A label-free, quantitative assay of amyloid fibril growth based on intrinsic fluorescence.

Kinetic assay of seeded growth: The graph shows the variation in intrinsic fluorescence intensity of amyloid fibrils. Fluorescence increases during the seeded aggregation of α-synuclein seeds with α-synuclein monomeric protein (blue curve) but not when α-synuclein seeds are incubated with β-synuclein monomeric protein (black curve), thus showing that no seeded growth occurred in this case

Physical determinants of the self-replication of protein fibrils

The ability of biological molecules to replicate themselves, achieved with the aid of a complex cellular machinery, is the foundation of life. However, a range of aberrant processes involve the selfreplication of pathological protein structures without any additional factors. A dramatic example is the autocatalytic replication of pathological protein aggregates, including amyloid fibrils and prions, involved in neurodegenerative disorders. Here, we use computer simulations to identify the necessary requirements for the self-replication of fibrillar assemblies of proteins. We establish that a key physical determinant for this process is the affinity of proteins for the surfaces of fibrils. We find that self-replication can only take place in a very narrow regime of inter-protein interactions, implying a high level of sensitivity to system parameters and experimental conditions. We then compare our theoretical predictions with kinetic and biosensor measurements of fibrils formed from the Aβ peptide associated with Alzheimer’s disease. Our results show a quantitative connection between the kinetics of self-replication and the surface coverage of fibrils by monomeric proteins. These findings reveal the fundamental physical requirements for the formation of supra-molecular structures able to replicate themselves, and shed light on mechanisms in play in the proliferation of protein aggregates in nature.We acknowledge support from the Human Frontier Science Program and Emmanuel College (A.Š), Leverhulme Trust and Magdalene College (A.K.B), St. John’s College (T.C.T.M), the Biotechnology and Biological Sciences Research Council (T.P.J.K. and C. M. D.), the Frances and Augustus Newman Foundation (T.P.J.K.), the European Research Council (T.P.J.K., S.L. and D.F), and the Engineering and Physical Sciences Research Council (D.F.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/nphys382

Direct observation of heterogeneous amyloid fibril growth kinetics via two-color super-resolution microscopy.

The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson's disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures

Expanding the solvent chemical space for self-assembly of dipeptide nanostructures.

Nanostructures composed of short, noncyclic peptides represent a growing field of research in nanotechnology due to their ease of production, often remarkable material properties, and biocompatibility. Such structures have so far been almost exclusively obtained through self-assembly from aqueous solution, and their morphologies are determined by the interactions between building blocks as well as interactions between building blocks and water. Using the diphenylalanine system, we demonstrate here that, in order to achieve structural and morphological control, a change in the solvent environment represents a simple and convenient alternative strategy to the chemical modification of the building blocks. Diphenylalanine (FF) is a dipeptide capable of self-assembly in aqueous solution into needle-like hollow micro- and nanocrystals with continuous nanoscale channels that possess advantageous properties such as high stiffness and piezoelectricity and have so emerged as attractive candidates for functional nanomaterials. We investigate systematically the solubility of diphenylalanine in a range of organic solvents and probe the role of the solvent in the kinetics of self-assembly and the structures of the final materials. Finally, we report the crystal structure of the FF peptide in microcrystalline form grown from MeOH solution at 1 Å resolution and discuss the structural changes relative to the conventional materials self-assembled in aqueous solution. These findings provide a significant expansion of the structures and morphologies that are accessible through FF self-assembly for existing and future nanotechnological applications of this peptide. Solvent mediation of molecular recognition and self-association processes represents an important route to the design of new supramolecular architectures deriving their functionality from the nanoscale ordering of their components.We thank the Newman Foundation (T.O.M., T.P.J.K.), the FEBS and the Tel Aviv University Center for Nanoscience and Nanotechnology (A.L.), the BBSRC (T.P.J.K.), and the Leverhulme Trust and Magdalene College (A.K.B.) for financial support. A.L. thanks Or Berger for his assistance with the HR-SEM imaging. The X-ray diffraction data collection experiments were performed in the crystallographic X-ray facility at the Department of Biochemistry, University of Cambridge. The authors thank Pavel Afonin for help with PHENIX software suite in the refinement of the structures.This is the accepted manuscript for a paper published in ACS Nano, 2014, 8 (2), pp 1243–1253 DOI: 10.1021/nn404237f , Publication Date (Web): January 14, 201

Nanoscopic insights into seeding mechanisms and toxicity of α-synuclein species in neurons.

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.We thank Dr Q. Jeng and Dr A. Stephens for technical assistance and Dr J. Skepper for TEM imaging. This work was funded by grants from the U.K. Medical Research Council (MR/K015850/1 and MR/K02292X/1), Alzheimer’s Research UK (ARUK-EG2012A-1), U.K. Engineering and Physical Sciences Research Council (EPSRC) (EP/H018301/1) and the Wellcome Trust (089703/Z/09/Z). D.P. wishes to acknowledge support from the Swiss National Science Foundation and the Wellcome Trust through personal fellowships. A.K.B thanks Magdalene College, Cambridge and the Leverhulme Trust for support.This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.1516546113

Quantitative thermophoretic study of disease-related protein aggregates

Amyloid fibrils are a hallmark of a range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. A detailed understanding of the physico-chemical properties of the different aggregated forms of proteins, and of their interactions with other compounds of diagnostic or therapeutic interest, is crucial for devising effective strategies against such diseases. Protein aggregates are situated at the boundary between soluble and insoluble structures, and are challenging to study because classical biophysical techniques, such as scattering, spectroscopic and calorimetric methods, are not well adapted for their study. Here we present a detailed characterization of the thermophoretic behavior of different forms of the protein a-synuclein, whose aggregation is associated with Parkinson's disease. Thermophoresis is the directed net diffusional flux of molecules and colloidal particles in a temperature gradient. Because of their low volume requirements and rapidity, analytical methods based on this effect have considerable potential for high throughput screening for drug discovery. In this paper we rationalize and describe in quantitative terms the thermophoretic behavior of monomeric, oligomeric and fibrillar forms of a-synuclein. Furthermore, we demonstrate that microscale thermophoresis (MST) is a valuable method for screening for ligands and binding partners of even such highly challenging samples as supramolecular protein aggregates

Cancer cell migration on straight, wavy, loop and grid microfibre patterns

Cell migration plays an important role in physiological and pathological processes where the fibrillar morphology of extracellular matrices (ECM) could regulate the migration dynamics. To mimic the morphological characteristics of fibrillar matrix structures, low-voltage continuous electrospinning was adapted to construct straight, wavy, looped and gridded fibre patterns made of polystyrene (of fibre diameter ca. 3 μm). Cells were free to explore their different shapes in response to the directly-adhered fibre, as well as to the neighbouring patterns. For all the patterns studied, analysing cellular migration dynamics of MDA-MB-231 (a highly migratory breast cancer cell line) demonstrated two interesting findings: first, although cells dynamically adjust their shapes and migration trajectories in response to different fibrillar environments, their average step speed is minimally affected by the fibre global pattern; secondly, a switch in behaviour was observed when the pattern features approach the upper limit of the cell body's minor axis, reflecting that cells' ability to divert from an existing fibre track is limited by the size along the cell body's minor axis. It is therefore concluded that the upper limit of cell body's minor axis might act as a guide for the design of microfibre patterns for different purposes of cell migration.</p

Nanobodies Raised against Monomeric α-Synuclein Distinguish between Fibrils at Different Maturation Stages

AbstractNanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric αSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble αSyn, comprising approximately amino acids 118–131 and 137–140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when αSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of αSyn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation

Dissidents and God-Talk in the Johannine Epistles

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