EWI-2 regulates α3β1 integrin–dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (α2β1 integrin ligand). However, on laminin-5 (α3β1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to α3β1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced α3β1–CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance α3β1–CD81 complex formation. These results show how laterally associated EWI-2 might regulate α3β1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes

β1 Integrins Show Specific Association with CD98 Protein in Low Density Membranes

Background: The CD98 (4F2, FRP-1) is a widely expressed cell surface protein heterodimer composed of a glycosylated heavy chain and a non-glycosylated light chain. Originally described as a T cell activation antigen, it was later shown to function in amino acid transport, cell fusion and homotypic cell aggregation. Several lines of evidence suggest its functional interaction with integrins but the biochemical basis for this interaction has been unclear. Results: We demonstrate that CD98 constitutively and specifically associates with β1 integrins (α2β1,α3β1, α5β1 and α6β1), but minimally with α4β1. Integrin-CD98 association was established by reciprocal immunoprecipitation experiments, and confirmed by CD98-induced clustering of α3β1 but not α4β1 on the surface of rhabdomyosarcoma cells. Integrin-CD98 association is independent of the α subunit cytoplasmic tail, is maintained in α3β1 ligand-interaction deficient mutants, and is not inhibited by EDTA. Within the CD98 heavy chain, a C109S mutation (but not a C330S mutation) caused a loss of β1 integrin association. The same C109S mutation also caused a loss of CD98 light chain association. Importantly, CD98 associated selectively with β1 integrins present in low density "light membrane" fractions on a sucrose gradient. CD98 was not present in dense fractions that contained the majority of β1 integrins. Notably, the C109S mutant of CD98, that did not associate with β1 integrins, showed also a reduced localization into light membrane fractions. Conclusions: We demonstrate that CD98 association with β1 integrins is specific, occurs in the context of low density membranes, and may require the CD98 light chain

Evidence for specific tetraspanin homodimers: inhibition of palmitoylation makes cysteine residues available for cross-linking.

It is a well-established fact that tetraspanin proteins, a large family of integral membrane proteins involved in cell motility, fusion and signalling, associate extensively with one another and with other transmembrane and membrane-proximal proteins. In this study, we present results strongly suggesting that tetraspanin homodimers are fundamental units within larger tetraspanin complexes. Evidence for constitutive CD9 homodimers was obtained using several cell lines, utilizing the following four methods: (1) spontaneous cross-linking via intermolecular disulphide bonds, (2) use of a cysteine-reactive covalent cross-linking agent, (3) use of an amino-reactive covalent cross-linking agent, and (4) covalent cross-linking via direct intermolecular disulphide bridging between unpalmitoylated membrane-proximal cysteine residues. In the last case, incubation of cells with the palmitoylation inhibitor 2-bromopalmitate exposed membrane-proximal cysteine residues, thus effectively promoting 'zero-length' cross-linking to stabilize homodimers. Similar to CD9, other tetraspanins (CD81 and CD151) also showed a tendency to homodimerize. Tetraspanin homodimers were assembled from newly synthesized proteins in the Golgi, as evidenced by cycloheximide and Brefeldin A inhibition studies. Importantly, tetraspanin homodimers appeared on the cell surface and participated in typical 'tetraspanin web' interactions with other proteins. Whereas homodimers were the predominant cross-linked species, we also observed some higher-order complexes (trimers, tetramers or higher) and a much lower level of cross-linking between different tetraspanins (CD81-CD9, CD9-CD151, CD81-CD151). In conclusion, our results strongly suggest that tetraspanin homodimers, formed in the Golgi and present at the cell surface, serve as building blocks for the assembly of larger, multicomponent tetraspanin protein complexes

Pedagogical tact as a component of a teacher’s moral culture

The research objective was to describe the phenomenon of pedagogical tact in the context of the cultural and moral traditions of Russian and foreign pedagogical practices. The authors used comparative historical analysis of scientific-pedagogical and historical-pedagogical sources, chronological and comparative methods, and generalization of historical and pedagogical data. According to deontological approach, pedagogical tact is a professional agreement on teacher’s standards of conduct, while the moral-cultural approach views pedagogical tact as a component of teacher’s moral culture and manners. Thus, there is a contradiction in these interpretations of pedagogical tact, which is aggravated in Russia by standardization of pedagogical activityLa meta de la investigación fue describir el tacto pedagógico en el marco de las tradiciones pedagógicas culturales y éticas de Rusia y otros países. Los autores hicieron un análisis comparativo histórico de recursos científicos históricos y pedagógicos, usaron métodos cronológicos y comparativos y generalizaron los datos pedagógicos. En el enfoque deontológico el tacto pedagógico es un acuerdo profesional sobre las normas de conducta de maestros, mientras que en el enfoque ético cultural es un componente del ademán y de la cultura de maestros. Así hay una contradicción entre las interpretaciones y en Rusia eso se empeora por la estandarización de actividades pedagógica

The Obesogenic Gut Microbiota as a Crucial Factor Defining the Depletion of Predicted Enzyme Abundance for Vitamin B12 Synthesis in the Mouse Intestine

Currently, obesity is a critical global public health burden. Numerous studies have demonstrated the regulation of the pathogenesis of obesity and metabolic abnormalities by the gut microbiota and microbial factors; however, their involvement in the various degrees of obesity is not yet well understood. Previously, obesity has been shown to be associated with decreased levels of vitamin B12. Considering exclusive microbial production of vitamin B12, we hypothesized that a decrease in cobalamin levels in obese individuals may be at least partially caused by its depleted production in the intestinal tract by the commensal microbiota. In the present study, our aim was to estimate the abundance of enzymes and metabolic pathways for vitamin B12 synthesis in the gut microbiota of mouse models of alimentary and genetically determined obesity, to evaluate the contribution of the obesogenic microbiome to vitamin B12 synthesis in the gut. We have defined a significantly lower predicted abundance of enzymes and metabolic pathways for vitamin B12 biosynthesis in obese mice compared to non-obese mice, wherein enzyme depletion was more pronounced in lepr(−/−) (db/db) mice, which developed severe obesity. The predicted abundance of enzymes involved in cobalamin synthesis is strongly correlated with the representation of several microbes in high-fat diet-fed mice, while there were almost no correlations in db/db mice. Therefore, the degree of obesity and the composition of the correspondent microbiota are the main contributors to the representation of genes and pathways for cobalamin biosynthesis in the mouse gut

Towards Zebrafish Models of CNS Channelopathies

Channelopathies are a large group of systemic disorders whose pathogenesis is associated with dysfunctional ion channels. Aberrant transmembrane transport of K+, Na+, Ca2+ and Cl− by these channels in the brain induces central nervous system (CNS) channelopathies, most commonly including epilepsy, but also migraine, as well as various movement and psychiatric disorders. Animal models are a useful tool for studying pathogenesis of a wide range of brain disorders, including channelopathies. Complementing multiple well-established rodent models, the zebrafish (Danio rerio) has become a popular translational model organism for neurobiology, psychopharmacology and toxicology research, and for probing mechanisms underlying CNS pathogenesis. Here, we discuss current prospects and challenges of developing genetic, pharmacological and other experimental models of major CNS channelopathies based on zebrafish

Glioblastoma Inhibition by Cell Surface Immunoglobulin Protein EWI-2, In Vitro and In Vivo1,2

EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP), which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP) known to affect proliferation and/or invasion of astrocytes and/or glioblastomas