Fig. 2

Extracellular Tau species activate TNT formation in neuronal CAD cells. a Characterization of Tau fibrils. Electron micrographs of recombinant hTau1N4R (40 μM) fibrils assembled in the presence of heparin (10 μM), left panel, Sup35NM (20 μM) fibrils, right panel). Scale bars: 0.1 μm. b Confocal imaging of TNTs in neuronal CAD cells in basal conditions. Cells were infected with LVs encoding GFP-Actin (red) and mCherry-Tubulin (green), and confocal imaging was performed 24 h later. Inset: high magnification of TNTs (white arrows). c Confocal imaging of TNTs in neuronal CAD cells after activation with Tau. Cells were infected with LVs encoding GFP-Actin (red) and mCherry-Tubulin (green). At 24 h later, non-labeled hTau1N4R fibrils were added to the extracellular medium, and cells were immediately imaged. Inset: high magnification of TNTs (white arrows). d Quantification of CAD cells with TNTs under basal conditions (CTL) and after activation with 1 μM hTau1N4R fibrils (Tau); n = 4 independent experiments; 200 cells per experiment (*, p < 0.1; Mann-Whitney test). e Quantification of CAD cells with TNTs under basal conditions (CTL) and after activation with the amyloid fibril prion Sup35NM (Sup35); n = 3 independent experiments; 200 cells per experiment (NS, non significant; Mann-Whitney test). For (b), (c), (d) and (e), cells were observed via laser-scanning confocal microscopy using a 40× oil-immersion lens (NA 1.3) and processed with ZEN and ImageJ software. A focal plane was collected for each specimen. Scale bars: 10 μm