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. 2019 Feb 4;8:e39695. doi: 10.7554/eLife.39695

Figure 2. Super-saturation can be tuned by the levels of endogenous aggregative polypeptides.

and RuvBL-dependent mechanism clears super-critical clusters: (A–F) Representative super-resolved reconstruction and free energy functional fit for AZC- (A and B), MG132-(C and D), and Rapamycin- (E and F) treated cells. Distribution functions were computed from 2500 to 10,000 clusters from 7 to 10 cells in each condition. Red-hot colour code is used to indicate the relative density of detections in A, C, E. Gray plot in B, D, F represents the functional fit for untreated cells for comparison. (G) Representative super-resolution reconstruction for an untreated cell showing many sub-diffractive aggregates (dark red) but few large aggregates (red hot). (H) Schematic of the observed effects of the different pharmacological treatments. (I) Representative super-resolution reconstruction for a RuvBL depleted cell and zoomed view of large (red hot) aggregates. (J) –Log(P(n)) versus n curve from 7000 clusters from 9 RuvBL depletion cells shows almost no effect on the sub-critical distribution. (K–M), quantification of the effect of the perturbations and comparisons of relative changes in the distributions of aggregate size (violin plot, (K), Rc (L) and the measure nucleation barrier; (M) the range of values in L and M is chosen to illustrate the main differences. Error bars in L and M represent s.e.m in fit estimation (Materials and methods). All cells imaged in this figure were fixed cells. Log refers to the natural log (base ‘e’) (See Figure 2 – figure supplement 1 for combined effect of AZC and MG).

Figure 2.

Figure 2—figure supplement 1. The effect of aggregation promoting amino acid substitute (AZC), proteasome inhibitor (MG132), translation inhibitor cycloheximide and the HSP70 inhibitor (Ver155008):

Figure 2—figure supplement 1.

(A) Violin plots of data from untreated (Black) and amino acid substitute AZC treated (Blue) cells. Data is from 10,000 untreated clusters (10 cells) and 4000 AZC clusters (seven cells). (B) Violin plots of data from untreated (Black), proteasome inhibitor MG132 (grey) and the combination of AZC +MG132 (Blue) cells. AZC +MG data is from 8000 clusters from seven cells. (C) Violin plots of cluster sizes, with HSP70 inhibitor Ver155008 in DMSO (grey), and DMSO control (black). Data is of 4000 clusters from five cells. (D) Sub-critical cluster size distribution and bar graph of critical radius for cycloheximide treated cells. Data is from 4500 clusters from 5 cells. (E–G) The sub-critical cluster size distribution is plotted for various times after MG132 treatments. Data is from 4000 to 7000 clusters from 5 to 10 cells at the various time points. In (C–G) Log refers to the natural log (base ‘e’).