Single Fibril Level Research Articles

Fibrillar adhesives with unprecedented adhesion strength, switchability and scalability.

Bio-inspired fibrillar adhesives have received worldwide attention but their potentials have been limited by a trade-off between adhesion strength and adhesion switchability, and a size scale effect that restricts the fibrils to micro/nanoscales. Here, we report a class of adhesive fibrils that achieve unprecedented adhesion strength (∼2MPa), switchability (∼2000), and scalability (up to millimeter-scale at the single fibril level), by leveraging the rubber-to-glass (R2G) transition in shape memory polymers (SMPs). Moreover, R2G SMP fibrillar adhesive arrays exhibit a switchability of >1000 (with the aid of controlled buckling) and an adhesion efficiency of 57.8%, with apparent contact area scalable to 1000mm2, outperforming existing fibrillar adhesives. We further demonstrate that the SMP fibrillar adhesives can be used as soft grippers and reusable superglue devices that are capable of holding and releasing heavy objects >2000 times of their own weight. These findings represent significant advances in smart fibrillar adhesives for numerous applications, especially those involving high-payload scenarios.

Quantitative Seed Amplification Assay: A Proof-of-Principle Study.

Amyloid fibrils of the protein α-synuclein (αS) have recently been identified as a biomarker for Parkinson's disease (PD). To detect the presence of these amyloid fibrils, seed amplification assays (SAAs) have been developed. SAAs allow for the detection of αS amyloid fibrils in biomatrices such as cerebral spinal fluid and are promising for PD diagnosis by providing a dichotomous (yes/no) response. The additional quantification of the number of αS amyloid fibrils may enable clinicians to evaluate and follow the disease progression and severity. Developing quantitative SAAs has been shown to be challenging. Here, we report on a proof-of-principle study on the quantification of αS fibrils in fibril-spiked model solutions of increasing compositional complexity including blood serum. We show that parameters derived from standard SAAs can be used for fibril quantification in these solutions. However, interactions between the monomeric αS reactant that is used for amplification and biomatrix components such as human serum albumin have to be taken into account. We demonstrate that quantification of fibrils is possible even down to the single fibril level in a model sample consisting of fibril-spiked diluted blood serum.

Single-molecule FRET imaging and deep learning reveal concentration dependence of aggregation pathways during Aβ aggregation.

Protein aggregation into amyloid fibrils is the hallmark of several devasting neurodegenerative diseases. Gaining an understanding of disease etiology hinges on our ability to understand the molecular mechanics of how soluble monomers assemble to form insoluble fibrils consisting of thousands of constituent monomers. Although amyloid fibril formation is a highly specific self-assembly process, growth patterns and resultant fibril morphologies are highly dependent on solution conditions. Using fluorescence lifetime imaging and deep learning, we have recently shown that amyloid assembly occurs via heterogeneous aggregation pathways resulting in a mixture of co-present mature fibrils with unique morphologies and physicochemical properties (Meng et al., PNAS_2022_e2116736119). Bulk biophysical methods are unable to fully characterize these mixtures of fibril polymorphs. Here, we further develop and use Förster resonance energy transfer (FRET) imaging to monitor the entire aggregation pathway of the Alzheimer's Disease related peptide amyloid β 42 (Aβ42) at the single fibril level in real time. We incubated a mixture of donor-labeled, acceptor-labeled, and unlabeled Aβ42 monomers, which resulted in the formation of fibrils with diverse FRET efficiency values, indicating structural heterogeneity. Single-fibril images reveal that increasing monomer concentration promotes the formation of more homogeneous fibrils. Fibrils formed at lower concentrations show assemble via highly heterogeneous pathways. Deep learning methods (https://github.com/hoisunglab/FNet) enable segmentation of single fibrils within images of highly overlapping fibrils, allowing for quantitative analysis of the aggregation process in terms of fibril growth rate and photon density for each fibril over the course of fibril assembly.

Probing physical properties of single amyloid fibrils using nanofluidic channels.

Amyloid fibril formation is central to the pathology of many diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Amyloid fibrils can also have functional and scaffolding roles, for example in bacterial biofilms, and have also been exploited as useful biomaterials. Despite being linear protein homopolymers, amyloid fibrils can exhibit significant structural and morphological polymorphism, making it relevant to study them on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently-labeled amyloid fibrils in solution, monitoring the extension and emission intensity of individual fibrils confined in nanochannels with a depth of 300 nm and a width that gradually increases from 300 to 3000 nm. The change in fibril extension with channel width permitted accurate determination of the persistence length of individual fibrils using Odijk's theory for strongly confined polymers. The technique was applied to amyloid fibrils prepared from the Alzheimer's related peptide amyloid-β(1-42) and the Parkinson's related protein α-synuclein, obtaining mean persistence lengths of 5.9 ± 4.5 μm and 3.0 ± 1.6 μm, respectively. The broad distributions of fibril persistence lengths indicate that amyloid fibril polymorphism can manifest in their physical properties. Interestingly, the α-synuclein fibrils had lower persistence lengths than the amyloid-β(1-42) fibrils, despite being thicker. Furthermore, there was no obvious within-sample correlation between the fluorescence emission intensity per unit length of the labelled fibrils and their persistence lengths, suggesting that stiffness may not be proportional to thickness. We foresee that the nanofluidics methodology established here will be a useful tool to study amyloid fibrils on the single fibril level to gain information on heterogeneity in their physical properties and interactions.

Mechanical properties of collagen fibrils determined by buckling analysis

The mechanical properties of biological nanofibers such as collagen fibrils are important in many applications, ranging from tissue-engineering to cancer treatment. However, mechanical testing is not straightforward at the nanometer scale. Here, we use the theory of column-buckling to determine the bending properties of individual collagen fibrils. To achieve this, fibrils were deposited on a manually pre-stretched foil, which was then released with the fibrils attached. Atomic Force Microscopy (AFM) imaging was used to determine the tensile modulus by measuring the buckling-wavelength and the radius for each fibril. Comparison with data obtained by AFM nanoindentation and other, more sophisticated methods, shows that our results are in very good agreement. The great advantage of this simple approach is that it can be used to quickly determine mechanical properties without force or stress-strain measurements, which are challenging to obtain accurately and at high throughput at the nanoscale. The method could be applied to any nanofibers, not just collagen fibrils. Statement of significanceCollagen fibrils are the main constituent of the extracellular matrix, and alterations of their mechanical properties can have significant effects on cell adhesion and motility. This has, ultimately, implications in age-related diseases and cancer. Furthermore, tuning the mechanical properties of collagen fibrils could be an important tool in the design of artificial cell scaffolds in tissue-engineering. For these reasons, it is important to have methods that can be used to determine the mechanical properties of fibrils at the single-fibril level and, therefore, at the nanometer scale. The method presented here has the advantage of being easy to use and avoids some of the fundamental issues of more established methods.

Impact of Collagen Crosslinking on Dislocated Human Shoulder Capsules-Effect on Structural and Mechanical Properties.

Classical treatments of shoulder instability are associated with recurrence. To determine whether the modification of the capsule properties may be an alternative procedure, the effect of crosslinking treatment on the structure and mechanical properties of diseased human shoulder capsules was investigated. Joint capsules harvested from patients during shoulder surgery (n = 5) were treated or not with UV and/or riboflavin (0.1%, 1.0% and 2.5%). The structure and the mechanical properties of the capsules were determined by atomic force microscopy. The effect of treatments on cell death was investigated. Collagen fibrils were well-aligned and adjacent to each other with a D-periodicity of 66.9 ± 3.2 nm and a diameter of 71.8 ± 15.4 nm in control untreated capsules. No effect of treatments was observed on the organization of the collagen fibrils nor on their intrinsic characteristics, including D-periodicity or their mean diameter. The treatments also did not induce cell death. In contrast, UV + 2.5% riboflavin induced capsule stiffness, as revealed by the increased Young’s modulus values (p < 0.0001 for each patient). Our results showed that the crosslinking procedure changed the biomechanics of diseased capsules, while keeping their structural organisation unchanged at the single fibril level. The UV/riboflavin crosslinking procedure may be a promising way to preserve the functions of collagen-based tissues and tune their elasticity for clinically relevant treatments.

Mechanism of secondary nucleation from the direct observation of Aβ42 aggregation at the single fibril level

The formation of amyloid fibrils and oligomers is linked to several neurodegenerative disorders, including Alzheimer’s disease (AD). To tackle these diseases, we need to progress our understanding at the molecular level, including the determination of the mechanisms and rates of fibril formation and self-replication. In the context of AD, the facilitation of the formation of new Aβ42 fibrils by existing ones through secondary nucleation is a major source of both new fibrils and toxic oligomeric species.

Mechanism of Secondary Nucleation at the Single Fibril Level from Direct Observations of Aβ42 Aggregation.

The formation of amyloid fibrils and oligomers is a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD), and contributes to the disease pathway. To progress our understanding of these diseases at a molecular level, it is crucial to determine the mechanisms and rates of amyloid formation and replication. In the context of AD, the self-replication of aggregates of the Aβ42 peptide by secondary nucleation, leading to the formation of new aggregates on the surfaces of existing ones, is a major source of both new fibrils and smaller toxic oligomeric species. However, the core mechanistic determinants, including the presence of intermediates, as well as the role of heterogeneities in the fibril population, are challenging to determine from bulk aggregation measurements. Here, we obtain such information by monitoring directly the time evolution of individual fibrils by TIRF microscopy. Crucially, essentially all aggregates have the ability to self-replicate via secondary nucleation, and the amplification of the aggregate concentration cannot be explained by a small fraction of "superspreader" fibrils. We observe that secondary nucleation is a catalytic multistep process involving the attachment of soluble species to the fibril surface, followed by conversion/detachment to yield a new fibril in solution. Furthermore, we find that fibrils formed by secondary nucleation resemble the parent fibril population. This detailed level of mechanistic insights into aggregate self-replication is key in the rational design of potential inhibitors of this process.

Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly

Amyloid fibrils are highly polymorphic structures formed by many different proteins. They provide biological function but also abnormally accumulate in numerous human diseases. The physicochemical principles of amyloid polymorphism are not understood due to lack of structural insights at the single-fibril level. To identify and classify different fibril polymorphs and to quantify the level of heterogeneity is essential to decipher the precise links between amyloid structures and their functional and disease associated properties such as toxicity, strains, propagation and spreading. Employing gentle, force-distance curve-based AFM, we produce detailed images, from which the 3D reconstruction of individual filaments in heterogeneous amyloid samples is achieved. Distinctive fibril polymorphs are then classified by hierarchical clustering, and sample heterogeneity is objectively quantified. These data demonstrate the polymorphic nature of fibril populations, provide important information regarding the energy landscape of amyloid self-assembly, and offer quantitative insights into the structural basis of polymorphism in amyloid populations.

Unique and outstanding quantum dots (QD)/tunicate cellulose nanofibrils (TCNF) nanohybrid platform material for use as 1D ink and 2D film

Quantum dots (QD)/polymer materials have wide applications in biological imaging, clinical diagnostics, anti-counterfeiting materials, light-emitting devices and solar cells. The development of QD/cellulose nanofibrils (CNF) hybrids with a more perfect structure and excellent properties is important for improving known applications. A unique tunicate CNF (TCNF) was homogeneously blended with outstanding CdSe/CdS core/shell QD to prepare a novel QD/TCNF hybrid. The QD were monodispersed on a single TCNF fibril surface as an evenly distributed monolayer with an extremely high packing density and no visible aggregation. The prepared hybrid is an excellent platform nanomaterial which was demonstrated by its good writing fidelity when applied as a 1D ink and by its good processability in the preparation of 2D films with acceptable transparency and flexibility. This one-step direct blending approach provides a facile shortcut to effectively fabricate cellulose-based high-performance functional QD nanomaterials at the single-fibril level.

Direct Observation of Prion Protein Fibril Elongation Kinetics

It is widely accepted that prion diseases are caused by the conversion of cellular prion protein (PrPC) to the misfolded form PrPSc, which is self-propagating by seeded aggregation. The molecular mechanism and propagation kinetics are not well understood. Here, we use Total Internal Reflection Fluorescence and Transient Amyloid Binding super-resolution microscopy to investigate the elongation kinetics of mouse prion protein at single fibril level. Recombinant mouse prion protein (recMoPrP 91-231) fibrils formed under partially denaturing conditions were deposited on a glass slide, incubated with recMoPrP 91-231 monomer solution and imaged for up to 70h. We found that PrP fibrils elongated by an intermittent ‘stop-and-go’ mechanism. The elongation rates of growing phase are obtained under different temperature, various concentrations of monomer, denaturant, and sodium chloride, which are then used to calculate the kinetic parameters. This study sheds light on the mechanism of prion protein seeded propagation in vitro and provides a useful system to study the influence of PrP mutants and screening of inhibitors.

Real-Time Monitoring of Self-Aggregation of β-Amyloid by a Fluorescent Probe Based on Ruthenium Complex.

Self-accumulation of amyloid-β protein (Aβ) into insoluble fibrils is the major hallmark of Alzheimer's disease. Real-time monitoring of fibril growth is essential for clarifying the mechanism underlying aggregation and discovering therapeutic targets. A variety of approaches including NMR, electron microscopy (EM), atomic force microscopy (AFM), and total internal reflection fluorescence microscopy (TIRFM) have been explored to monitor the fibril growth or reveal morphology of Aβ aggregates. However, none of the methods allow real-time observation under physiological conditions while without any perturbations. Here, we present a fluorescent probe [Ru(phen)2(fipc)]2+ (Ru-fipc) (phen = 1,10-phenanthroline, fipc = 5-fluoro-N-(1,10-phenanthrolin-5-yl)-1H-indole-2-carboxamide) that can bind to all the Aβ forms, i.e., monomers, oligomers, and fibrils, while not perturbing aggregation. Using this probe in combination with laser confocal microscopy, the entire aggregation process could be clearly and exactly imaged at the single fibril level. The reliability of Ru-fipc was confirmed based on colocalization with thioflavin T (ThT). Importantly, Ru-fipc can be used to monitor the very early nucleation and oligomerization process, which is thought to be a critical step in the development of neurotoxicity while it cannot be visualized with ThT. To our knowledge, this is the first fluorescent probe developed for real-time monitoring of Aβ aggregation, especially for the very early assembly stage, in solution with minimal perturbation. This method is suitable for in vitro and in vivo studies. We believe this would provide a valuable complementary tool for the study of pathogenesis and discovery of therapeutic targets of neurodegenerative diseases.

Evaluation of surface charge shift of collagen fibrils exposed to glutaraldehyde

Collagen fibrils are a major component of the extracellular matrix. They form nanometer-scale “cables” acting as a scaffold for cells in animal tissues and are widely used in tissue-engineering. Besides controlling their structure and mechanical properties, it is crucial to have information of their surface charge, as this affects how cells attach to the scaffold. Here, we employed Kelvin-probe Force Microscopy to determine the electrostatic surface potential at the single-fibril level and investigated how glutaraldehyde, a well-established protein cross-linking agent, shifts the surface charge to more negative values without disrupting the fibrils themselves. This shift can be interpreted as the result of the reaction between the carbonyl groups of glutaraldehyde and the amine groups of collagen. It reduces the overall density of positively charged amine groups on the collagen fibril surface and, ultimately, results in the observed negative shift of the surface potential measured. Reactions between carbonyl-containing compounds and proteins are considered the first step in glycation, the non-enzymatic reaction between sugars and proteins. It is conceivable that similar charge shifts happen in vivo caused by sugars, which could have serious implications on age-related diseases such as diabetes and which has been hypothesised for many years.

Confinement-induced liquid crystalline transitions in amyloid fibril cholesteric tactoids.

Chirality is ubiquitous in nature and plays crucial roles in biology, medicine, physics and materials science. Understanding and controlling chirality is therefore an important research challenge with broad implications. Unlike other chiral colloids, such as nanocellulose or filamentous viruses, amyloid fibrils form nematic phases but appear to miss their twisted form, the cholesteric or chiral nematic phases, despite a well-defined chirality at the single fibril level. Here we report the discovery of cholesteric phases in amyloids, using β-lactoglobulin fibrils shortened by shear stresses. The physical behaviour of these new cholesteric materials exhibits unprecedented structural complexity, with confinement-driven ordering transitions between at least three types of nematic and cholesteric tactoids. We use energy functional theory to rationalize these results and observe a chirality inversion from the left-handed amyloids to right-handed cholesteric droplets. These findings deepen our understanding of cholesteric phases, advancing their use in soft nanotechnology, nanomaterial templating and self-assembly.

Large-Area Au-Nanoparticle-Functionalized Si Nanorod Arrays for Spatially Uniform Surface-Enhanced Raman Spectroscopy.

In this study, large-area hexagonal-packed Si nanorod (SiNR) arrays in conjunction with Au nanoparticles (AuNPs) were fabricated for surface-enhanced Raman spectroscopy (SERS). We have achieved ultrasensitive molecular detection with high reproducibility and spatial uniformity. A finite-difference time-domain simulation suggests that a wide range of three-dimensional electric fields are generated along the surfaces of the SiNR array. With the tuning of the gap and diameter of the SiNRs, the produced long decay length (>130 nm) of the enhanced electric field makes the SERS substrate a zero-gap system for ultrasensitive detection of large biomolecules. In the detection of R6G molecules, our SERS system achieved an enhancement factor of >107 with a relative standard deviation as small as 3.9-7.2% over 30 points across the substrate. More significantly, the SERS substrate yielded ultrasensitive Raman signals on long amyloid-β fibrils at the single-fibril level, which provides promising potentials for ultrasensitive detection of amyloid aggregates that are related to Alzheimer's disease. Our study demonstrates that the SiNRs functionalized with AuNPs may serve as excellent SERS substrates in chemical and biomedical detection.

B6 Super-resolution fluorescence imaging of the seeding and polymerizatoin of the huntingtin exon 1 protein

Background Huntington’s disease (HD) is caused by the expansion of the CAG repeat in exon 1 of the gene coding for the huntingtin (HTT) protein. The mutant version of the polyglutamine tract can result in misfolding and aggregation of N-terminal fragments of HTT under physiological conditions. Therefore, it’s crucial to understand the mechanisms underlying the seeding and polymerization of HTT exon 1. However, the formation of HTT aggregates has been difficult to investigate by conventional fluorescence microscopy due to their small size. Recent advances in super-resolution microscope have made this possible by permitting researchers to investigate protein aggregation in much greater detail. Aims To use highly novel super-resolution microscope techniques to understand the mechanisms underlying the seeding and polymerization of HTT exon 1 Methods/techniques Purify native HTT exon 1 protein from newly designed constructs and use two-colour TIRF (total internal reflection fluorescence microscope)-SIM (Structured illumination microscopy) microscope to directly observe the growth of HTT exon 1 at the single fibril level. Results/outcome We have designed and cloned new constructs ideal for making native HTT exon 1 protein by precise protease cleavage and fluorescent dye labelling for microscope imaging. The characterisation and application of these constructs will be presented. Conclusions Our methods will enable us to directly visualise the dynamics of the HTT exon 1 aggregation and study the aggregation kinetics which is important to HD therapeutics.

Understanding nanocellulose chirality and structure-properties relationship at the single fibril level.

Nanocellulose fibrils are ubiquitous in nature and nanotechnologies but their mesoscopic structural assembly is not yet fully understood. Here we study the structural features of rod-like cellulose nanoparticles on a single particle level, by applying statistical polymer physics concepts on electron and atomic force microscopy images, and we assess their physical properties via quantitative nanomechanical mapping. We show evidence of right-handed chirality, observed on both bundles and on single fibrils. Statistical analysis of contours from microscopy images shows a non-Gaussian kink angle distribution. This is inconsistent with a structure consisting of alternating amorphous and crystalline domains along the contour and supports process-induced kink formation. The intrinsic mechanical properties of nanocellulose are extracted from nanoindentation and persistence length method for transversal and longitudinal directions, respectively. The structural analysis is pushed to the level of single cellulose polymer chains, and their smallest associated unit with a proposed 2 × 2 chain-packing arrangement.

New Strategies for Cellular‐Scale Imaging of Bone and Cartilage

Confocal microscopy can be used to obtain detailed, 3D‐resolved images of cells inside bone and cartilage. However the high scattering coefficients of dense connective tissues often necessitate tissue sectioning for resolving cellular scale detail inside. Multiphoton microscopy enables clearer imaging and greater depth penetration into these tissues and the related technique of second harmonic generation enables acquisition of collagen images at the single‐fibril level in 3D and in some cases in vivo. New improvements in clearing technologies and optical methods such as light sheet microscopy continue to advance the depth in to which dense tissues can be imaged using highly‐specific fluorescent probes. Concurrently, improvements in X‐ray computed tomography, the standard method for 3D medical assessment of bone morphology and lesions, have led to resolutions rivaling those obtained with optical microscopy, in some cases with specifically targeted electron dense contrast agents. Different techniques excel for different specific questions to be addressed within this diverse and powerful imaging landscape.

Single Fibril Growth Kinetics of α-Synuclein

Neurodegenerative disorders associated with protein misfolding are fatal diseases that are caused by fibrillation of endogenous proteins such as α-synuclein (α-syn) in Parkinson's disease (PD) or amyloid-β in Alzheimer's disease. Fibrils of α-syn are a major pathological hallmark of PD and certain aggregation intermediates are postulated to cause synaptic failure and cell death of dopaminergic neurons in the substantia nigra. For the development of therapeutic approaches, the mechanistic understanding of the fibrillation process is essential. Here we report real-time observation of α-syn fibril elongation on a glass surface, imaged by total internal reflection fluorescence microscopy using thioflavin T fluorescence. Fibrillation on the glass surface occurred in the same time frame and yielded fibrils of similar length as fibrillation in solution. Time-resolved imaging of fibrillation on a single fibril level indicated that α-syn fibril elongation follows a stop-and-go mechanism; that is, fibrils either extend at a homogenous growth rate or stop to grow for variable time intervals. The fibril growth kinetics were compatible with a model featuring two states, a growth state and a stop state, which were approximately isoenergetic and interconverted with rate constants of ~1.5×10−4s−1. In the growth state, α-syn monomers were incorporated into the fibril with a rate constant of 8.6×103M−1s−1. Fibril elongation of α-syn is slow compared to other amyloidogenic proteins.

Self-Assembly of Polysaccharides Gives Rise to Distinct Mechanical Signatures in Marine Gels

Marine-gel biopolymers were recently visualized at the molecular level using atomic force microscopy (AFM) to reveal fine fibril-forming networks with low to high degrees of cross-linking. In this work, we use force spectroscopy to quantify the intra- and intermolecular forces within the marine-gel network. Combining force measurements, AFM imaging, and the known chemical composition of marine gels allows us to identify the microscopic origins of distinct mechanical responses. At the single-fibril level, we uncover force-extension curves that resemble those of individual polysaccharide fibrils. They exhibit entropic elasticity followed by extensions associated with chair-to-boat transitions specific to the type of polysaccharide at high forces. Surprisingly, a low degree of cross-linking leads to sawtooth patterns that we attribute to the unraveling of polysaccharide entanglements. At a high degree of cross-linking, we observe force plateaus that arise from unzipping, as well as unwinding, of helical bundles. Finally, the complex 3D network structure gives rise to force staircases of increasing height that correspond to the hierarchical peeling of fibrils away from the junction zones. In addition, we show that these diverse mechanical responses also arise in reconstituted polysaccharide gels, which highlights their dominant role in the mechanical architecture of marine gels.