α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes
Fig 1
α-Syn fibrils affect the morphology of lysosomes.
(A) Representative confocal images of control and Alexa 568–tagged α-syn fibril-treated (18 hours) CAD cells, immunolabeled for LAMP1 (green). Colocalization between LAMP1+ puncta and α-syn fibrils is indicated by arrows in the inset of a selected region delimited by a square. % of α-syn fibrils colocalizing with LAMP1+ puncta (74 ± 1%) and % of LAMP1+ puncta colocalizing with α-syn fibrils (60 ± 1%) performed by object-based 3D colocalization method (Imaris software) is presented. Mean ± SEM, n = 3 (30 cells per condition). Scale bar: 10 μm (for inset: 2 μm). (B) SR images of control (left panel) and Alexa 568–tagged α-syn fibril-treated CAD cells for 18 hours (right panel), immunolabeled for LAMP1 (far-red) antibody (pseudo colored in gray). Merge images of each condition are presented with additional HCS CellMask Blue staining. Magenta and green arrows indicate the selected lysosomes having α-syn fibrils inside the lysosomal lumen and on the lysosomal membrane, respectively, where higher magnifications of these lysosomes and 3D reconstructions are represented in magenta and green squares, respectively. Scale bar: 10 μm (for insets: 2 μm, for magenta and green insets: 0.5 μm). Average diameter (μm) of lysosomes in control (0.43 ± 0.01) and in α-syn fibril-treated cells, the latter subgrouped as the following: Lysosomes without α-syn fibrils (0.43 ± 0.01) and lysosomes with α-syn fibrils (0.63 ± 0.01) are presented. Mean ± SEM, lysosomes’ diameters were measured in SR images of 8 α-syn fibril-treated cells and 7 control cells (155 lysosomes per condition). Images were acquired by spinning disk microscopy with SR module. ns = not significant, ****P < 0.0001 by Kruskal–Wallis nonparametric ANOVA test followed by Dunn multiple comparison tests. (C) FM image of LAMP1-GFP transfected and α-syn fibril-treated CAD cells for 18 hours (left panel). Enlarged merged images of correlative resin EM and FM images of 4 selected lysosomes not containing (number 1, indicated by green square) and containing α-syn fibrils (numbers 2, 3, and 4, indicated by red squares) are presented. Lysosomes number 1 and 2 are further magnified (blue squares) and presented within the same image. Scale bar: 10 μm (for red and green insets: 0.5 μm, for blue insets: 0.2 μm). (D) Overlay of on-section correlative EM images immunogold labeled for LAMP110 and FM images of LAMP1-GFP transfected and α-syn fibril-treated CAD cells (18 hours). Insets of 3 selected regions (indicated by red, purple, and black squares) are presented. Average perimeter (μm) of lysosomes in control (1.16 ± 0.04) and in α-syn fibril-treated cells: Lysosomes without α-syn fibrils (1.16 ± 0.06) and lysosomes with α-syn fibrils (2.24 ± 0.14; upper graph) are presented. ns = not significant, ****P < 0.0001 by 1-way ANOVA followed by Tukey multiple comparison tests. Average area (μm2) of lysosomes in control (0.08 ± 0.01) and in α-syn fibril-treated cells: Lysosomes without α-syn fibrils (0.08 ± 0.01) and lysosomes with α-syn fibrils (0.16 ± 0.01; lower graph) are presented. Mean ± SEM (50 cells per condition). ns = not significant, ****P < 0.0001 by Kruskal–Wallis nonparametric ANOVA test followed by Dunn multiple comparison tests. Scale bar: 10 μm (for insets: 0.5 μm). The data underlying this figure may be found in S1 Data. α-syn, α-synuclein; CAD, Cath.a-differentiated; EM, electron microscopy; FM, fluorescence microscopy; LAMP, lysosome-associated membrane protein; LD, lipid droplet; Lyso and lys, lysosome; PM, plasma membrane; SR, super-resolution; TEM, transmission electron microscopy.