Atomic force microscopy-indentation demonstrates that alginate beads are mechanically stable under cell culture conditions.

Journal of the mechanical behavior of biomedical materials 93 (2019) 61-69

C-Y Chui, A Bonilla-Brunner, J Seifert, S Contera, H Ye

Alginate microbeads are extensively used in tissue engineering as microcarriers and cell encapsulation vessels. In this study, we used atomic force microscopy (AFM) based indentation using 20 µm colloidal probes to assess the local reduced elastic modulus (E * ) using a novel method to detect the contact point based on the principle of virtual work, to measure microbead mechanical stability under cell culture conditions for 2 weeks. The bead diameter and swelling were assessed in parallel. Alginate beads swelled up to 150% of their original diameter following addition of cell culture media. The diameter eventually stabilized from day 2 onwards. This behaviour was mirrored in E * where a significant decrease was observed at the start of the culture period before stabilization was observed at ~ 2.1 kPa. Furthermore, the mechanical properties of freeze dried alginate beads after re-swelling them in culture media were measured. These beads displayed vastly different structural and mechanical properties compared those that did not go through the freeze drying process, with around 125% swelling and a significantly higher E * at values over 3 kPa.

A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Science (New York, N.Y.) 363 (2019) 875-880

M Schewe, H Sun, Ü Mert, A Mackenzie, ACW Pike, F Schulz, C Constantin, KS Vowinkel, LJ Conrad, AK Kiper, W Gonzalez, M Musinszki, M Tegtmeier, DC Pryde, H Belabed, M Nazare, BL de Groot, N Decher, B Fakler, EP Carpenter, SJ Tucker, T Baukrowitz

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes.

Microbial cell (Graz, Austria) 6 (2019) 65-101

HL Klein, KKH Ang, MR Arkin, EC Beckwitt, Y-H Chang, J Fan, Y Kwon, MJ Morten, S Mukherjee, OJ Pambos, H El Sayyed, ES Thrall, JP Vieira-da-Rocha, Q Wang, S Wang, H-Y Yeh, JS Biteen, P Chi, W-D Heyer, AN Kapanidis, JJ Loparo, TR Strick, P Sung, B Van Houten, H Niu, E Rothenberg

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

CHAP: a versatile tool for the structural and functional annotation of ion channel pores


G Klesse, S Rao, MSP Sansom, S Tucker

The regulation of ion channel and transporter function requires the modulation of energetic barriers or "gates" within their transmembrane pathways. However, despite the ever-increasing number of available structures, our understanding of these barriers is often simply determined from calculating the physical dimensions of the pore. Such approaches (e.g. the HOLE program) have worked very well in the past, but there is now considerable evidence that the unusual behaviour of water within the narrow hydrophobic spaces found within many ion channel pores can also produce energetic barriers to ion conduction without requiring physical occlusion of the permeation pathway. Several different classes of ion channels have now been shown to exploit this principle of "hydrophobic gating" to regulate ion flow. However, measurement of pore radius alone is unable to identify such barriers and new tools are required for more accurate functional annotation of an exponentially increasing number of ion channel structures. We have previously shown how molecular dynamics simulations of water behaviour can be used as a proxy to accurately predict hydrophobic gates. Here we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology to predict the conductive status of new ion channel structures.

A simple mathematical model of allometric exponential growth describes the early three-dimensional growth dynamics of secondary xylem in Arabidopsis roots

Royal Society Open Science The Royal Society 6 (2019) 190126-190126

A Thamm, S Sanegre-Sans, J Paisley, S Meader, A Milhinhos, S ANTORANZ CONTERA, J Agusti

A multi-mode digital holographic microscope.

The Review of scientific instruments 90 (2019) 023705-

JL Flewellen, IM Zaid, RM Berry

We present a transmission-mode digital holographic microscope that can switch easily between three different imaging modes: inline, dark field off-axis, and bright field off-axis. Our instrument can be used: to track through time in three dimensions microscopic dielectric objects, such as motile micro-organisms; localize brightly scattering nanoparticles, which cannot be seen under conventional bright field illumination; and recover topographic information and measure the refractive index and dry mass of samples via quantitative phase recovery. Holograms are captured on a digital camera capable of high-speed video recording of up to 2000 frames per second. The inline mode of operation can be easily configurable to a large range of magnifications. We demonstrate the efficacy of the inline mode in tracking motile bacteria in three dimensions in a 160 μm × 160 μm × 100 μm volume at 45× magnification. Through the use of a novel physical mask in a conjugate Fourier plane in the imaging path, we use our microscope for high magnification, dark field off-axis holography, demonstrated by localizing 100 nm gold nanoparticles at 225× magnification up to at least 16 μm from the imaging plane. Finally, the bright field off-axis mode facilitates quantitative phase microscopy, which we employ to measure the refractive index of a standard resolution test target and to measure the dry mass of human erythrocytes.

A Heuristic Derived from Analysis of the Ion Channel Structural Proteome Permits the Rapid Identification of Hydrophobic Gates


S Rao, G Klesse, P Stansfeld, S Tucker, MSP Sansom

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined. However, these newly-resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore and it is now known that more accurate annotation of their conductive state requires an additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavour liquid-phase water, leading to local de-wetting which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore de-wetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but may also facilitate the design of novel nanopores controlled by hydrophobic gates.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria.

Nature communications 9 (2018) 1478-

D Dulin, DLV Bauer, AM Malinen, JJW Bakermans, M Kaller, Z Morichaud, I Petushkov, M Depken, K Brodolin, A Kulbachinskiy, AN Kapanidis

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation.

Methods in molecular biology (Clifton, N.J.) 1729 (2018) 233-246

D Di Paolo, RM Berry

For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates. Electroporation allows for internalization of purified CheY proteins labeled with organic dyes into E. coli cells in controllable concentrations. Using fluorescence video microscopy, it is possible to observe single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.

Single-molecule techniques in biophysics: a review of the progress in methods and applications.

Reports on progress in physics. Physical Society (Great Britain) 81 (2018) 024601-024601

H Miller, Z Zhou, J Shepherd, AJM Wollman, MC Leake

Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k B T, where k B is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.

Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Molecular cell (2018)

W Lin, K Das, D Degen, A Mazumder, D Duchi, D Wang, YW Ebright, RY Ebright, E Sineva, M Gigliotti, A Srivastava, S Mandal, Y Jiang, Y Liu, R Yin, Z Zhang, ET Eng, D Thomas, S Donadio, H Zhang, C Zhang, AN Kapanidis, RH Ebright

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Coming together during viral assembly.

Nature reviews. Microbiology 16 (2018) 721-721

C Hepp, NC Robb

Multifrequency AFM reveals lipid membrane mechanical properties and the effect of cholesterol in modulating viscoelasticity.

Proceedings of the National Academy of Sciences of the United States of America 115 (2018) 2658-2663

Z Al-Rekabi, S Contera

The physical properties of lipid bilayers comprising the cell membrane occupy the current spotlight of membrane biology. Their traditional representation as a passive 2D fluid has gradually been abandoned in favor of a more complex picture: an anisotropic time-dependent viscoelastic biphasic material, capable of transmitting or attenuating mechanical forces that regulate biological processes. In establishing new models, quantitative experiments are necessary when attempting to develop suitable techniques for dynamic measurements. Here, we map both the elastic and viscous properties of the model system 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers using multifrequency atomic force microscopy (AFM), namely amplitude modulation-frequency modulation (AM-FM) AFM imaging in an aqueous environment. Furthermore, we investigate the effect of cholesterol (Chol) on the DPPC bilayer in concentrations from 0 to 60%. The AM-AFM quantitative maps demonstrate that at low Chol concentrations, the lipid bilayer displays a distinct phase separation and is elastic, whereas at higher Chol concentration, the bilayer appears homogenous and exhibits both elastic and viscous properties. At low-Chol contents, the Estorage modulus (elastic) dominates. As the Chol insertions increases, higher energy is dissipated; and although the bilayer stiffens (increase in Estorage), the viscous component dominates (Eloss). Our results provide evidence that the lipid bilayer exhibits both elastic and viscous properties that are modulated by the presence of Chol, which may affect the propagation (elastic) or attenuation (viscous) of mechanical signals across the cell membrane.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 669-676

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, S Rezaei Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes

Nucleic Acids Research 46 (2018) 677-688

D Duchi, K Gryte, NC Robb, Z Morichaud, C Sheppard, K Brodolin, S Wigneshweraraj, AN Kapanidis

© The Author(s) 2017. Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex sub-sequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia colitranscription open complexes. Our results revealed the existence of two stable states within RNAP-DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

Self-propulsion of catalytic nanomotors synthesised by seeded growth of asymmetric platinum-gold nanoparticles.

Chemical communications (Cambridge, England) 54 (2018) 1901-1904

I Santiago, L Jiang, J Foord, AJ Turberfield

Asymmetric bimetallic nanomotors are synthesised by seeded growth in solution, providing a convenient and high-throughput alternative to the usual top-down lithographic fabrication of self-propelled catalytic nanoparticles. These synthetic nanomotors catalyse H2O2 decomposition and exhibit enhanced diffusion that depends on fuel concentration, consistent with their chemical propulsion.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 984-984

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, SR Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Hydrophobic Gating: Examination of Recent Ion Channel Structures


S Rao, G Klesse, SJ Tucker, MSP Sansom

Rare NaV1.7 variants associated with painful diabetic peripheral neuropathy.

Pain 159 (2018) 469-480

I Blesneac, AC Themistocleous, C Fratter, LJ Conrad, JD Ramirez, JJ Cox, S Tesfaye, PR Shillo, ASC Rice, SJ Tucker, DLH Bennett

Diabetic peripheral neuropathy (DPN) is a common disabling complication of diabetes. Almost half of the patients with DPN develop neuropathic pain (NeuP) for which current analgesic treatments are inadequate. Understanding the role of genetic variability in the development of painful DPN is needed for improved understanding of pain pathogenesis for better patient stratification in clinical trials and to target therapy more appropriately. Here, we examined the relationship between variants in the voltage-gated sodium channel NaV1.7 and NeuP in a deeply phenotyped cohort of patients with DPN. Although no rare variants were found in 78 participants with painless DPN, we identified 12 rare NaV1.7 variants in 10 (out of 111) study participants with painful DPN. Five of these variants had previously been described in the context of other NeuP disorders and 7 have not previously been linked to NeuP. Those patients with rare variants reported more severe pain and greater sensitivity to pressure stimuli on quantitative sensory testing. Electrophysiological characterization of 2 of the novel variants (M1852T and T1596I) demonstrated that gain of function changes as a consequence of markedly impaired channel fast inactivation. Using a structural model of NaV1.7, we were also able to provide further insight into the structural mechanisms underlying fast inactivation and the role of the C-terminal domain in this process. Our observations suggest that rare NaV1.7 variants contribute to the development NeuP in patients with DPN. Their identification should aid understanding of sensory phenotype, patient stratification, and help target treatments effectively.

How to probe the spin contribution to momentum relaxation in topological insulators.

Nature communications 9 (2018) 56-

M-S Nam, BH Williams, Y Chen, S Contera, S Yao, M Lu, Y-F Chen, GA Timco, CA Muryn, REP Winpenny, A Ardavan

Topological insulators exhibit a metallic surface state in which the directions of the carriers' momentum and spin are locked together. This characteristic property, which lies at the heart of proposed applications of topological insulators, protects carriers in the surface state from back-scattering unless the scattering centres are time-reversal symmetry breaking (i.e. magnetic). Here, we introduce a method of probing the effect of magnetic scattering by decorating the surface of topological insulators with molecules, whose magnetic degrees of freedom can be engineered independently of their electrostatic structure. We show that this approach allows us to separate the effects of magnetic and non-magnetic scattering in the perturbative limit. We thereby confirm that the low-temperature conductivity of SmB6 is dominated by a surface state and that the momentum of quasiparticles in this state is particularly sensitive to magnetic scatterers, as expected in a topological insulator.