T-cell receptors (TCRs) upon binding to peptide-MHC ligands transduce signals in T lymphocytes.Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (γδε) and ζ subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP-70 kinase and other signaling components. We examined the role of the low-density detergent-insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/ CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine-phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM-associated receptor complexes are highly enriched in the hyperphosphorylated p23 ζ chains, contain most of the TCR/CD3-associated, phosphorylation-activated ZAP-70 kinases and seem to integrate into higher order, multiple tyrosine-phosphorylated substrate-containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.

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Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre‐clinical stages of Alzheimer's disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer's disease. After self‐proteolysis, the N‐terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three‐dimensional X‐ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple‐helical filament (QHF‐litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

Although the detailed mechanisms of cell migration remain largely unknown, it is now clear that growth factors and cell adhesion molecules are crucial for this process. We have shown that type I insulin-like growth factor (IGF-I) promotes migration of human colonic tumour cells. Since morphological analysis suggested an involvement of adhesion molecules, we have now examined the role of integrins (cell-matrix adhesion molecules) and E-cadherin/catenins complex (cellcell adhesion molecules) in the IGF-I-induced migration. Using a monolayer wounding assay, we have determined that, except for ␣2␤1, all of the integrins expressed in HT29-D4 cells are involved in the induced cell migration. Immunofluorescence studies revealed that upon IGF-I stimulation the integrins reorganized at the leading edge of migrating cells. We also demonstrate that E-cadherin is involved in cell migration. A rapid tyrosine phosphorylation of E-cadherin and ␤-catenin was detected upon IGF-I stimulation. Tyrosine phosphorylation was associated with reduced membranous expression of E-cadherin and promotion of cell motility, suggesting a regulation of the E-cadherin/catenins complex. This effect can be reversed by incubating cells with tyrosine kinase inhibitors. Taken together, our results suggest that IGF-I promotes colonic cell migration through reorganization of integrin receptors and through modulation of E-cadherin/catenins complex function. Int.

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