DNAJA2 and Hero11 suppress large immobile aggregates of TDP-43. (A) Representative microscope images of GFP-TDP43ΔNLS signals in HEK293T cells. Cells were cotransfected with plasmids for GFP-TDP43ΔNLS and the indicated proteins, and after 48 h imaged at 100× optical magnification. (Top row) GFP-TDP43ΔNLS signals. (Bottom row) Brightfield image. (B) Total area (µm²) of GFP-TDP43ΔNLS puncta in the cytoplasm per cell. For each image, total puncta area was measured and normalized by dividing over the number of cells. Each dot represents one image. The error bars represent the means and standard deviations calculated from the 30 images per cotransfection condition. (C) FRAP recovery curves of GFP-TDP43ΔNLS puncta. Cells were cotransfected with plasmids for GFP-TDP43ΔNLS and GST, and after 48 h FRAP was performed via bleaching of an area in the middle of each punctum. Each trace represents the recovery curve for a punctum from −5 sec prebleach to 150 sec postbleach (where the area within the punctum was bleached at 0 sec) and colored according to puncta size. (D) Punctum internal mobility binned according to punctum size. The mobile fractions were extracted from the fits of the exponential recovery functions to the FRAP recovery curves for each punctum. Each dot represents a punctum. Boxplots represent the median and interquartile range, and whiskers represent the 1.5× interquartile range. (E) Cytoplasmic punctum sizes in each of the cotransfections. Each dot represents a punctum. Error lines represent the median and 95% confidence intervals, chosen due to the asymmetrical distribution, calculated from all puncta of a cotransfection. The dotted line indicates 20 µm², and the red text indicates the percentage of aggregates in each cotransfection above this line. There are 1156, 1107, 79, and 281 puncta for the GST, Hero11KR→G, DNAJA2, and Hero11 cotransfections, respectively.
