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Fig. 3 | BMC Biology

Fig. 3

From: Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold

Fig. 3

Dissection of ApCPEB PLD amyloid-like assembly pathway. a Secondary structure content of soluble ApCPEB PLD conformers at 0h derived from the far-UV CD spectrum deconvolution. b Raw far-UV CD spectra of ApCPEB PLD over time. At zero time, the spectrum shows minima at 222 and 208 nm and a maximum at around 195 nm, characteristic of the α-helical structure. A loss of signal with time is accompanying an increase in the θ222/208nm, which is associated with CCs formation. c Concentration-independent CD spectra normalized by the ellipticity signal value at 222 nm. The change of shape with time can be better appreciated when the relative intensities of the spectra, with relation to the respective value at 222 nm, are compared to compensate for the loss of signal. Normalized spectra do not overlap, particularly at 72 h, showing the occurrence of structural changes over time. d Dot blot analysis of on-pathway ApCPEB multimers formed during the assembly. SDS-sensitive A11-reactive oligomeric species appear from ~ 15 to 75 h and evolve to SDS-resistant OC-reactive, amyloid-like aggregates beyond that time point. e Concentration (μM) of the azo-dye CR bound to ApCPEB PLD amyloid-like species. ApCPEB PLD amyloid-like formation is lowered in the presence of QBP1. Data are expressed as mean ± SEM: ***p< 0.001 (One-way ANOVA and Tukey post-test). f Representative electron micrographs of ApCPEB PLD oligomers and amyloid-like filaments. Scale bar: 50 nm. g Nile Red fluorescence emission spectra of ApCPEB PLD, ApCPEB PLD:AmB, and ApCPEB PLD:EGCG complexes. While treatment with AmB increases the Nile red fluorescence intensity, EGCG causes the opposite effect. h Fluorescence micrographs of COS-7 cells 24 h after the microinjection of indicated samples along with fluorescein-labeled dextran. ApCPEB PLD:AmB microinjection decreases the number of live cells after 24 h. Scale bars: 100 μm. i Survival curves of COS-7 microinjected cells. None of the samples but the ApCPEB PLD species trapped with AmB cause toxicity. The toxic phenotype was rescued by the A11 antibody. Data are expressed as the mean ± SEM: ***p < 0.00 (red asterisks) ApCPEB PLD:AmB versus remaining samples; two-way ANOVA and Bonferroni post-test. Ubi-MCS and the cytoplasmic region of VAMP2 are used as controls of a folded and disordered protein, respectively. No. of cells microinjected per sample: n = 100–200 cells, 3 replicates

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