Super-resolution imaging of alpha-synuclein polymorphisms and their potential role in neurodegeneration

The conversion of soluble, functional proteins into amyloid fibrils has been linked to the development of neurodegenerative disorders, including Parkinson's and Alzheimer's disease. In the brains of patients with these disorders, the increasing presence of amyloid-containing plaques corresponds to neuronal cell death and the worsening of symptoms. However, protein amyloids are not merely confined to dying cells. Rather, some show a propensity to be transmitted to, and enter adjacent cells and induce the polymerization of the native monomer population. Whether this process is directly associated with toxicity or not is still highly debated. In this mini review, we will discuss structural polymorphisms of α-synuclein, as determined by super-resolution imaging techniques, and how these may be related to neuronal toxicity.This work was funded by grants from the UK Medical Research Council (MR/K015850/1 and MR/K02292X/1), Alzheimer’s Research UK (ARUK-EG2012A-1), the UK Engineering and Physical Sciences Research Council (EP/H018301/1), and the Wellcome Trust (089703/ Z/09/Z)

Heparin acts as a structural component of β-endorphin amyloid fibrils rather than a simple aggregation promoter.

The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide β-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in the presence of heparin leads to a loss of function. Applying correlative optical super-resolution microscopy methods, we show that heparin incorporates into emerging β-endorphin fibrils forming an integral component and is essential for amyloid templating. This will have direct implications on β-endorphin's normal physiological function and raises concerns on the biological relevance of heparin-promoted amyloid models.This work was funded by grants from the Wellcome Trust, the Medical Research Council UK, the Alzheimer Research UK Trust, the Engineering and Physical Sciences Research Council UK, and the Biotechnology and Biological Sciences Research Council. NN was supported through Early PostDoc.Mobility personal fellowship from Swiss National Science Foundation

Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration

The plasticity of the cytoskeleton architecture and membrane properties is important for the establishment of cell polarity, adhesion and migration. Here, we present a method which combines stimulated emission depletion (STED) super-resolution imaging and atomic force microscopy (AFM) to correlate cytoskeletal structural information with membrane physical properties in live astrocytes. Using STED compatible dyes for live cell imaging of the cytoskeleton, and simultaneously mapping the cell surface topology with AFM, we obtain unprecedented detail of highly organized networks of actin and microtubules in astrocytes. Combining mechanical data from AFM with optical imaging of actin and tubulin further reveals links between cytoskeleton organization and membrane properties. Using this methodology we illustrate that scratch-induced migration induces cytoskeleton remodeling. The latter is caused by a polarization of actin and microtubule elements within astroglial cell processes, which correlates strongly with changes in cell stiffness. The method opens new avenues for the dynamic probing of the membrane structural and functional plasticity of living brain cells. It is a powerful tool for providing new insights into mechanisms of cell structural remodeling during physiological or pathological processes, such as brain development or tumorigenesis.This work was supported by grants from College de France and ERC to NR, Paris 6 University doctoral school ED3C and Labex Memolife to GG. CFK acknowledges funding from the Engineering and Physical Sciences Research council (EPSRC, UK), the Wellcome Trust, UK, the Medical Research Council (MRC, UK) and Infinitus Ltd. GSKS acknowledges funding from the Wellcome Trust, UK and the MRC

Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro. Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies

A Highly Porous Metal-Organic Framework System to Deliver Payloads for Gene Knockdown

© 2019 Elsevier Inc. Gene knockdown is an advantageous therapeutic strategy to lower dangerous genetic over-expression. However, the molecules responsible for initiating this process are unstable. Porous nanoparticles called metal-organic frameworks can encapsulate, protect, and deliver these compounds efficaciously without the need for chemical modifications—commonly done to enhance stability. By applying this platform technology, this work demonstrates the successful reduction in expression of a gene by avoiding retention and subsequent degradation in cellular compartments.This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (NanoMOFdeli), ERC-2016-COG 726380, and (SUPUVIR) no. 722380. M.H.T. thanks the Gates Cambridge Trust for funding, S. Haddad for helpful discussions, and A. Li for assistance with data visualization. D.F.-J. thanks the Royal Society for funding through a University Research Fellowship. S.B.d.Q.F., F.M.R., and D.I.J. were funded by Cancer Research UK Senior Group Leader Grant CRUK/A15678. O.K.F. gratefully acknowledges DTRA for financial support (grant HDTRA-1-14-1-0014). C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z) and the UK Medical Research Council (grants MR/K015850/1 and MR/K02292X/1), and Infinitus (China) Ltd. Computational work was supported by the Cambridge High Performance Computing Cluster, Darwin

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in C. elegans

Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging. Until now, an extensive characterization of the nature of age-dependent protein aggregation has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and have an amyloid-like structure resembling that of protein aggregates observed in disease. We then demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these findings imply that amyloid-like aggregates contribute to the aging process and therefore could be important targets for strategies designed to maintain physiological functions in the late stages of life

Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control

Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar ‘superfolded’ proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues