α-Synuclein strains cause distinct synucleinopathies after local and systemic administration

W. Peelaerts, L. Bousset, A. Van der Perren, A. Moskalyuk, R. Pulizzi, M. Giugliano, C. Van den Haute, R. Melki and V. Baekelandt

Misfolded protein aggregates represent a continuum with overlapping features in neurodegenerative diseases, but differences in protein components and affected brain regions1. The molecular hallmark of synucleinopathies such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are megadalton α-synuclein-rich deposits suggestive of one molecular event causing distinct disease phenotypes. Glial α-synuclein (α-SYN) filamentous deposits are prominent in multiple system atrophy and neuronal α-SYN inclusions are found in Parkinson’s disease and dementia with Lewy bodies2. The discovery of α-SYN assemblies with different structural characteristics or ‘strains’ has led to the hypothesis that strains could account for the different clinico-pathological traits within synucleinopathies3, 4. In this study we show that α-SYN strain conformation and seeding propensity lead to distinct histopathological and behavioural phenotypes. We assess the properties of structurally well-defined α-SYN assemblies (oligomers, ribbons and fibrils) after injection in rat brain. We prove that α-SYN strains amplify in vivo. Fibrils seem to be the major toxic strain, resulting in progressive motor impairment and cell death, whereas ribbons cause a distinct histopathological phenotype displaying Parkinson’s disease and multiple system atrophy traits. Additionally, we show that α-SYN assemblies cross the blood–brain barrier and distribute to the central nervous system after intravenous injection. Our results demonstrate that distinct α-SYN strains display differential seeding capacities, inducing strain-specific pathology and neurotoxic phenotypes.

DOI

Journal:Nature

A DNA-based molecular probe for optically reporting cellular traction forces

Brandon L Blakely, Christoph E Dumelin, Britta Trappmann, Lynn M McGregor, Colin K Choi, Peter C Anthony, Van K Duesterberg, Brendon M Baker, Steven M Block, David R Liu, and Christopher S Chen

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

DOI

Journal: Nature Methods

Protein folding: Turbo-charged crosslinking

David J. Craik

The efficient production of stable bioactive proteins often requires the selective formation of several disulfide crosslinks. Two recent studies have now shown that replacing cysteine with selenocysteine in the unfolded protein can autocatalyse the formation of the desired crosslinks.

DOI

Journal: Nature Chemistry

HSF-1–mediated cytoskeletal integrity determines thermotolerance and life span

Nathan A. Baird, Peter M. Douglas1, Milos S. Simic, Ana R. Grant, James J. Moresco, Suzanne C. Wolff, John R. Yates III, Gerard Manning, Andrew Dillin

The conserved heat shock transcription factor–1 (HSF-1) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, we engineered a modified HSF-1 strain that increased stress resistance and longevity without enhanced chaperone induction. This health assurance acted through the regulation of the calcium-binding protein PAT-10. Loss of pat-10 caused a collapse of the actin cytoskeleton, stress resistance, and life span. Furthermore, overexpression of pat-10 increased actin filament stability, thermotolerance, and longevity, indicating that in addition to chaperone regulation, HSF-1 has a prominent role in cytoskeletal integrity, ensuring cellular function during stress and aging.

DOI

Journal: Science

A long noncoding RNA protects the heart from pathological hypertrophy

Pei Han, Wei Li, Chiou-Hong Lin, Jin Yang, Ching Shang, Sylvia T. Nuernberg, Kevin Kai Jin, Weihong Xu, Chieh-Yu Lin, Chien-Jung Lin, Yiqin Xiong, Huan-Chieh Chien, Bin Zhou, Euan Ashley, Daniel Bernstein, Peng-Sheng Chen, Huei-Sheng Vincent Chen, Thomas Quertermous, and  Ching-Pin Chang

The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription1, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction2. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA–chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1–Hdac–Parp chromatin repressor complex3 to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy3. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized—but not naked—DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt–Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA–chromatin interaction.

DOI

Journal: Nature

DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate

Bian Liu, Ronald J. Baskin, and Stephen C. Kowalczykowski

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules1, 2, 3. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour4, 5, 6. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably6, 7, 8, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme–DNA complex by depleting Mg2+-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.

DOI

Journal: Nature

Ultrastable atomic force microscopy: Improved force and positional stability

Allison B. Churnside, Thomas T. Perkins

Atomic force microscopy (AFM) is an exciting technique for biophysical studies of single molecules, but its usefulness is limited by instrumental drift. We dramatically reduced positional drift by adding two lasers to track and thereby actively stabilize the tip and the surface. These lasers also enabled label-free optical images that were spatially aligned to the tip position. Finally, sub-pN force stability over 100 s was achieved by removing the gold coating from soft cantilevers. These enhancements to AFM instrumentation can immediately benefit research in biophysics and nanoscience.

DOI

Journal: Febs Letters

Artificial Muscles from Fishing Line and Sewing Thread

Carter S. Haines, Márcio D. Lima, Na Li, Geoffrey M. Spinks, Javad Foroughi, John D. W. Madden, Shi Hyeong Kim, Shaoli Fang, Mônica Jung de Andrade, Fatma Göktepe, Özer Göktepe, Seyed M. Mirvakili, Sina Naficy, Xavier Lepró, Jiyoung Oh, Mikhail E. Kozlov, Seon Jeong Kim, Xiuru Xu, Benjamin J. Swedlove, Gordon G. Wallace, Ray H. Baughman

The high cost of powerful, large-stroke, high-stress artificial muscles has combined with performance limitations such as low cycle life, hysteresis, and low efficiency to restrict applications. We demonstrated that inexpensive high-strength polymer fibers used for fishing line and sewing thread can be easily transformed by twist insertion to provide fast, scalable, nonhysteretic, long-life tensile and torsional muscles. Extreme twisting produces coiled muscles that can contract by 49%, lift loads over 100 times heavier than can human muscle of the same length and weight, and generate 5.3 kilowatts of mechanical work per kilogram of muscle weight, similar to that produced by a jet engine. Woven textiles that change porosity in response to temperature and actuating window shutters that could help conserve energy were also demonstrated. Large-stroke tensile actuation was theoretically and experimentally shown to result from torsional actuation.

DOI

Journal: Science

Chemical mapping of a single molecule by plasmon-enhanced Raman scattering

R. Zhang, Y. Zhang, Z. C. Dong, S. Jiang, C. Zhang, L. G. Chen, L. Zhang, Y. Liao, J. Aizpurua, Y. Luo,  J. L. Yang, and J. G. Hou

Visualizing individual molecules with chemical recognition is a longstanding target in catalysis, molecular nanotechnology and biotechnology. Molecular vibrations provide a valuable ‘fingerprint’ for such identification. Vibrational spectroscopy based on tip-enhanced Raman scattering allows us to access the spectral signals of molecular species very efficiently via the strong localized plasmonic fields produced at the tip apex1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. However, the best spatial resolution of the tip-enhanced Raman scattering imaging is still limited to 3−15 nanometres5, 12, 13, 14, 15, 16, which is not adequate for resolving a single molecule chemically. Here we demonstrate Raman spectral imaging with spatial resolution below one nanometre, resolving the inner structure and surface configuration of a single molecule. This is achieved by spectrally matching the resonance of the nanocavity plasmon to the molecular vibronic transitions, particularly the downward transition responsible for the emission of Raman photons. This matching is made possible by the extremely precise tuning capability provided by scanning tunnelling microscopy. Experimental evidence suggests that the highly confined and broadband nature of the nanocavity plasmon field in the tunnelling gap is essential for ultrahigh-resolution imaging through the generation of an efficient double-resonance enhancement for both Raman excitation and Raman emission. Our technique not only allows for chemical imaging at the single-molecule level, but also offers a new way to study the optical processes and photochemistry of a single molecule.

DOI

Journal: Nature

Single-Molecule Reconstruction of Oligonucleotide Secondary Structure by Atomic Force Microscopy

Alice Pyne, Ruth Thompson, Carl Leung, Debdulal Roy and Bart W. Hoogenboom

Based on soft-touch atomic force microscopy, a method is described to reconstruct the secondary structure of single extended biomolecules, without the need for crystallization. The method is tested by accurately reproducing the dimensions of the B-DNA crystal structure. Importantly, intramolecular variations in groove depth of the DNA double helix are resolved, which would be inaccessible for methods that rely on ensemble-averaging.

DOI

Journal: Small

Observation of Brownian Motion in Liquids at Short Times: Instantaneous Velocity and Memory Loss

Simon Kheifets, Akarsh Simha, Kevin Melin, Tongcang Li, Mark G. Raizen

Measurement of the instantaneous velocity of Brownian motion of suspended particles in liquid probes the microscopic foundations of statistical mechanics in soft condensed matter. However, instantaneous velocity has eluded experimental observation for more than a century since Einstein’s prediction of the small length and time scales involved. We report shot-noise–limited, high-bandwidth measurements of Brownian motion of micrometer-sized beads suspended in water and acetone by an optical tweezer. We observe the hydrodynamic instantaneous velocity of Brownian motion in a liquid, which follows a modified energy equipartition theorem that accounts for the kinetic energy of the fluid displaced by the moving bead. We also observe an anticorrelated thermal force, which is conventionally assumed to be uncorrelated.

DOI

Journal: Science

Radio-Wave Oscillations of Molecular-Chain Resonators

Stefan Müllegger, Mohammad Rashidi, Karlheinz Mayr, Michael Fattinger, Andreas Ney, and Reinhold Koch

We report a new type of nanomechanical resonator system based on one-dimensional chains of only 4 to 7 weakly coupled small molecules. Experimental characterization of the truly nanoscopic resonators is achieved by means of a novel radio-frequency scanning tunneling microscopy detection technique at cryogenic temperatures. Above 20 K we observe concerted oscillations of the individual molecules in chains, reminiscent of the first and second eigenmodes of a one-dimensional harmonic resonator. Radio-frequency scanning tunneling microscopy based frequency measurement reveals a characteristic length dependence of the oscillation frequency (between 51 and 127 MHz) in reasonable agreement with one-dimensional oscillator models. Our study demonstrates a new strategy for investigating and controlling the resonance properties of nanomechanical oscillators.

DOI

Journal:Physical Review Letters

Single-molecule resolution of protein structure and interfacial dynamics on biomaterial surfaces

Sean Yu McLoughlin, Mark Kastantin, Daniel K. Schwartz, and Joel L. Kaar

A method was developed to monitor dynamic changes in protein structure and interfacial behavior on surfaces by single-molecule Förster resonance energy transfer. This method entails the incorporation of unnatural amino acids to site-specifically label proteins with single-molecule Förster resonance energy transfer probes for high-throughput dynamic fluorescence tracking microscopy on surfaces. Structural changes in the enzyme organophosphorus hydrolase (OPH) were monitored upon adsorption to fused silica (FS) surfaces in the presence of BSA on a molecule-by-molecule basis. Analysis of >30,000 individual trajectories enabled the observation of heterogeneities in the kinetics of surface-induced OPH unfolding with unprecedented resolution. In particular, two distinct pathways were observed: a majority population (∼ 85%) unfolded with a characteristic time scale of 0.10 s, and the remainder unfolded more slowly with a time scale of 0.7 s. Importantly, even after unfolding, OPH readily desorbed from FS surfaces, challenging the common notion that surface-induced unfolding leads to irreversible protein binding. This suggests that protein fouling of surfaces is a highly dynamic process because of subtle differences in the adsorption/desorption rates of folded and unfolded species. Moreover, such observations imply that surfaces may act as a source of unfolded (i.e., aggregation-prone) protein back into solution. Continuing study of other proteins and surfaces will examine whether these conclusions are general or specific to OPH in contact with FS. Ultimately, this method, which is widely applicable to virtually any protein, provides the framework to develop surfaces and surface modifications with improved biocompatibility.

DOI

Journal: Proceedings of the National Academy of Sciences

High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations

Felix Rico, Laura Gonzalez, Ignacio Casuso, Manel Puig-Vidal, Simon Scheuring

The mechanical unfolding of the muscle protein titin by atomic force microscopy was a landmark in our understanding of single-biomolecule mechanics. Molecular dynamics simulations offered atomic-level descriptions of the forced unfolding. However, experiment and simulation could not be directly compared because they differed in pulling velocity by orders of magnitude. We have developed high-speed force spectroscopy to unfold titin at velocities reached by simulation (~4 millimeters per second). We found that a small β-strand pair of an immunoglobulin domain dynamically unfolds and refolds, buffering pulling forces up to ~100 piconewtons. The distance to the unfolding transition barrier is larger than previously estimated but is in better agreement with atomistic predictions. The ability to directly compare experiment and simulation is likely to be important in studies of biomechanical processes.

DOI

Journal: Science

Amyloid-β Dynamics Are Regulated by Orexin and the Sleep-Wake Cycle

Jae-Eun Kang, Miranda M. Lim, Randall J. Bateman, James J. Lee, Liam P. Smyth, John R. Cirrito, Nobuhiro Fujiki, Seiji Nishino, David M. Holtzman

Amyloid-β (Aβ) accumulation in the brain extracellular space is a hallmark of Alzheimer’s disease. The factors regulating this process are only partly understood. Aβ aggregation is a concentration-dependent process that is likely responsive to changes in brain interstitial fluid (ISF) levels of Aβ. Using in vivo microdialysis in mice, we found that the amount of ISF Aβ correlated with wakefulness. The amount of ISF Aβ also significantly increased during acute sleep deprivation and during orexin infusion, but decreased with infusion of a dual orexin receptor antagonist. Chronic sleep restriction significantly increased, and a dual orexin receptor antagonist decreased, Aβ plaque formation in amyloid precursor protein transgenic mice. Thus, the sleep-wake cycle and orexin may play a role in the pathogenesis of Alzheimer’s disease.

DOI

Journal: Science

Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus

Robert H. Vass and Peter Chien

Chromosome replication relies on sliding clamps that are loaded by energy-dependent complexes. In Escherichia coli, the ATP-binding clamp loader subunit DnaX exists as both long (τ) and short (γ) forms generated through programmed translational frameshifting, but the need for both forms is unclear. Here, we show that in Caulobacter crescentus, DnaX isoforms are unexpectedly generated through partial proteolysis by the AAA+ protease casein lytic proteinase (Clp) XP. We find that the normally processive ClpXP protease partially degrades DnaX to produce stable fragments upon encountering a glycine-rich region adjacent to a structured domain. Increasing the sequence complexity of this region prevents partial proteolysis and generates a τ-only form of DnaX in vivo that is unable to support viability on its own. Growth is restored when γ is provided in trans, but these strains are more sensitive to DNA damage compared with strains that can generate γ through proteolysis. Our work reveals an unexpected mode of partial processing by the ClpXP protease to generate DnaX isoforms, demonstrates that both τ and γ forms of DnaX are required for Caulobacter viability, and identifies a role for clamp loader diversity in responding to DNA damage. The conservation of distinct DnaX isoforms throughout bacteria despite fundamentally different mechanisms for producing them suggests there may be a conserved need for alternate clamp loader complexes during DNA damaging conditions.

DOI

Journal: Proceedings of the National Academy of Sciences

Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

Gabriel Žoldáka, Johannes Stigler, Benjamin Pelz, Hongbin Li, and Matthias Rief

In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.

DOI

Journal: Proceedings of the National Academy of Sciences

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology

Mahmood Haj-Yahya, Bruno Fauvet, Yifat Herman-Bachinsky, Mirva Hejjaoui, Sudhir N. Bavikara, Subramanian Vedhanarayanan Karthikeyan, Aaron Ciechanover, Hilal A. Lashuel, and Ashraf Brik

Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di- or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications.

DOI

Journal: Proceedings of the National Academy of Sciences

Single-molecule fluorescence probes dynamics of barrier crossing

Hoi Sung Chung, and William A. Eaton

Kramers developed the theory on how chemical reaction rates are influenced by the viscosity of the medium1. At the viscosity of water, the kinetics of unimolecular reactions are described by diffusion of a Brownian particle over a free-energy barrier separating reactants and products. For reactions in solution this famous theory extended Eyring’s transition state theory, and is widely applied in physics, chemistry and biology, including to reactions as complex as protein folding. Because the diffusion coefficient of Kramers’ theory is determined by the dynamics in the sparsely populated region of the barrier top, its properties have not been directly measured for any molecular system. Here we show that the Kramers diffusion coefficient and free-energy barrier can be characterized by measuring the temperature- and viscosity-dependence of the transition path time for protein folding. The transition path is the small fraction of an equilibrium trajectory for a single molecule when the free-energy barrier separating two states is actually crossed. Its duration, the transition path time, can now be determined from photon trajectories for single protein molecules undergoing folding/unfolding transitions5. Our finding of a long transition path time with an unusually small solvent viscosity dependence suggests that internal friction as well as solvent friction determine the Kramers diffusion coefficient for α-helical proteins, as opposed to a breakdown of his theory, which occurs for many small-molecule reactions2. It is noteworthy that the new and fundamental information concerning Kramers’ theory and the dynamics of barrier crossings obtained here come from experiments on a protein rather than a much simpler chemical or physical system.

DOI

Journal: Nature

Nanomechanics of HaloTag Tethers

Ionel Popa , Ronen Berkovich, Jorge Alegre-Cebollada, Carmen L. Badilla, Jaime Andrés Rivas-Pardo, Yukinori Taniguchi , Masaru Kawakami , and Julio M. Fernandez 

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to 2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding–unfolding cycles at high forces, without interference from the HaloTag tether.

DOI

Journal: Journal of the American Chemical Society