Fig. 2: Site-specific alanine substitutions within the TDP-43 CR reveal key residues in TDP-43 phase separation, helix assembly, and function. | Nature Communications

Fig. 2: Site-specific alanine substitutions within the TDP-43 CR reveal key residues in TDP-43 phase separation, helix assembly, and function.

From: Structural details of helix-mediated multimerization of the conserved region of TDP-43 C-terminal domain

Fig. 2

A Saturation concentration (csat) at 150 mM NaCl for WT CTD (gray box with dashed lines covering the mean ± standard deviation from independent experiments shown in Supplementary Fig. 1) and single alanine substitution variants within CR and its adjacent residues. Data obtained from n  =  3 technical replicates, mean ± SD. B DIC micrographs of CTD variants at 80 μM in 150 mM NaCl. Scale bar, 10 μm. All variants form round liquid-like droplets except N319A (morphologically irregular), Q327A/S333A (slightly less round), and W334A (no droplets), highlighted with a blue box. C 15N chemical shift perturbations (CSP) (Δδ15N) and csat (mean ± SD) for the single alanine substitution variants exhibit a strong correlation (Pearson correlation coefficient, r = −0.94). Both axes are normalized by WT CTD values. D Pairwise intermolecular contact map from two-chain AAMD simulations of TDP-43310–350. Contacts averaged over 18 trajectories; CR contacts highlighted (cyan). E Snapshots from two-chain AAMD simulations show heterogeneous helix-helix contacts, involving a multitude of residues. F Total number of contacts (Ncontact) for each residue position (mean ± SEM from 18 replicas) computed by one-dimensional summation of pairwise contacts from (D). G Ncontact (mean ± SEM) in F show a good correlation (Pearson correlation coefficient, r = 0.7) with the csat (mean ± SD) of single alanine substitution variants in A (including G335A and G338A csat values from our previous work5), normalized to WT. H TDP-43 nuclear retention was quantified by measuring the ratio of HA-TDP-43 in nuclear/cytoplasmic fractionation experiments. Nuclear/cytoplasmic fractionation ratio of WT TDP-43 and its single alanine substitution variants within CR in HEK293HA-TDP-43 were measured by immunoblotting. Nuclear/cytoplasmic fractionation ratio of helix-disrupting mutant, A326P is included as a control of reduced nuclear retention. Statistical analysis was assessed using a two-sided Mann–Whitney U test. ****p < 0.0001, ***p < 0.0005, **p = 0.001, *p = 0.01, ns non-significant. Mean ± SD is shown for ≥5 independent replicates. Source data are provided as a Source Data file.

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