Direct three-dimensional visualization of membrane disruption by amyloid fibrils

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer’s, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from β₂-microglobulin (β₂m). Using cryoelectron tomography, we demonstrate that fragmented β₂m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15–25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid–protein interactions in membrane remodelling.The failure of molecular chaperones to prevent the accumulation of misfolded proteins results in protein aggregation and amyloid formation, processes associated with severe human degenerative diseases (1, 2). Despite the attention focused on these problems during the century since these disorders were first identified (3–5) and advances in understanding the structure of the cross-β conformation of amyloid fibrils in atomic detail (6, 7), the basic pathological mechanisms of amyloidosis remain poorly understood and therapeutic intervention is lacking. The identity of the toxic species and the mechanisms of cytotoxicity remain major unsolved problems. In some systems, there is evidence suggesting that prefibrillar oligomers, rather than the fully formed fibrils, are the source of toxicity (8, 9). In these cases, cytotoxicity is thought to result from the formation of specific membrane pores (10, 11) although alternative models including membrane destabilization or membrane thinning have also been proposed (12–15). In other cases, toxicity may reside with the amyloid fibrils themselves. Evidence that toxicity correlates with fibrillar assemblies has been reported for yeast and mammalian prion proteins (16, 17), human lysozyme (18), Huntingtin exon 1, α-synuclein (19), and Amyloid-β (Aβ) (20, 21). Furthermore, Aβ plaques have been shown to form rapidly in vivo and to precede neuropathological changes in a mouse model (22). The end surfaces of fibrils (herein termed “fibril ends”) are unusually reactive entities: they play a key role in catalyzing recruitment and conformational conversion of amyloid-forming proteins (23, 24) and provide the sites for templated elongation of amyloid fibril growth (25, 26). Recently, Xue et al. (27) showed that short fibrils of β₂-microglobulin (β₂m), α-synuclein, and hen lysozyme, each prepared by fragmentation of longer fibrils, cause increased damage to membranes and disruption to cellular function compared with the initial long fibrils. Short and long fibril preparations differ in the number of fibril ends at a given protein concentration. Because these are known to be reactive sites, the above observations suggest a role for fibril ends in amyloid–lipid interaction and possibly in amyloid pathogenesis (23, 24, 27). Fragmented fibrils of all three proteins were also found to induce dye leakage from negatively charged liposomes, the most susceptible of which contain a mixture of the cellular lipids phosphatidylcholine (PC) and phosphatidylglycerol (PG), but liposomes with a variety of compositions were damaged by the fibrils in all cases (27).Here, we use β₂m amyloid fibrils formed in vitro as a model system to investigate the structural basis of membrane damage by amyloid fibrils (27, 28). Previous studies have shown that these fibrils possess a parallel in register cross-β structure (29, 30) assembled into multidomain filaments coiled together, described by cryo-EM (28). These fibrils bind amyloid-specific ligands such as serum amyloid P component with a similar affinity to their ex vivo counterparts (31). Using the conditions under which β₂m amyloid fibrils induce dye release from liposomes (pH 7.4), we examined the effects of both long (∼1,400 nm) and fragmented (∼400 nm) β₂m fibrils (27), as well as various control preparations, on the 3D structures of the liposomes by confocal microscopy, cryo-EM, and tomography. We found pronounced distortions in the liposomes, interruptions to the bilayer structure, and extraction of lipids that were induced by the presence of amyloid fibrils. The most severe distortions were seen in proximity to the fibril ends, which are enriched in the fragmented fibril samples. This type of membrane remodelling appears distinct from the actions of other previously described proteins that induce membrane breakage, as in the action of membrane pore-forming proteins (32). The results suggest a role of fibrils in membrane damage that could contribute to the cellular dysfunction associated with amyloid disease.