Fig. 1.

Intracellular fibrils seed α-Syn aggregation. (A–F) Monomeric fluorescently labeled α-Syn (α-Syn⁵⁹⁴) or α-Syn⁵⁹⁴ PFFs were delivered into QBI-WT-Syn cells by using cationic-liposome reagent (Bioporter). Cells were passaged after 4 h and visualized under DIC and fluorescence-microscopy after fixation at 24 h. Both transduced α-Syn⁵⁹⁴ monomer (A–C, arrowheads) and fibrils (D–F, arrows) could readily be detected within cell boundaries (dashed lines) indicating efficient intracellular delivery. (G–L) QBI cells stably expressing A53T-Syn were transduced with WT-α-Syn PFFs, fixed at 48 h, and immunostained with either a pan-α-Syn antibody (SNL4; G) or a monoclonal antibody specifically recognizing misfolded α-Syn (Syn506; J). Colabeling with Alexa Fluor 488 conjugated PHΑ-L (PHA) was used to reveal the plasma membrane. Confocal microscopy shows large α-Syn-positive intracellular inclusions in fibril-seeded cells (G–I and J–L). (M–O) A 3D view of an inclusion-bearing cell reconstructed from serial confocal images. Removal of the PHA signal (green) reveals the juxtanuclear position of a single prominent α-Syn aggregate and confirmed its intracellular location. (P) Quantification of α-Syn inclusions in QBI-WT-Syn cells seeded with either WT-Syn monomer or PFFs (data from three separate transductions from two independent experiments; n > 500 cells per condition). [Scale bars, 10 μm (F); 6 μm (G); 15 μm (j).]