Figure 5. hnRNPA2 LC co-phase separates and aggregates with TDP-43 CTD.

a) In 0 mM NaCl, neither hnRNPA2 LC (AlexaFluor-488, green) nor TDP-43 CTD (AlexaFluor-555, red) phase separate on their own even at 40 μM (2:0 and 0:2). However, mixing them at equal concentrations (20 μM, 1:1), induces co-localization to structures resembling droplets (see d).

b) In 50 mM NaCl, TDP-43 CTD does not phase separate on its own even at 40 μM (0:2) but hnRNPA2 LC phase separates at both concentrations. Upon mixing, hnRNPA2 LC and TDP-43 CTD co-localize in droplets.

c) After 2 hours in 50 mM NaCl, TDP-43 CTD shows faint elongated species distinct from droplets. When mixed 1:1 and left to incubate for 2 hours, hnRNPA2 LC and TDP-43 CTD form spherical structures connected by elongated assemblies.

d) Brightfield images of 1:1 hnRNPA2 LC:TDP-43 CTD at each salt concentration. At 0 mM NaCl, 1:1 hnRNPA2:TDP-43 form structures wetting the surface of the coverslip. At 50 mM NaCl, liquid droplets form initially and convert to aggregates.

e) Intermolecular PRE experiments mixing 15 μM ¹⁵N hnRNPA2 LC with 10 μM (black) or 5 μM (gray) natural abundance TDP-43 CTD conjugated to an MTSL at S273C, S317C, S350C, or S387C show that hnRNPA2 interacts with TDP-43 CTD primarily in the region of the helix (S317C) and the C-terminal region (S387C). These interactions are weaker than those observed for homotypic interaction.

f) Intermolecular PRE experiments mixing 10 μM ¹⁵N TDP-43 CTD with 15 μM natural abundance hnRNPA2 LC conjugated to an MTSL at S285C or S329C show that hnRNPA2 interacts with TDP-43 CTD primarily in the region of the helix (residues 320–340).

Data are mean ± SD. Scale bar represents 4 μm. See also Figure S5.