FIGURE 4.

Transfer of TDP-43 and TDP-43 fragments requires physical connectivity between cells. Extracellular vesicles isolated from stably expressing TDP-43 cell lines and human tissues indicate that the TDP-43 fragments are incorporated into EVs, albeit with a low degree of efficiency (A). The TDP-43 antibody used here recognizes an epitope at the extreme C-terminus of the WT-TDP-GFP^{1– 414}, which is absent in some constructs or obfuscated by the C-terminal GFP tags but can be observed with the anti-GFP antibody (e.g., construct 6^{1– 314}). Isolated EVs were added directly onto WT SH-SY5Y cells but failed to transfer any appreciable protein (B). Further, donor and acceptor cells were physically separated during coculture using Transwell inserts, allowing the distribution of cell products within a shared medium while creating a physical barrier between the cells (C,D), but proteins also fail to transfer by this method. Graphical representations of the experimental setup are shown in (E,F). Full ANOVA tables of (B–D) are available in Supplementary Tables S7–S9. Data in (B) are presented as the proportion of all detected cells that displayed detectable fluorescence, provided as mean ± SEM. Data in (C,D) are presented as the proportion of all detected acceptor cells that are double labeled, provided here mean ± SEM. Significance determined by one-way ANOVA with Tukey post hoc test, n = 6–9 (B), and n = 3 (C,D). *p < 0.05, **p < 0.0, ***p < 0.001, ****p < 0.0001, ns, not significant.