known as helix-breakers and are highly represented within the transmembrane helices of membrane transport proteins, where they can mediate the formation of structures essential for protein stability and transport function. The importance of these 10 intramembranous proline residues for QacA-mediated transport function was determined by examining the functional effect of substituting these residues with glycine, alanine or serine. Several proline-substituted QacA mutants failed to confer high-level resistance to selected QacA substrates. However, no single proline mutation, including those at conserved positions, significantly disrupted QacA protein expression or QacA-mediated resistance to all representative substrates, suggesting that these residues are not essential for the formation of structures requisite to the QacA substrate transport mechanism.     

RGS2 determines short-term synaptic plasticity in hippocampal neurons by regulating Gi/o-mediated inhibition of presynaptic Ca2+ channels

RGS2, one of the small members of the regulator of G protein signaling (RGS) family, is highly expressed in brain and regulates G(i/o) as well as G(q)-coupled receptor pathways. RGS2 modulates anxiety, aggression, and blood pressure in mice, suggesting that RGS2 regulates synaptic circuits underlying animal physiology and behavior. How RGS2 in brain influences synaptic activity is unknown. We therefore analyzed the synaptic function of RGS2 in hippocampal neurons by comparing electrophysiological recordings from RGS2 knockout and wild-type mice. Our study provides a general mechanism of the action of the RGS family containing RGS2 by demonstrating that RGS2 increases synaptic vesicle release by downregulating the G(i/o)-mediated presynaptic Ca(2+) channel inhibition and therefore provides an explanation of how regulation of RGS2 expression can modulate the function of neuronal circuits underlying behavior.     

Methods to control ectomycorrhizal colonization: effectiveness of chemical and physical barriers

We conducted greenhouse experiments using Douglas-fir (Pseudotsuga menziesii var. glauca) seedlings where chemical methods (fungicides) were used to prevent ectomycorrhizal colonization of single seedlings or physical methods (mesh barriers) were used to prevent formation of mycorrhizal connections between neighboring seedlings. These methods were chosen for their ease of application in the field. We applied the fungicides, Topas (nonspecific) and Senator (ascomycete specific), separately and in combination at different concentrations and application frequencies to seedlings grown in unsterilized forest soils. Additionally, we assessed the ability of hyphae to penetrate mesh barriers of various pore sizes (0.2, 1, 20, and 500 microm) to form mycorrhizas on roots of neighboring seedlings. Ectomycorrhizal colonization was reduced by approximately 55% with the application of Topas at 0.5 g l(-1). Meshes with pore sizes of 0.2 and 1 microm were effective in preventing the formation of mycorrhizas via hyphal growth across the mesh barriers. Hence, meshes in this range of pore sizes could also be used to prevent the formation of common mycorrhizal networks in the field. Depending on the ecological question of interest, Topas or the employment of mesh with pore sizes <1 microm are suitable for restricting mycorrhization in the field.     

Spatial and temporal organization of the Bacillus subtilis replication cycle

DNA replication occurs at discrete sites in the cell. To gain insight into the spatial and temporal organization of the Bacillus subtilis replication cycle, we simultaneously visualized replication origins and the replication machinery (replisomes) inside live cells. We found that the origin of replication is positioned near midcell prior to replication. After initiation, the replisome colocalizes with the origin, confirming that replication initiates near midcell. The replisome remains near midcell after duplicated origins separate. Artificially mispositioning the origin region leads to mislocalization of the replisome indicating that the location of the origin at the time of initiation establishes the position of the replisome. Time-lapse microscopy revealed that a single replisome focus reversibly splits into two closely spaced foci every few seconds in many cells, including cells that recently initiated replication. Thus, sister replication forks are likely not intimately associated with each other throughout the replication cycle. Fork dynamics persisted when replication elongation was halted, and is thus independent of the relative movement of DNA through the replisome. Our results provide new insights into how the replisome is positioned in the cell and refine our current understanding of the spatial and temporal events of the B. subtilis replication cycle.     

Vertical jumping performance of bonobo (Pan paniscus) suggests superior muscle properties

Vertical jumping was used to assess muscle mechanical output in bonobos and comparisons were drawn to human jumping. Jump height, defined as the vertical displacement of the body centre of mass during the airborne phase, was determined for three bonobos of varying age and sex. All bonobos reached jump heights above 0.7 m, which greatly exceeds typical human maximal performance (0.3-0.4m). Jumps by one male bonobo (34 kg) and one human male (61.5 kg) were analysed using an inverse dynamics approach. Despite the difference in size, the mechanical output delivered by the bonobo and the human jumper during the push-off was similar: about 450 J, with a peak power output close to 3000 W. In the bonobo, most of the mechanical output was generated at the hips. To account for the mechanical output, the muscles actuating the bonobo's hips (directly and indirectly) must deliver muscle-mass-specific power and work output of 615 Wkg-1 and 92 Jkg-1, respectively. This was twice the output expected on the basis of muscle mass specific work and power in other jumping animals but seems physiologically possible. We suggest that the difference is due to a higher specific force (force per unit of cross-sectional area) in the bonobo.