Fig. 3

Extracellular hTau1N4R species favor the establishment of TNTs between primary neurons. a Imaging of TNTs in primary neurons. Cells were infected with LVs encoding mCherry-Actin (red). At 72 h post-infection, cells were processed for immunostaining analysis using anti-acetylated tubulin antibodies visualized with an Alexa 647-labeled secondary antibody (green, polymerized tubulin). Nuclei were labeled with DAPI (blue). b Real-time snapshots of TNTs in primary neurons co-infected with LVs encoding GFP-Actin (red) and mCherry-Tubulin (green, monomeric and polymerized tubulin). (c) Myo10 is present in TNTs in primary neurons. Neurons were plated in Lab-Tek chambers, infected with LVs encoding mCherry-Actin (red) and processed for immunocytochemistry analysis using anti-myosin 10 antibodies visualized with an Alexa 488-labeled secondary antibody (green) and anti-acetylated tubulin visualized with an Alexa 647-labeled secondary antibody (white). Images for (a), (b) and (c) were acquired 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. TNTs are shown in enlargements. For (a), (b) and (c), to observe TNTs in primary neurons, 1 μM hTau1N4R fibrils were added in extracellular medium. TNTs were never observed in primary neurons without addition of exogenous Tau fibrils. Scale bars: 10 μm