Tau is a microtubule-associated protein in mammalian brain. In Alzheimer's disease, this protein is present in the somatodendritic compartment of certain nerve cells, where it forms a portion of paired helical filament, the major constituent of the neurofibrillary tangle. For clarification of the mechanism of this formation, recombinant human tau and its fragments (N-terminal half, C-terminal half, and 4-repeats) expressed in Escherichia coli were prepared, eight peptide fragments (C-tails 1-8) of the C-tail region were synthesized, and the conformation and capacity for aggregation essential for filamentous structure formation in vitro were examined. Recombinant full-length tau, the N-terminal half, 4-repeats, and the C-terminal half did not form filamentous structures in aqueous solution after standing at 20 degrees C. Peptides corresponding to the C-tail region of tau, C-tail 5, C-tail 7, and C-tail 8, produced the paired filament or single straight filament in acidic solution. The rate of filament formation by each peptide was followed by circular dichroism, which showed the C-tails to have predominantly random coil structures immediately following dissolution in aqueous solution and be gradually converted to the beta-sheet structure. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, whose negative peak intensity at 218 nm in circular dichroism increased due to filamentous structure formation. This delay was eliminated by seeding supersaturated solution of preformed filaments. C-tails interacted with recombinant full-length tau to form definite single straight filament. The C-tail region of tau is thus shown indispensable to the formation of paired helical filament and nucleation to reduce the rate of paired helical filament formation in amyloidogenesis in vitro. These findings may provide some clarification of the pathogenesis of Alzheimer's disease.
E-cadherin is a key Ca-dependent cell adhesion molecule, which is expressed on many cell surfaces and involved in cell morphogenesis, embryonic development, EMT, etc. The fusion protein E-cad-Fc consists of the extracellular domain of E-cadherin and the IgG Fc domain. On plates coated with this chimeric protein, ES/iPS cells are cultivated particularly well and induced to differentiate. The cells adhere to the plate via E-cad-Fc in the presence of Ca2+ and detach by a chelating agent. For the purpose of clarifying the structures of E-cad-Fc in the presence and absence of Ca2+, we analyzed the molecular structure of E-cad-Fc by AFM in liquid. Our AFM observations revealed a rod-like structure of the entire extracellular domain of E-cad-Fc in the presence of Ca2+ as well as trans-binding of E-cad-Fc with adjacent molecules, which may be the first, direct confirmation of trans-dimerization of E-cadherin. The observed structures were in good agreement with an X-ray crystallographic model. Furthermore, we succeeded in visualizing the changes in the rod-like structure of the EC domains with and without calcium. The biomatrix surface plays an important role in cell culture, so the analysis of its structure and function may help promote cell engineering based on cell recognition.
This paper reports direct imaging of cells with a new scanning mode for atomic force microscopy (AFM). a tapping mode for AFM (TMAFM). Two kinds of cells. Escherichia coli (E. coli) and cultured hamster ovary (CHO) cells. prepared on indium tin oxide (ITO) glasses were observed in air. The TMAFM yielded very reproducible images without appreciable modifications of the sample surfaces. The images were examined in two modes of the data representation; normal height-mode and deflection-mode (similar to a first derivative of height-mode data) representations. The deflection-mode image of the E. coli sample showed much finer surface details than the conventional height-mode one. while the height-mode image of the CHO sample provided more detailed structure than the deflection-mode one.
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