Figure 3. HspB8 prevents hardening and fiber formation of FUS droplets and keeps them dynamic.

(A) FRAP experiment of fresh FUS_m condensates (0 hr) and condensates incubated for 5 hr. A kymograph shown below illustrates the kinetics of the process. (B) FRAP experiment of fresh FUS_m condensates mixed with HspB8 (0 hr) and condensates incubated for 5 hr. A kymograph shown below illustrates the kinetics of the process. (C) Kinetics of the FUS_m aging process. Plotted are the initial slopes of the FRAP recovery curves for FUS_m condensates in the absence (black) or presence of HspB8 (red). (D) Successful complete fusion events were registered over time, demonstrating the aging process of the FUS_m sample in the absence (turquoise, N=40) or the presence of HspB8 (yellow, N=330). The half-life of liquid-like FUS_m condensates alone was estimated to be around 1.5 hr from logistic regression. (E) The size-normalized coalescence relaxation time is an indicator for the material state of the condensates. While it increases for FUS_m condensates during the hardening process, it stays constant over 6 hr in the presence of HspB8. Tweezer experiments were performed with fresh samples of 5 μM FUS_m with or without 20 μM HspB8. (F) Aging process of 5 µM FUS_m condensates in the absence and presence of 5 µM HspB8. In the presence of the chaperone, the droplet morphology is maintained over the whole timeframe of the experiment (12 hr). Scale bar is 10 µm. (G) Left panel: shown is the total area of droplet material (green line) or fibrous material (red line) within FUS_m droplets as a function of time after FUS_m droplets were added to the existing fibrous material. Spacing between data points is 30 min. Right panel: shown is the total area of droplet material (green line) and fibrous material (red line) within FUS_m droplets as a function of time after FUS_m droplets and HspB8 were added to the existing fibrous material. Spacing between data points is 30 min. (H) The onset of 5 µM FUS_m fiber formation as a function of HspB8 concentration. FRAP, fluorescence recovery after photobleaching.

Figure 3—figure supplement 1. HspB8 prevents hardening and fiber formation of FUS droplets and keeps them dynamic.

(A) Quantification of a half-bleach FRAP experiment of fresh FUS_m condensates (black) and condensates incubated for 5 hr (red). (B) Same experiment as in (A) in the presence of 5 µM HspB8. (C) HspB8-dependent decrease in mobility has an EC₅₀ of 1.5 µM. Plotted are the initial slopes of the FRAP recovery curves for 5 µM FUS_m condensates in the presence of different concentrations of HspB8. (D) Quantification of a full-bleach FRAP experiment of fresh FUS_m condensates in the absence (black) and presence of 5 µM HspB8 (red). (E) Representative images of attempted fusion events at early and late stages. Attempts to fuse several droplets of FUS_m condensates after hardening result in large amorphous assemblies (red arrow). Scale bar is 3 μm. (F) Colocalization of gels or fibers formed by FUS_m and Cy3-labeled HspB8. Scale bar is 10 µm. (G) Crosslink abundance plot from reconstituted FUS_m:HspB8 condensates. Plotted are the relative enrichment (droplet vs. non-droplet) for each unique crosslinking site (y-axis) sorted according to the known domain structure within FUS_m and HspB8 (x-axis). Shown are only high confidence crosslinking sites (see Materials and methods for details) from five biologically independent sets of experiments (n=5; circles in different shades of gray). Crosslinking sites that were consistently upregulated or downregulated twofold or more (log2ratio≥1 or ≤−1 and FDR≤0.05) in at least two out of five biological replicate sets of experiments and in addition contained no opposing regulation in any replicate set were considered significant and are highlighted with a green (enriched in droplets) or red background rectangle (decreased in droplets). All other changes in crosslinking abundances were considered insignificant and are shown on gray background. The significance threshold of twofold enrichment is indicated as dashed red lines. Dimeric links are indicated by an additional ‘−d’, loop-links by a ‘−l’, and mono-links by an ‘-mono’ at their respective unique crosslinking site. (H) Overall crosslinking pattern of FUS_m:HspB8 condensates that were allowed to mature and that were crosslinked 3 hr after their initial formation. (I) Focus on the RRM domain. A comparison of the crosslinks within the RRM (intra, loop, and mono-links) shows no change in abundance when FUS_m:HspB8 condensates of fresh droplets were compared with those that were crosslinked 3 hr after their initial formation. FRAP, fluorescence recovery after photobleaching; RRM, RNA recognition motif.