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The molecular mechanism of protein aggregation is of both fundamental and clinical importance as amyloid aggregates are linked to a number of neurodegenerative disorders. Such protein aggregates include macroscopic insoluble fibrils as well as small soluble oligomeric species. Time-dependent resolution of these species is prerequisite for a detailed quantitative understanding of protein aggregation; this remains challenging due to the lack of methods for detecting and characterizing transient and heterogeneous protein oligomers. Here we have used single molecule fluorescence techniques combined with mechanistic modeling to study the heparin-induced aggregation of the repeat region of tau, which forms the core region of neurofibrillary tangles found in Alzheimer's disease. We distinguish several subpopulations of oligomers with different stability and follow their evolution during aggregation reactions as a function of temperature and concentration. Employment of techniques from chemical kinetics reveals that the two largest populations are structurally distinct from fibrils and are both kinetically and thermodynamically unstable. The first population is in rapid exchange with monomers and held together by electrostatic interactions; the second is kinetically more stable, dominates at later times, and is probably off-pathway to fibril formation. These more stable oligomers may contribute to other oligomer induced effects in the cellular environment, for example, by overloading protein quality control systems. We also show that the shortest growing filaments remain suspended in aqueous buffer and thus comprise a third, smaller population of transient oligomers with cross-β structure. Overall our data show that a diverse population of oligomers of different structures and half-lives are formed during the aggregation reaction with the great majority of oligomers formed not going on to form fibrils. ; DK acknowledges funding from the Royal Society and ERC Advanced Grant (669237). MK acknowledges fellowships from the Danish Research Council and the Lundbeck Foundation (R165-2013-15269). AJD acknowledges a studentship from the Schiff Foundation. GM acknowledges a fellowship from Sidney Sussex College, Cambridge. TPJK acknowledges financial support from the Wellcome Trust, the Cambridge Centre for Misfolding Diseases, the BBSRC, and the Frances and Augustus Newman foundation. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) through the ERC grant PhysProt (agreement no. 337969).

Microcapsules are a key class of microscale materials with applications in areas ranging from personal care to biomedicine, and with increasing potential to act as extracellular matrix (ECM) models of hollow organs or tissues. Such capsules are conventionally generated from non-ECM materials including synthetic polymers. Here, we fabricated robust microcapsules with controllable shell thickness from physically- and enzymatically-crosslinked gelatin and achieved a core-shell architecture by exploiting a liquid-liquid phase separated aqueous dispersed phase system in a one-step microfluidic process. Microfluidic mechanical testing revealed that the mechanical robustness of thicker-shell capsules could be controlled through modulation of the shell thickness. Furthermore, the microcapsules demonstrated environmentally-responsive deformation, including buckling by osmosis and external mechanical forces. A sequential release of cargo species was obtained through the degradation of the capsules. Stability measurements showed the capsules were stable at 37 {\deg}C for more than two weeks. Finally, all-aqueous liquid-liquid phase separated and multiphase liquid-liquid phase separated systems were generated with the gel-sol transition of microgel precursors. These smart capsules are promising models of hollow biostructures, microscale drug carriers, and building blocks or compartments for active soft materials and robots. ; Cambridge Trust (Y.X.; B.L.), the Jardine Foundation (Y.X.), Trinity College Cambridge (Y.X.), Peterhouse College Cambridge (T.C.T.M.), the Swiss National Science Foundation (T.C.T.M.), the Engineering and Physical Sciences Research Council (K.L.S.), the Schmidt Science Fellowship program in partnership with the Rhodes Trust (K.L.S.), St John's College Cambridge (K.L.S.), China Scholarship Council (H.Z.; B.L.), EPSRC Cambridge NanoDTC (EP/037221/1; A.P.M.G.), the Newman Foundation (T.P.J.K.), the Wellcome Trust (T.P.J.K.), and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) through the ERC grant PhysProt (agreement n◦ 337969; T.P.J.K.).

Support from the Wellcome Trust, Medical Research Council, European Union Seventh Framework Programme (FP7/2007-2013) under grant agreements no. 604102 (HBP), 241995 (GENCODYS), 242498 (EUROSPIN), 242167 (SYNSYS). ; The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity. ; Publisher PDF ; Peer reviewed

Authors thank the Royal Society for the University Research Fellowship of S. F. L. (UF120277). This work was funded in part by the Michael J Fox Foundation and Indiana University. We thank the EPSRC for the Doctoral Prize of L. M. N., J. W, and S. E. B. are funded by Cancer Research UK (C14303/A17197, C47594/A16267), the EPSRC-CRUK Cancer Imaging Centre in Cambridge and Manchester (C197/A16465) and the European Union's Seventh Framework Programme (FP7-PEOPLE-2013- CIG-630729). We thank the European Reseach council for the Starting Grant of J.A.V. (804581). ; Neurodegenerative diseases such as Alzheimer's and Parkinson's are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging. ; Publisher PDF ; Peer reviewed

Correction to: Cell Death & Differentiation https://doi.org/10.1038/s41418-020-0542-z This article was originally published under a non-CC-BY licence, but has now been made available under a CC-BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly. Correction to: Cell Death & Differentiation https://doi.org/10.1038/s41418-020-0542-z The Affiliation 'UCL Queen Square Institute of Neurology' appeared incorrectly in the original article as 'UCL Institute of Neurology' This has been corrected in the PDF and HTML versions. Acknowledgements This work was supported by the Wellcome/MRC Parkinson's Disease Consortium grant (grant number WT089698), the Leverhulme Trust and the National Institute of Health Research University College London Hospitals Biomedical Research Centre. S.G. was supported by Wellcome, and is an MRC Senior Clinical Fellow. KSD and TK were supported by a Parkinson's UK Senior Fellowship (ref. F-0902). RY and TK were supported by MRC grant MR/J012831/1. The study was funded additionally by RFBR, project number 20-34-70074. M. Schepinov is employed by Retrotope Inc. This work was supported by the grant of the Russian Federation Government no. 075-15-2019-1877. ; Peer reviewed ; Postprint ; Publisher PDF

This is the author accepted manuscript. The final version is avialble via PNAS at http://www.pnas.org/content/112/16/E1994.long#ack-1. ; We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species. ; We thank Dr. Katherine Stott, from the Biophysics Facility, Department of Biochemistry, University of Cambridge, for her assistance in using these facilities. This work was supported by the Agency for Science, Technology and Research, Singapore (S.W.C.), the "La Caixa" foundation (S.D.), Wellcome/MRC (Medical Research Council) Parkinson's Disease Consortium Grant WT089698 (to E.D. and N.W.W.), National Institute for Health Research Biomedical Research Centres funding at University College London (to N.W.W.), the BBSRC through Grants BB/H003843/1 (to M.O.) and BB/E019927/1 (to C.M.D.), the Spanish Ministry of Economy and Competitiveness through Grants SAF 2012-39720 (to C.R.), BFU2013-44202 (to J.M.V.), and BIO2011-28941-C03-03 (to C.A. and G.R.), the Spanish Ministry of Health with cofunding by The European Regional Development Fund through Grant CP10/00527 (to C.R.), the Madrid Regional Government through Grant S2013/MIT-2807 (to J.M.V.), Parkinson's UK through Grant H-0903 (to T.G.), the Wellcome Trust, the Leverhulme Trust, the European Commission through project LSHM-CT-2006-037525 (to C.M.D.), the Medical Research Council through Grant MRC G1002272 (to E.J.D.-G. and C.M.D.), and the Engineering and Physical Sciences Research Council (C.M.D.). A.Y.A. was a Parkinson's UK Senior Research Fellow. N.C. is a Royal Society Research Fellow and also acknowledges financial support by the Human Frontier Science Program from Long-Term Fellowship LT000795/2009.