Fig. 4

Point mutations at oligomerization interface abrogate TDP-43 polymerization in vitro and in cells. a Transmission electron microscopy (TEM) image of TDP-43 NTD fibrillar oligomers following negative staining. One region (dotted black box) from the image is shown magnified on the right. b Position of the six amino acids that were substituted to abolish oligomerization are shown on the cartoon of TDP-43 NTD crystal structure with side chains shown as sticks and labeled in red. c Overlay of 2D ¹H-¹⁵N HSQC spectra of wild type TDP-43 NTD (blue) and oligomerization mutant TDP-43 NTD-6M (red) at protein concentration of 100 μM at 30 °C. d Overlay of 2D¹H-¹⁵N HSQC spectra of TDP-43 oligomerization mutant TDP-43 NTD-6M at increasing protein concentration of 100, 200, 400, and 800 μM in red, green, magenta and black, respectively at 30 °C. e TEM image showing the lack of fibrillar structures for the oligomerization mutant TDP-43 NTD-6M. One region (dotted black box) from the image is shown magnified on the right. f Immunoblots (from 4–12% denaturing polyacrylamide gel) with anti-GFP antibody (upper panel) of lysates obtained from cells transiently expressing wild-type GFP-tagged human TDP-43 (GFP-TDP-43) or oligomerization mutants (GFP-TDP-43-Tm/Hm/4 M/5 M/6 M/ΔNTD) followed by DSG-mediated cross-linking. Oligomerization mutants display significantly reduced high-molecular weight oligomeric TDP-43 compared to wild-type TDP-43. Actin is used as protein loading control (lower panel). Immunoblots are representative of three independent experiments