Multiplexed Near-Field Optical Trapping Exploiting Anapole States.

Optical tweezers have had a major impact on bioscience research by enabling the study of biological particles with high accuracy. The focus so far has been on trapping individual particles, ranging from the cellular to the molecular level. However, biology is intrinsically heterogeneous; therefore, access to variations within the same population and species is necessary for the rigorous understanding of a biological system. Optical tweezers have demonstrated the ability of trapping multiple targets in parallel; however, the multiplexing capability becomes a challenge when moving toward the nanoscale. Here, we experimentally demonstrate a resonant metasurface that is capable of trapping a high number of nanoparticles in parallel, thereby opening up the field to large-scale multiplexed optical trapping. The unit cell of the metasurface supports an anapole state that generates a strong field enhancement for low-power near-field trapping; importantly, the anapole state is also more angle-tolerant than comparable resonant modes, which allows its excitation with a focused light beam, necessary for generating the required power density and optical forces. We use the anapole state to demonstrate the trapping of 100's of 100 nm polystyrene beads over a 10 min period, as well as the multiplexed trapping of lipid vesicles with a moderate intensity of <250 µW/µm2. This demonstration will enable studies relating to the heterogeneity of biological systems, such as viruses, extracellular vesicles, and other bioparticles at the nanoscale.

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo.

In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

Crowding-induced morphological changes in synthetic lipid vesicles determined using smFRET.

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found utility among bio-inspired technologies, including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under-explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering, and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol, and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of the vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.

Sticker-and-spacer model for amyloid beta condensation and fibrillation.

A major pathogenic hallmark of Alzheimer's disease is the presence of neurotoxic plaques composed of amyloid beta (Aß) peptides in patients' brains. The pathway of plaque formation remains elusive, though some clues appear to lie in the dominant presence of Aß1 - 42 in these plaques despite Aß1-40 making up approximately 90% of the Aß pool. We hypothesize that this asymmetry is driven by the hydrophobicity of the two extra amino acids that are incorporated in Aß1-42. To investigate this hypothesis at the level of single molecules, we have developed a molecular "sticker-and-spacer lattice model" of unfolded Aß. The model protein has a single sticker that may reversibly dimerise and elongate into semi-flexible linear chains. The growth is hampered by excluded-volume interactions that are encoded by the hydrophilic spacers but are rendered cooperative by the attractive interactions of hydrophobic spacers. For sufficiently strong hydrophobicity, the chains undergo liquid-liquid phase-separation (LLPS) into condensates that facilitate the nucleation of fibers. We find that a small fraction of Aß1-40 in a mixture of Aß1-40 and Aß1-42 shifts the critical concentration for LLPS to lower values. This study provides theoretical support for the hypothesis that LLPS condensates act as a precursor for aggregation and provides an explanation for the Aß1-42-enrichment of aggregates in terms of hydrophobic interactions.

Ligand-induced transmembrane conformational coupling in monomeric EGFR.

Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.

Tween-20 Induces the Structural Remodeling of Single Lipid Vesicles.

The solubilization of lipid membranes by Tween-20 is crucial for a number of biotechnological applications, but the mechanistic details remain elusive. Evidence from ensemble assays supports a solubilization model that encompasses surfactant association with the membrane and the release of mixed micelles to solution, but whether this process also involves intermediate transitions between regimes is unanswered. In search of mechanistic origins, increasing focus is placed on identifying Tween-20 interactions with controllable membrane mimetics. Here, we employed ultrasensitive biosensing approaches, including single-vesicle spectroscopy based on fluorescence and energy transfer from membrane-encapsulated molecules, to interrogate interactions between Tween-20 and submicrometer-sized vesicles below the optical diffraction limit. We discovered that Tween-20, even at concentrations below the critical micellar concentration, triggers stepwise and phase-dependent structural remodeling events, including permeabilization and swelling, in both freely diffusing and surface-tethered vesicles, highlighting the substantial impact the surfactant has on vesicle conformation and stability prior to lysis.

Single-molecule FRET dynamics of molecular motors in an ABEL trap.

Single-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap's inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. In this work we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 seconds with sub-millisecond time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

Amyloid-ß oligomerization monitored by single-molecule stepwise photobleaching.

A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- ß peptide (Aß). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aß oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aß aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aß(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aß oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.

The Mechanism of Vesicle Solubilization by the Detergent Sodium Dodecyl Sulfate.

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotechnological applications, including viral inactivation and protein extraction. Although the ensemble thermodynamics have been thoroughly explored, the underlying molecular dynamics have remained inaccessible, owing to major limitations of traditional measurement tools. Here, we integrate multiple advanced biophysical approaches to gain multiangle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single submicron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on intact vesicles triggers biphasic solubilization kinetics comprising an initial vesicle expansion event followed by rapid lipid loss and micellization. Our findings support a general mechanism of detergent-induced membrane solubilization, and we expect that the framework of correlative biophysical technologies presented here will form a general platform for elucidating the complex kinetics of membrane perturbation induced by a wide variety of surfactants and disrupting agents.

Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs.

The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.

Optical detection of gadolinium(iii) ions via quantum dot aggregation.

A rapid, sensitive and selective optical readout of the presence of gadolinium(iii) ions would have a wide range of applications for clinical and environmental monitoring. We demonstrate that water-soluble CdTe quantum dots (QDs) are induced to aggregate by Gd3+ ions in aqueous solution. By using a combination of photoluminescence spectroscopy, dynamic light scattering and fluorescence correlation spectroscopy (FCS) to monitor quantum dot aggregation kinetics, we correlate the efficiency of the self-quenching process with the degree of aggregation across a broad range of conditions, including different sizes of QDs. We attribute the aggregation to metal binding to the QD's surface ligands and the quenching to intra-aggregate energy transfer between QDs. When the strategy was applied to additional trivalent ions, the aggregation rate varied according to the particular trivalent metal ion used, suggesting that the selectivity can be enhanced and controlled by appropriate design of the capping ligands and solution conditions.

Single-Molecule Fluorescence Detection of a Synthetic Heparan Sulfate Disaccharide.

The first single-molecule fluorescence detection of a structurally-defined synthetic carbohydrate is reported: a heparan sulfate (HS) disaccharide fragment labeled with Alexa488. Single molecules have been measured whilst freely diffusing in solution and controlled encapsulation in surface-tethered lipid vesicles has allowed extended observations of carbohydrate molecules down to the single-molecule level. The diverse and dynamic nature of HS-protein interactions means that new tools to investigate pure HS fragments at the molecular level would significantly enhance our understanding of HS. This work is a proof-of-principle demonstration of the feasibility of single-molecule studies of synthetic carbohydrates which offers a new approach to the study of pure glycosaminoglycan (GAG) fragments.

Morphology-Specific Inhibition of ß-Amyloid Aggregates by 17ß-Hydroxysteroid Dehydrogenase Type 10.

A major hallmark of Alzheimer's disease (AD) is the formation of toxic aggregates of the ß-amyloid peptide (Aß). Given that Aß peptides are known to localise within mitochondria and interact with 17ß-HSD10, a mitochondrial protein expressed at high levels in AD brains, we investigated the inhibitory potential of 17ß-HSD10 against Aß aggregation under a range of physiological conditions. Fluorescence self-quenching (FSQ) of Aß(1-42) labelled with HiLyte Fluor 555 was used to evaluate the inhibitory effect under conditions established to grow distinct Aß morphologies. 17ß-HSD10 preferentially inhibits the formation of globular and fibrillar-like structures but has no effect on the growth of amorphous plaque-like aggregates at endosomal pH 6. This work provides insights into the dependence of the Aß-17ß-HSD10 interaction with the morphology of Aß aggregates and how this impacts enzymatic function.

Crowding-Induced Hybridization of Single DNA Hairpins.

It is clear that a crowded environment influences the structure, dynamics, and interactions of biological molecules, but the complexity of this phenomenon demands the development of new experimental and theoretical approaches. Here we use two complementary single-molecule FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biological role of DNA and its technological applications, are strongly modulated by a crowded environment. We directly observed single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in solution or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concentrations of crowder [10% (w/w) polyethylene glycol (PEG)]. These experiments serve both to unambiguously highlight the impact of a crowded environment on a fundamental biological process, DNA base pairing, and to illustrate the benefits of single-molecule approaches to probing the structure and dynamics of complex biomolecular systems.

The phosphorylation of Hsp20 enhances its association with amyloid-ß to increase protection against neuronal cell death.

Up-regulation of Hsp20 protein levels in response to amyloid fibril formation is considered a key protective response against the onset of Alzheimer's disease (AD). Indeed, the physical interaction between Hsp20 and Aß is known to prevent Aß oligomerisation and protects neuronal cells from Aß mediated toxicity, however, details of the molecular mechanism and regulatory cell signalling events behind this process have remained elusive. Using both conventional MTT end-point assays and novel real time measurement of cell impedance, we show that Hsp20 protects human neuroblastoma SH-SY5Y cells from the neurotoxic effects of Aß. In an attempt to provide a mechanism for the neuroprotection afforded by Hsp20, we used peptide array, co-immunoprecipitation analysis and NMR techniques to map the interaction between Hsp20 and Aß and report a binding mode where Hsp20 binds adjacent to the oligomerisation domain of Aß, preventing aggregation. The Hsp20/Aß interaction is enhanced by Hsp20 phosphorylation, which serves to increase association with low molecular weight Aß species and decrease the effective concentration of Hsp20 required to disrupt the formation of amyloid oligomers. Finally, using a novel fluorescent assay for the real time evaluation of morphology-specific Aß aggregation, we show that phospho-dependency of this effect is more pronounced for fibrils than for globular Aß forms and that 25mers corresponding to the Hsp20 N-terminal can be used as Aß aggregate inhibitors. Our report is the first to provide a molecular model for the Hsp20/Aß complex and the first to suggest that modulation of the cAMP/cGMP pathways could be a novel route to enhance Hsp20-mediated attenuation of Aß fibril neurotoxicity.

Real-time probing of ß-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes.

The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting ß-amyloid aggregates (Aß) in solution. However, it is accepted that ThT-based methods suffer from important drawbacks. Some of these are due to the cationic structure of ThT, which limits its application at slightly acidic conditions; whereas some limitations are related to the general use of an extrinsic-dye sensing strategy and its intrinsic requirement for the formation of a sensor-binding site during the aggregation process. Here, we introduce fluorescence-self-quenching (FSQ) between N-terminally tagged peptides as a strategy to overcome some of these limitations. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, we characterize the fluorescence response of HiLyte fluor 555-labelled Aß peptides and demonstrate that Aß self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, we prove that N-terminal tagging of ß-amyloid peptides does not alter the self-assembly kinetics or the resulting aggregated structures. We also tested the ability of FSQ-based methods to monitor the inhibition of Aß1-42 aggregation using the small heat-shock protein Hsp20 as a model system. Overall, FSQ-based strategies for amyloid-sensing fill the gap between current morphology-specific protocols using extrinsic dyes, and highly-specialized single-molecule techniques that are difficult to implement in high-throughput analytical determinations. When performed in Förster resonance energy transfer (FRET) format, the method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid aggregation.