Fig. 8: Sizing of nanoscale clusters by smMDS.

a Schematic of TDP-43 phase separation and the formation of nanoscale clusters in the pre-phase separating regime. b Phase separation behavior of GFP-tagged TDP-43 as a function of KCl concentration as observed by widefield microscopy imaging. The phase diagram (left panel) was generated from measurements at five KCl concentrations and at 0.5 µM protein concentration. Representative images at 100 mM and 25 mM KCl are shown (right panels). c_crit denotes the critical KCl concentration. Experiments were repeated at least three times with similar results. Scale bar is 10 µm. c Continuous scan diffusion profiles for 0.5 µM GFP-tagged TDP-43 at 100 mM KCl. The upper panel shows the diffusion profile as obtained from a continuous scan measurement. Bright bursts indicate nanoclusters passing through the confocal detection volume. The bottom panel is a re-binned diffusion profile to extract the size of monomeric TDP-43. Diffusion profiles are shown as blue lines, experimental fits as orange lines, and error as green bands. The extracted R_H [with errors] is given as an inset. d Exemplary fluorescence time trace (1-ms binning) from a step scan measurement at channel position 960 µm, as indicated in panel e. Nanoclusters were detected as bursts that exhibit a signal >5 standard deviations above the mean. Detection events are highlighted in red. e Total intensity of a segmented step scan across the chip (top panel) and histogram of detected nanocluster events as a function of chip position (bottom panel). Gaussian distributions were fit to each peak to extract a mean diffusion distance at each channel position. f Plot of mean diffusion distance versus time of travel within the channel. The inset graphically shows how diffusion distances were determined. The diffusion distance corresponds to the half of the full-width half maximum (FWHM) of the Gaussian distributions at each measurement point. The width at timepoint zero was used for normalization. Data points (mean) are from three repeats; error bars indicate standard deviations. The orange line shows the fit according to Eq. 1. The extracted average R_H of TDP-43 nanoclusters is given as an inset (mean ± standard deviation).