Fig 1.
LSNMR characterization of the PrLD of TDP-43’s aggregation at low pH.
(a) The 1H-15N HSQC spectra of TDP-43 PrLD at pH 4 and 25°C (110 μM), showing a narrow 1H amide chemical shift range typical of a disordered region. Black peaks correspond to a spectrum recorded within 1 hour from sample preparation, the time elapsed between column elution and the start of the NMR experiments (see Methods). The spectrum in red was the last recorded on this sample (S1 and S2 Tables), where signals corresponding to some but not all residues disappear due to self-association. (b) Signals whose intensity is lost over time correspond to the central region spanning residues 311–360 (delimited by blue arrows), which contain an α-helix as evinced by 13Cα chemical shifts (top panel). In addition, several short segments within the 2 regions N-terminal and carboxyl terminus to the 311–360 core (i.e., 267–310 and 361–414) also exhibit moderate signal loss, which are consistently observed at 55 μM (middle panel) and at 110 μM (bottom panel). This is illustrated by plotting the intensity values from the first 1H-15N HSQC (I0, black bars, 1 hour from sample preparation) and those from a 1H-15N HSQC recorded approximately 20 hours later, plotted as I/I0 (red bars, I and I0 correspond to intensity values for the second and first 1H-15N HSQC, respectively, normalized to the most intense peak). Note that average values for the 2 regions 267–310 and 361–414 N-terminal and carboxyl terminus to the central segment are shown as dashed horizontal lines (black and red colors for the I0 and I/I0 data sets, respectively), which highlights that the regions with lower than the average intensities mostly map to segments containing aromatic residues: Phe (yellow boxes), Tyr (gray box), and Trp (green boxes). (c) TDP-43’s PrLD sequence illustrating the central region composed of residues 311–360 (blue lines, to match blue arrows in panel b), which was the focus of some earlier studies and contains the helical segment important for assembly, as well as the aromatic residues outside this core (Phe in yellow, Tyr in gray, and Trp in green). (d) At the end of the NMR experiments, the samples were found aggregated (left panel) and displayed extensive droplet formation (right panel, scale bar: 100 μm). This was imaged on the 110 μM sample. The 15N-1H HSQC spectra are available from Mendeley Data (http://dx.doi.org/10.17632/gyy4tc5nky.1), and the NMR chemical shifts are deposited in the BMRB under accession code 50154. Numerical data for the 3 plots in panel B can be found in S1 Data. BMRB, Biological Magnetic Resonance Bank; LSNMR, liquid-state NMR; PrLD, prion-like domain; TDP-43, Transactive response DNA-binding Protein of 43 kDa.
Fig 2.
Droplet assembly is coupled to fibril formation.
(a) Light scattering following the signal at 280 nm over time (top, blue circles), maximum emission wavelength (top, red circles), and fluorescence anisotropy (bottom, green circles) over time during droplet and fibril formation. (b) The TDP-43’s PrLD sample eluted from the column is free of droplets (top). To test for the presence of amyloid fibrils, ThT was added to the sample and visualized by fluorescence microscopy (bottom). The lack of fluorescence indicates that no amyloid fibrils are present at the beginning of the experiments. (c) Aggregated samples (i.e., samples where no signal is detected in LSNMR experiments) present extensive droplet formation, as well as irregularly shaped clumps (top, light transmitted image). Interestingly, both the droplets and the clumps were recognized by ThT (bottom, fluorescent image). These images were collected using fluorescence microscopy on 110 μM TDP-43 PrLD. The scale bars in panels a and b are 100 μm. (d) Confocal microscopy images of the 55 μM aggregated sample (transmitted light, top; fluorescence, middle; overlay, bottom) showing ThT bound to droplets surface as well as to the irregularly shaped clamps, suggesting that the latter are made by amyloid fibrils which are formed and released from the droplet surface. Scale bar is 50 μm. (e) TEM micrographs confirmed the presence of amyloid fibrils, in agreement with ThT fluorescence (scale bar: 2 μm; at inset image: 200 nm). Numerical data for panel A can be found in S1 Data. PrLD, prion-like domain; TDP-43, Transactive response DNA-binding Protein of 43 kDa; TEM, transmission electron microscopy; ThT, Thioflavin T.
Fig 3.
ThT-positive, TDP-43’s PrLD fibrils emerge from protein droplets.
(a) Visualization of a ThT-stained droplet from the 110 μM aggregated sample by confocal microscopy (top: transmitted light image, bottom: overlay with the ThT fluorescent image; scale bar: 50 μm). The nonhomogeneous staining suggests that fibril assembly occurs on droplet surface, and not its interior. Scale bar is 50 μm. (b) Sections of the droplet evince that the droplet interior is devoid of fibrils, whereas ThT fluorescence maps the droplet surface, at the droplet/solvent interface boundaries, and suggests detachment of fibrils once a certain size is reached. Images 1–24 are selected sections from a collection of 42 slices passing from pole to equator to pole that illustrate the distribution of ThT-positive aggregates across the droplet surface. PrLD, prion-like domain; TDP-43, Transactive response DNA-binding Protein of 43 kDa; ThT, Thioflavin T.
Fig 4.
Fibrils from the PrLD of TDP-43 are stabilized by Phe-Gly motifs.
(a) Two-dimensional 13C-13C DARR spectrum recorded with a 250 ms mixing time on fibrils formed by the PrLD of TDP-43. This experiment detects residues immobilized within the fibril core. Fibrils are rich in β-sheet content, as indicated by the presence of Ser residues showing differences of 13Cβ-13Cα > 8 ppm (black boxes). Cross-peaks between Ser and Gly (black ellipses) indicate that Ser-Gly motifs, which are located outside the helical region, are relevant in the fibril form. Signals around 130–140 ppm (yellow shaded region) correspond to aromatic 13C from Phe/Tyr residues. Considering that there is only 1 Tyr in the PrLD sequence, most cross-peaks arise from Phe (13Cδ/13Cε)–Gly (13Cα) (black circles). (b) Horizontal slices extracted from the 250 ms DARR shown in a, taken at δ1 = 27.03 ppm (in black), which illustrates a crowded region that covers aliphatic, aromatic, and carbonyl peaks; δ1 = 45.07 ppm (in green), which is distinctive of Gly 13Cα chemical shift values, illustrating signals contributed by aromatic 13C nuclei from Phe/Tyr, as well as by 13Cβ of Ser, as identified by their distinctive chemical shift values. This supports the presence of immobilized Gly, Ser, and Phe residues in the fibril core; and δ1 = 49.69 ppm (in blue), which lacks strong resonances thus facilitating an appreciation of the overall signal-to-noise of the SSNMR data. (c) A short mixing time (20 ms) 2D 13C-13C DARR spectrum (in cyan) overlapped onto the 250-ms spectrum from panel a (in red). At this short mixing, only intra-residue contacts are detected, which supports the assignment of sequential contacts that build up at larger mixing times. All these spectra are available from Mendeley Data (http://dx.doi.org/10.17632/gyy4tc5nky.1). DARR, Dipolar Assisted Rotational Resonance; PrLD, prion-like domain; SSNMR, solid-state NMR; TDP-43, Transactive response DNA-binding Protein of 43 kDa.