Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy

Abstract

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.

Key Nucleation Stages and Associated Molecular Determinants and Processes in pH-Induced Formation of Amyloid Beta Oligomers as Revealed by High-Speed AFM

“…only observed in ~ 2 consecutive frames before leaving the scan area, and show an indistinct morphology likely owing to artefact when tracking fast moving objects [8]. Recently, HS-AFM imaging at 0.05 sec intervals of fast diffusing yeast prion Sup35 NM monomers show similar difficulty in delineating an accurate morphology [9]. Eventually the prion Sup35 NM monomers become 'invisible' at slower imaging intervals of > 0.2 secs.…”

“…One such single molecule technique, High-Speed Atomic Fore Microscopy (HS-AFM), is enabling nanometer resolution imaging on millisecond timescales to observe structural-dynamics of single A monomers, oligomers and fibrils. In particular, HS-AFM studies are implementing approaches such as injecting bio-reagents, adjusting salt or monomer concentration and changing temperature on the "fly" during imaging to induce real-time dynamic changes in A, primarily with a focus on fibril growth [9,[19][20][21]. During the time-course of imaging fibrils, some studies have observed the preceding formation of oligomers on the substrate.…”

High-Speed Atomic Force Microscopy Reveals the Structural Dynamics of the Amyloid-β and Amylin Aggregation Pathways

“…The dimers were less flexible and basically maintained a dumbbell structure in which two spherical structures were connected [110]. Konno and Watanabe-Nakayama et al observed yeast prion Sup35 monomer, oligomer, and fiber elongation [114]. HS-AFM revealed the structural dynamics of the intrinsically disordered (IDR) and partially folded regions of the Sup35 monomer, differences in the core structure and in the IDR between Sup35 oligomers and fibrils, the stepwise growth of oligomers with distinct core size, and the continuous unidirectional elongation of Sup35 fibrils [114].…”

“…α-synuclein fibrils with different structures exhibited different toxicities [7] and caused different AFM was used to capture structural images and measure the nanomechanical properties of individual amyloid aggregates [85][86][87] in the ongoing heterologous aggregation processes of Aβ [20,[88][89][90][91][92][93][94][95][96][97][98], synuclein [90,[99][100][101][102][103][104][105][106], and amylin [107,108]. High-speed AFM (HS-AFM) enabled the kinetic measurement of the structural dynamics of biological molecular processes [79][80][81][82][83][84] including amyloid aggregation [93,[108][109][110][111][112][113][114][115][116]. Here, we show that HS-AFM links structural and dynamics studies, reviewing recent HS-AFM studies and including our findings for Aβ42 [93] and amylin [116], which is associated with ...…”

“…AFM was used to capture structural images and measure the nanomechanical properties of individual amyloid aggregates [ 85 , 86 , 87 ] in the ongoing heterologous aggregation processes of Aβ [ 20 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 ], synuclein [ 90 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 ], and amylin [ 107 , 108 ]. High-speed AFM (HS-AFM) enabled the kinetic measurement of the structural dynamics of biological molecular processes [ 79 , 80 , 81 , 82 , 83 , 84 ] including amyloid aggregation [ 93 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 ]. Here, we show that HS-AFM links structural and dynamics studies, reviewing recent HS-AFM studies and including our findings for Aβ42 [ 93 ] and amylin [ 116 ], which is associated with not only type II diabetes but also AD [ 117 , 118 , 119 , 120 , 121 , 122 , 123 ].…”