Figure 5(A) Subcellular localization of α-synucleinfibrils in donor cells after 24 hours co-culture. The left panels show representative images of co-localization of α-synuclein puncta with early endosomes (EEA1, top panel), recycling endosomes (Vamp3, middle panel) and lysosomal vesicles (Lamp1, bottom panel) (see arrows for co-localized spots); in red: α-synucleinfibrils, in green: EEA1, Vamp3 or Lamp1 and in blue: nuclei. Scale bar represents 10 μm. On the right, the bar graph represents the percentage of α-synucleinpuncta co-localized with endo-lysosomal vesicles in A. Data are mean ± s.e.m. from three independent experiments.(B) Confocal images showing a TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 immunostained with Lamp1 (in green). Labelling of TNTs is performed using a plasma membrane dye (WGAAlexa-488, in blue). The image represents a Z-projection of the several middle stacks where the TNT is located. The merged image with WGA-Alexa 633 (blue), the orthogonal view and insets showing 3D reconstructions of the TNT (with WGA in white) are in the right panel. Several α-synuclein fibrillar foci (in red) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles (in green). Of note, only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia. Scale bars represent 10 μm (confocal images) and 3 μm (insets). The merge of α-synucleinfibrils (red) and Lamp1 (green) channels and the respective single channels are showed on the left panel.(C) The top panels show representative images of GFP-transfected acceptor cells after 24 h co-culture with donor cells containing α-synucleinfibrilsATTO-550. Immunofluorescence was performed with endo-lysosomal organelle markers (top panel: EEA1, middle panel: Vamp3 and Lamp1: bottom panel) and revealed that α-synucleinfibrils co-localized with lysosomal vesicles (see arrow); in red: α-synucleinfibrils, in green: organelle markers and in blue: nuclei. Scale bar represents 10 μm, arrows pinpoint assemblies co-localizing with organelle markers. The bar graph at the bottom shows the quantification of the percentage of co-localization of α-synucleinfibrils with organelle markers, revealing that α-synuclein puncta co-localized mostly with lysosomal vesicles. Data show mean ± s.e.m. from three independent experiments.(D) Representative confocal pictures of control cells loaded with ATTO-550α-synucleinfibrils and LysoTracker Deep Red (top panel), and of donor (middle panel) and acceptor GFP-transfected acceptor cells (bottom panel, GFP-vector not shown) cells, loaded with α-synucleinfibrilsATTO-550 and LysoTracker Deep Red (middle panel) and co-cultured for 24 hours; in red: α-synucleinfibrils, in green: LysoTracker Deep Red and in blue: nuclei. Scale bar represents 10 μm, arrows in inset pinpoint fibrils co-localizing with LysoTracker positive vesicles.(E) The percentage of co-localization of LysoTracker positive vesicles with α-synucleinfibrils revealed that 80% of lysosomal vesicles that transferred from donor to acceptor cells contained α-synucleinfibrils thus demonstrating direct transfer of α-synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells. Data show mean ± s.e.m. from three independent experiments.