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
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
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
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
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
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
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
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
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
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
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
