Transmitted and target cell–derived TDP-43 interact and are found in the same cytoplasmic granule. (A) Coimmunoprecipitation of overexpressed TDP-L1 + TDP-L2 and endogenous TDP-43 using a polyclonal luciferase antibody demonstrates that the transfected TDP-L1 and TDP-L2 constructs interact with endogenous TDP-43 in HEK-293. Cell lysate of HEK-293 cells transfected with an empty vector (mock) was used as a negative control. One representative Western blot of three replicates is shown. (B) TDP-43 split VenusYFP assay principle and constructs are shown. Oligomerization of TDP-43 monomers fused to nonfluorescent VenusYFP halves (V1 and V2) restores VenusYFP fluorescence, which is visualized by microscopy. (C) Setup for the co-culture experiment using the VenusYFP split constructs. (D) Fluorescence micrographs of co-culture experiment. After 48 h of co-culturing, HEK-293 cells originally transfected with either TDP-V1 or TDP-V2 VenusYFP-positive cells were detected. Cell nuclei were stained with DAPI. Bar, 10 µm. (E) Setup for uptake experiment using TDP full-length Venus (TDP-Venus), followed by sorbitol stress (400 mM for 1 h) and immunostaining for TDP-43. The arrowhead indicates an aggregate. Bar, 5 µm. (F) Representative micrograph of a cell that took up TDP-Venus (green) from CM. After stressing the cell with sorbitol, total TDP-43 (red) and α–smooth muscle actin (cyan) were immunolabeled, and nuclei were stained with DAPI. Arrowheads indicate an aggregate with a core containing TDP-Venus (green; labeled also by the total TDP-43 immunostaining in red) surrounded by a solely red immunostaining signal, indicating endogenously expressed, untagged TDP-43. (Right) Orthogonal projections of confocal z stacks of the aggregate-containing cell. Bars, 5 µm. IP, immunoprecipitation.