Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.     

Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins

Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Gα or Gβγ subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gβγ subunits are likely to be involved since the expression of the C terminus of β-adrenergic receptor kinase 1, a Gβγ subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gβγ dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gβγ subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gβγ subunits and activation of MAPKs to repress skeletal muscle differentiation.     

The primate subarcuate fossa and its relationship to the semicircular canals part II: adult interspecific variation

Studies have reported an empirical link between the size of the semicircular canals and locomotor agility across adult primates. In this paper, we investigate the possibility that this relationship does not follow from the function of the semicircular canals to sense head rotations, but rather reflects spatial constraints imposed by the subarcuate fossa. The latter sits among the three canals and contains the petrosal lobule of the cerebellar paraflocculus, a structure involved in neural processing of locomotion-related eye movements. Hence, it is feasible that agility-related variations of lobule and fossa size affect the arc size of the surrounding semicircular canals. The present study tests such hypothetical correlations by evaluating canal size, fossa size, and agility among extant adult primates. Phylogenetically informed multivariate regression analyses show that, after controlling for body mass, the size of the subarcuate fossa has a significant positive effect on the overall size of the anterior canal and the width of the posterior canal. Multivariate regressions involving the height of the posterior canal and overall size of the lateral canal are not significant. Further bivariate analyses confirm that fossa size is unlikely to play a role in the previously reported link between agility and the size of the posterior and lateral canals. However, fossa size, especially its opening though the arc of the anterior canal, cannot be excluded as a factor that influences the size of the anterior canal more than agility. The findings show that the most reliable functional signals pertaining to locomotion in species that possess a patent subarcuate fossa are likely to come from the lateral canal and are least likely to come from the anterior canal.     

M-CSF potently augments RANKL-induced resorption activation in mature human osteoclasts

Macrophage-CSF (M-CSF) is critical for osteoclast (OC) differentiation and is reported to enhance mature OC survival and motility. However, its role in the regulation of bone resorption, the main function of OCs, has not been well characterised. To address this we analysed short-term cultures of fully differentiated OCs derived from human colony forming unit-granulocyte macrophages (CFU-GM). When cultured on dentine, OC survival was enhanced by M-CSF but more effectively by receptor activator of NFκB ligand (RANKL). Resorption was entirely dependent on the presence of RANKL. Co-treatment with M-CSF augmented RANKL-induced resorption in a concentration-dependent manner with a (200-300%) stimulation at 25 ng/mL, an effect observed within 4-6 h. M-CSF co-treatment also increased number of resorption pits and F-actin sealing zones, but not the number of OCs or pit size, indicating stimulation of the proportion of OCs activated. M-CSF facilitated RANKL-induced activation of c-fos and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but not NFκB nor nuclear factor of activated T-cells, cytoplasmic-1 (NFATc1). The mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 partially blocked augmentation of resorption by M-CSF. Our results reveal a previously unidentified role of M-CSF as a potent stimulator of mature OC resorbing activity, possibly mediated via ERK upstream of c-fos.     

Microbial laboratory evolution in the era of genome‐scale science

Laboratory evolution studies provide fundamental biological insight through direct observation of the evolution process. They not only enable testing of evolutionary theory and principles, but also have applications to metabolic engineering and human health. Genome‐scale tools are revolutionizing studies of laboratory evolution by providing complete determination of the genetic basis of adaptation and the changes in the organism's gene expression state. Here, we review studies centered on four central themes of laboratory evolution studies: (1) the genetic basis of adaptation; (2) the importance of mutations to genes that encode regulatory hubs; (3) the view of adaptive evolution as an optimization process; and (4) the dynamics with which laboratory populations evolve.     

Genetic stability of bone marrow-derived human mesenchymal stromal cells in the Quantum System

Background aimsThe Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality.MethodsA rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum.ResultsBM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation.ConclusionsQuantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates.     

Substrate recognition of type III secretion machines--testing the RNA signal hypothesis

Secretion by the type III pathway of Gram-negative microbes transports polypeptides into the extracellular medium or into the cytoplasm of host cells during infection. In pathogenic Yersinia spp., type III machines recognize 14 different Yop protein substrates via discrete signals genetically encoded in 7-15 codons at the 5' portion of yop genes. Although the signals necessary and sufficient for substrate recognition of Yop proteins have been mapped, a clear mechanism on how proteins are recognized by the machinery and then initiated into the transport pathway has not yet emerged. As synonymous substitutions, mutations that alter mRNA sequence but not codon specificity, affect the function of some secretion signals, recent work with several different microbes tested the hypothesis of an RNA-encoded secretion signal for polypeptides that travel the type III pathway. This review summarizes experimental observations and mechanistic models for substrate recognition in this field.     

Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.     

Bax inhibition protects against free fatty acid-induced lysosomal permeabilization

Lysosomal permeabilization is a key feature of hepatocyte lipotoxicity, yet the mechanisms mediating this critical cellular event are unclear. This study examined the mechanisms involved in free fatty acid (FFA)-induced lysosomal permeabilization and the role of Bax, a Bcl-2 family member, in this event. Exposure of liver cells to palmitate induced Bax activation and translocation to lysosomes. Studies to suppress Bax activation either by pharmacological approaches or small interfering-RNA-mediated inhibition of Bax expression showed that lysosomal permeabilization is Bax dependent. In addition, palmitate treatment resulted in a significant decrease in Bcl-X(L), a Bax antagonist. Moreover, forced Bcl-X(L) expression blocked lysosomal permeabilization. Lysosomal permeabilization by FFA was ceramide and caspase independent. Finally, paradigms that inhibit lysosomal permeabilization also reduced apoptosis. In conclusion, these data strongly support a regulatory role for Bax in FFA-mediated lysosomal permeabilization and subsequent cell death.