Agonist-stimulated beta-adrenergic receptor internalization requires dynamic cytoskeletal actin turnover

Stimulation of beta-adrenergic receptors (betaARs) leads to sequential recruitment of beta-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for betaAR endocytosis is not known. In this study, we have used beta(1)- and beta(2) ARs, two ubiquitously expressed members of the betaAR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of beta(2)ARs, beta(1)ARs, and mutants of beta(1)ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit beta-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for beta(2)AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of beta(2)AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated beta(2)AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of beta(2)AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal betaAR internalization.     

ProbFAST: Probabilistic Functional Analysis System Tool

Background  

The post-genomic era has brought new challenges regarding the understanding of the organization and function of the human genome. Many of these challenges are centered on the meaning of differential gene regulation under distinct biological conditions and can be performed by analyzing the Multiple Differential Expression (MDE) of genes associated with normal and abnormal biological processes. Currently MDE analyses are limited to usual methods of differential expression initially designed for paired analysis.     

BayGO: Bayesian analysis of ontology term enrichment in microarray data

Background  

The search for enriched (aka over-represented or enhanced) ontology terms in a list of genes obtained from microarray experiments is becoming a standard procedure for a system-level analysis. This procedure tries to summarize the information focussing on classification designs such as Gene Ontology, KEGG pathways, and so on, instead of focussing on individual genes. Although it is well known in statistics that association and significance are distinct concepts, only the former approach has been used to deal with the ontology term enrichment problem.     

Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.     

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin''s active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.     

ProbCD: enrichment analysis accounting for categorization uncertainty

Background  

As in many other areas of science, systems biology makes extensive use of statistical association and significance estimates in contingency tables, a type of categorical data analysis known in this field as enrichment (also over-representation or enhancement) analysis. In spite of efforts to create probabilistic annotations, especially in the Gene Ontology context, or to deal with uncertainty in high throughput-based datasets, current enrichment methods largely ignore this probabilistic information since they are mainly based on variants of the Fisher Exact Test.