Microtubule network is required for insulin signaling through activation of Akt/protein kinase B: evidence that insulin stimulates vesicle docking/fusion but not intracellular mobility

The microtubule network has been shown to be required for insulin-dependent GLUT4 redistribution; however, the precise molecular function has not been elucidated. In this article, we used fluorescence recovery after photobleaching (FRAP) to evaluate the role of microtubules in intracellular GLUT4 vesicle mobility. A comparison of the rate of fluorescence recovery (t((1/2))), and the maximum fluorescence recovered (F(max)) was made between basal and insulin-treated cells with or without nocodazole treatment to disrupt microtubules. We found that intracellular mobility of fluorescently tagged GLUT4 (HA-GLUT4-GFP) was high in basal cells. Mobility was not increased by insulin treatment. Basal mobility was dependent upon an intact microtubule network. Using a constitutively active Akt to signal GLUT4 redistribution, we found that microtubule-based GLUT4 vesicle mobility was not obligatory for GLUT4 plasma membrane insertion. Our findings suggest that microtubules organize the insulin-signaling complex and provide a surface for basal mobility of GLUT4 vesicles. Our data do not support an obligatory requirement for long range microtubule-based movement of GLUT4 vesicles for insulin-mediated GLUT4 redistribution to the cell surface. Taken together, these findings suggest a model in which insulin signaling targets membrane docking and/or fusion rather than GLUT4 trafficking to the cell surface.     

A multi-generation Calanus finmarchicus culturing system for use in long-term oil exposure experiments

Crustacean zooplankton form the keystone link between primary producers and fish stocks along the Norwegian shelf and in the southern part of the Barents Sea. We have established a multi-generation cultivation system for zooplankton in order to perform future experiments on the biological effects of drilling discharges from the offshore petroleum industry. A population of the cold-water species, Calanus finmarchicus, was collected in March 2004 and maintained in a static system of 100 l polypropylene containers through all stages (Eggs-CVI). The population exhibited an average developmental time of 105 days which corresponds to realistic sub-Arctic to Arctic conditions (water temperatures = 5 °C (spring) and 8 °C (late summer)). A series of experiments was performed to examine copepod egg production as a function of different food sources and feeding concentrations. Only minor differences in egg production were observed for C. finmarchicus females fed with varying concentrations of the two diatom species Chaetoceros socialis and Thalassiosira weissflogii. In a separate experiment, the response to food availability was examined over time by tracking egg production and fecal pellet production (used as an indicator of food ingestion). Both hatching success and food ingestion were positively correlated with the food concentrations offered to adult females. Through this work we have demonstrated that C. finmarchicus populations can be maintained in the laboratory through multiple generations. In addition, methods to control egg production through changes in food availability have been established making it feasible to control the start date of exposure experiments or the timing of the collection of eggs to initiate a new generation.     

Mode of action and properties of the beta-xylosidases from Talaromyces emersonii and Trichoderma reesei

Enzymatic hydrolysis of arabinoxylan is an important prerequisite for the utilization of hemicellulose for ethanol fermentation or for making the low calorie sweetener xylitol by catalytic hydrogenation of the generated xylose. This study focus on cloning and characterization of two industrial relevant beta-xylosidases (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from Talaromyces emersonii (betaXTE) and Trichoderma reesei (betaXTR) and a comparison of these in relation to hemicellulose hydrolysis using an industrial relevant substrate. Both beta-xylosidases were expressed in A. oryzae and subsequently purified. During the enzymatic hydrolysis of xylobiose, the reaction product of both enzymes was found to be beta-D-xylose proving that the hydrolysis is proceeding via a retaining reaction mechanism. Based on sequence similarities and glycosyl hydrolases family membership, the active site residues of betaXTE and betaXTR are predicted to be Asp 242 and Glu 441, and Asp 264 and Glu 464, respectively. The involvement in catalysis of these carboxyls was examined by modification using the carbodiimide-nucleophile procedure resulting in a complete inactivation of both enzymes. The degree of xylose release from vinasse, an ethanol fermentation by-product, by betaXTE and betaXTR was 12.1% and 7.7%, respectively. Using the beta-xylosidases in combination with the multicomponent enzyme product Ultraflo L, resulted in 41.9% and 40.8% release of xylose, respectively indicating a strong synergistic effect between the exo-acting beta-xylosidases and the endo-1,4-beta-xylanases and alpha-L-arabinofuranosidase in Ultraflo L. There seems to be no measurable differences between the two beta-xylosidases when used in this specific application despite the differences in specific activity and kinetic properties.     

A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus

The Wnt pathways are evolutionarily well-conserved signal transduction pathways that are known to play important roles in all Metazoans investigated to date. Here, we examine the Wnt pathway genes and target genes present in the genome of the echinoderm Strongylocentrotus purpuratus. Analysis of the Wnt genes revealed that eleven of the thirteen reported Wnt subfamilies are represented in sea urchin, with the intriguing identification of a Wnt-A ortholog thought to be absent in deuterostomes. A phylogenetic study of the Frizzled proteins, the Wnt receptors, performed throughout the animal kingdom showed that not all Frizzled subfamilies were present in the metazoan common ancestor, e.g. Fz3/6 emerged later during evolution. Using sequence analysis, orthologs of the vast majority of the cellular machinery involved in transducing the three types of Wnt pathways were found in the sea urchin genome. Furthermore, of about one hundred target genes identified in other organisms, more than half have clear echinoderm orthologs. Thus, these analyses produce new inputs in the evolutionary history of the Wnt genes in an animal occupying a position that offers great insights into the basal properties of deuterostomes.     

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.     

Selective export of HLA-F by its cytoplasmic tail

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.     

Direct and random routing of a molecular motor protein at a DNA junction

With the aim of investigating how motor proteins negotiate DNA nanostructures, we produced test circuits based on recombination intermediates in which 1D translocation across a Holliday junction (HJ) could be assessed by subsequent triplex displacement signals on each DNA arm. Using the EcoR124I restriction-modification enzyme, a 3′–5′ double-strand DNA (dsDNA) translocase, we could show that the motor will tend to follow its translocated strand across a junction. Nonetheless, as the frequency of junction bypass events increases, the motor will occasionally jump tracks.