RIAM and Vinculin Binding to Talin Are Mutually Exclusive and Regulate Adhesion Assembly and Turnover

Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1–R13) organized into a compact cluster of four-helix bundles (R2–R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.     

Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling

During recycling of synaptic vesicles (SVs), the retrieval machinery faces the challenge of recapturing SV proteins in a timely and precise manner. The significant dilution factor that would result from equilibration of vesicle proteins with the much larger cell surface would make recapture by diffusional encounter with the endocytic retrieval machinery unlikely. If SV proteins exchanged with counterparts residing at steady state on the cell surface, the dilution problem would be largely avoided. In this scenario, during electrical activity, endocytosis would be driven by the concentration of a pre-existing pool of SVs residing on the axonal or synaptic surface rather than the heavily diluted postfusion vesicular pool. Using both live cell imaging of endogenous synaptotagmin Ia (sytIa) as well as pHluorin-tagged sytIa and VAMP-2, we show here that synaptic vesicle proteins interchange with a large pool on the cell axonal surface whose concentration is approximately 10-fold lower than that in SVs.     

Fishes and salinities in the St Lucia estuarine system—a review

The recorded salinity ranges of freshwater, estuarine and marine fish species in Lake St Lucia, a Ramsar and World Heritage Site, are documented. The freshwater group is most diverse and abundant under oligohaline conditions, although the Mozambique tilapia (Oreochromis mossambicus) was common under all salinity regimes. Estuary resident species also favoured oligohaline conditions but, in contrast to the freshwater taxa, were well represented in salinities up to 40 ‰. The marine group was most diverse and abundant within the salinity range 10–40 ‰, but a large number of species could also be found in salinities up to 70 ‰. Very few fish species were able to tolerate salinities between 70 ‰ and 110 ‰, with only O. mossambicus surviving for extended periods in salinities above 110 ‰. All the aquatic macrophytes and most of the zoobenthos within the lake appear to die out within the salinity range of 50–60 ‰, thus creating additional stress to those fish present under such conditions. The food resources least affected by extreme hypersalinity are the microphytobenthos and detritus food chains, with detritivorous fishes being dominant when the lake is in this state. Mass mortalities of fishes in Lake St Lucia have been recorded under both low (<5 ‰) and high salinity (>70 ‰) conditions. The fish kills are often triggered by exceptionally low or high water temperatures which affect the osmoregulatory abilities of these species. Hypersaline conditions and fish mortalities under the most recent closed estuary mouth conditions (2002–2005) are reviewed. If the surface area of St Lucia (35,000 ha) is compared to the total surface area of all South African estuaries (approximately 70,000 ha), then the possibility exists that the loss of the Lake St Lucia nursery area for estuary-associated marine fish species over the past few years may cause significant short-term declines in the future abundance of these taxa on both a local and regional scale.     

Dynamics of neutral and selected alleles when the offspring distribution is skewed

We analyze the dynamics of two alternative alleles in a simple model of a population that allows for large family sizes in the distribution of offspring number. This population model was first introduced by Eldon and Wakeley, who described the backward-time genealogical relationships among sampled individuals, assuming neutrality. We study the corresponding forward-time dynamics of allele frequencies, with or without selection. We derive a continuum approximation, analogous to Kimura's diffusion approximation, and we describe three distinct regimes of behavior that correspond to distinct regimes in the coalescent processes of Eldon and Wakeley. We demonstrate that the effect of selection is strongly amplified in the Eldon-Wakeley model, compared to the Wright-Fisher model with the same variance effective population size. Remarkably, an advantageous allele can even be guaranteed to fix in the Eldon-Wakeley model, despite the presence of genetic drift. We compute the selection coefficient required for such behavior in populations of Pacific oysters, based on estimates of their family sizes. Our analysis underscores that populations with the same effective population size may nevertheless experience radically different forms of genetic drift, depending on the reproductive mechanism, with significant consequences for the resulting allele dynamics.     

Carbohydrate analysis of Acanthamoeba castellanii

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting “cyst wall” material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.     

An integrated study of acute effects of valproic acid in the liver using metabonomics, proteomics, and transcriptomics platforms

An integrated omics approach was undertaken in order to elucidate a systems biology level understanding of the acute hepatotoxcity of valproic acid (VPA). Metabonomics, proteomics and gene expression microarray platforms were employed in this systems biology study. CD-1 female pregnant mice were injected subcutaneously with 600 mg/kg VPA or vehicle control. Urine, serum, and liver tissue were collected at 6, 12, and 24 h after dosing. Principal component analysis (PCA) of the metabonomics data showed clustering of the dosed groups away from the controls for the urine samples. Looser clustering was seen in the other sample sets investigated. However, VPA administration resulted in altered glucose concentrations in urine samples at 12 and 24 h and in aqueous liver tissue extracts at 12 h after VPA administration. Proteomics studies identified two proteins, glycogen phosphorylase and amylo-1,6-glucosidase, which were increased in dosed animals relative to control. Both of these proteins are involved in converting glycogen to glucose. Examination of the expression of 20,000 liver genes did not reveal significantly altered expression at 6, 12, or 24 h after VPA exposure. The combined studies indicated a perturbation in the glycogenolysis pathway following administration of VPA.     

Genetic and phenotypic diversity in <Emphasis Type="Italic">Burkholderia</Emphasis>: contributions by prophage and phage-like elements

Background  

Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains.