10.1371/journal.pbio.1002361.g005 Rubén Hervás Rubén Hervás Liying Li Liying Li Amitabha Majumdar Amitabha Majumdar María del Carmen Fernández-Ramírez María del Carmen Fernández-Ramírez Jay R. Unruh Jay R. Unruh Brian D. Slaughter Brian D. Slaughter Albert Galera-Prat Albert Galera-Prat Elena Santana Elena Santana Mari Suzuki Mari Suzuki Yoshitaka Nagai Yoshitaka Nagai Marta Bruix Marta Bruix Sergio Casas-Tintó Sergio Casas-Tintó Margarita Menéndez Margarita Menéndez Douglas V. Laurents Douglas V. Laurents Kausik Si Kausik Si Mariano Carrión-Vázquez Mariano Carrión-Vázquez Orb2 cytotoxic oligomeric species rapidly evolve into mature amyloids and they can be kinetically trapped as well as sequestered by pathological amyloids. Public Library of Science 2016 RNA binding protein Orb 2 Orb 2 protein Memory Consolidation Amyloids Orb 2 shares Orb 2 amyloid Orb 2 Amyloidogenesis 2016-01-28 12:42:22 Figure https://plos.figshare.com/articles/figure/_Orb2_cytotoxic_oligomeric_species_rapidly_evolve_into_mature_amyloids_and_they_can_be_kinetically_trapped_as_well_as_sequestered_by_pathological_amyloids_/1642725 <p><b>(A)</b> Structural features during Orb2A assembly probed with conformational antibodies showed the formation of species reactive to A11 and OC antibodies. OC can exist in a SDS-resistant form, while A11-reactive oligomers are SDS-sensitive. <b>(B)</b> Lifespan of A<i>β</i>42 A11-reactive species. <b>(C)</b> Chemical structure of EGCG (left) and amphotericin B (AmB, right), used to trap OC- and A11-reactive oligomers, respectively. <b>(D)</b> Immunodot blot analysis of Orb2A:EGCG and Orb2A:AmB complexes probed with A11 and OC conformational antibodies show that oligomers trapped in the presence of EGCG interact with the OC antibody (and not with A11), whereas those formed in the presence of AmB react strongly with the A11 antibody (and not with OC antibody). <b>(E)</b> Survival curves of COS-7 cells microinjected with several samples (the A11-reactivity of which is indicated). Data are represented as the mean ± SEM: ***<i>p</i> < 0.001 (green asterisks, Orb2A:AmB versus remaining samples; Two-way ANOVA and Bonferroni post-test). Dextran, AmB, the intrinsically disordered region of Vamp2 (Vamp2Cyt) and dimethylsulfoxide (DMSO) are used as controls. The number of single-cells microinjected per sample was <i>n</i> = 100–200. <b>(F)</b> Fluorescence micrographs of COS-7 cells microinjected with different samples and fluorescein-labelled dextran (at 0 and 24 h after microinjection). Microinjection of the Orb2A:AmB complex resulted in a marked drop in the number of live cells at 24 h compared to those at 0 h. Notably, the A11 antibody rescued cells from apoptosis. Scale bars: 100 <i>μ</i>m. <b>(G)</b> Huntingtin protein-containing expanded polyQ repeats (HttQ128) enhances Orb2A aggregation, and this enhancement requires the Orb2A PLD. Orb2A and HttQ128 coaggregates. HttQ does not induce aggregation of other EGFP-tagged neuronal proteins. Substitution of Orb2A PLD with the NM region of yeast Sup35 showed similar HttQ-dependent aggregation. Scale bar: large panel 20 <i>μ</i>m, inset 10 <i>μ</i>m. The underlying data for panels in this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002361#pbio.1002361.s001" target="_blank">S1 Data</a>.</p>