Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy

Huhle, Alexander; Klaue, Daniel; Brutzer, Hergen; Daldrop, Peter; Joo, Sihwa; Otto, Oliver; Keyser, Ulrich F.; Seidel, Ralf

doi:10.1038/ncomms6885

Cited by 125 publications

(151 citation statements)

References 38 publications

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“…A pair of magnets placed above the cell was used to stretch the DNA and to supercoil it by rotating the magnets. Simultaneously, we measured the DNA length from the magnetic bead z-position using video microscopy (Otto et al, 2010;Huhle et al, 2015). DNA supercoiling at constant force results in a characteristic bell-shaped DNA length-versus-turn curve (gray curve in Figure 1B).…”

Section: Detection Of Single Cascade-induced R-loopsmentioning

confidence: 99%

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

Rutkauskas¹,

Šinkūnas²,

et al. 2015

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CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.

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Section: Detection Of Single Cascade-induced R-loopsmentioning

confidence: 99%

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

Rutkauskas¹,

Šinkūnas²,

et al. 2015

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“…Our high-resolution magnetic tweezers setup has been described previously (6). It consists of a home-built inverted microscope equipped with a 100Â (NA 1.25) objective (Olympus, Center Valley, PA).…”

Section: Magnetic Tweezersmentioning

confidence: 99%

“…Two permanent NeFeB magnets (W-05-N50-G, Supermagnete, Gottmadingen, Germany) mounted onto a motorized stage above the sample were used to generate the magnetic field gradient for the magnetic tweezers experiments. Images were streamed to a computer (Precision T7500 Work Station, Dell, Round Rock, TX) equipped with an image acquisition card (NI PCIe 1429, National Instruments, Austin, TX) and a fast GPU (GeForce GTX 480, Nvidia, Santa Clara, CA) and analyzed in real time (6).…”

Section: Magnetic Tweezersmentioning

confidence: 99%

“…In recent studies, tethers with reduced length (shorter than the bead diameter) have been used more frequently (6,24,26,33,34), particularly in high-resolution magnetictweezers measurements, due to their increased stiffness along the molecule axis. Such tethers represent a severe challenge for direct force calibration.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, to ascertain dynamical or force-induced conformational changes, the length of the biomolecular tether under tension is determined. For this, camera-based particle tracking is typically applied (3)(4)(5)(6)(7). Magnetic tweezers have been widely used to study the mechanical properties of biological (8,9) and synthetic (10) polymers, to investigate the response of DNA upon supercoiling (1,(11)(12)(13)(14)(15), and to resolve the real-time dynamics of DNA-interacting proteins, such as DNA-binding proteins (16)(17)(18), enzymes that regulate DNA supercoiling (19)(20)(21), and molecular motors (22)(23)(24)(25)(26)(27).…”

Section: Introductionmentioning

confidence: 99%

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Extending the Range for Force Calibration in Magnetic Tweezers

Daldrop

Brutzer

Huhle

et al. 2015

Biophysical Journal

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Magnetic tweezers are a wide-spread tool used to study the mechanics and the function of a large variety of biomolecules and biomolecular machines. This tool uses a magnetic particle and a strong magnetic field gradient to apply defined forces to the molecule of interest. Forces are typically quantified by analyzing the lateral fluctuations of the biomolecule-tethered particle in the direction perpendicular to the applied force. Since the magnetic field pins the anisotropy axis of the particle, the lateral fluctuations follow the geometry of a pendulum with a short pendulum length along and a long pendulum length perpendicular to the field lines. Typically, the short pendulum geometry is used for force calibration by power-spectral-density (PSD) analysis, because the movement of the bead in this direction can be approximated by a simple translational motion. Here, we provide a detailed analysis of the fluctuations according to the long pendulum geometry and show that for this direction, both the translational and the rotational motions of the particle have to be considered. We provide analytical formulas for the PSD of this coupled system that agree well with PSDs obtained in experiments and simulations and that finally allow a faithful quantification of the magnetic force for the long pendulum geometry. We furthermore demonstrate that this methodology allows the calibration of much larger forces than the short pendulum geometry in a tether-length-dependent manner. In addition, the accuracy of determination of the absolute force is improved. Our force calibration based on the long pendulum geometry will facilitate high-resolution magnetic-tweezers experiments that rely on short molecules and large forces, as well as highly parallelized measurements that use low frame rates.

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Force versus Response: Methods for Activating and Characterizing Mechanosensitive Ion Channels and GPCRs

Roeterink,

Casadevall i Solvas,

Collins

et al. 2024

Adv Healthcare Materials

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Mechanotransduction is the process whereby cells convert mechanical signals into electrochemical responses, where mechanosensitive proteins mediate this interaction. To characterize these critical proteins, numerous techniques have been developed that apply forces and measure the subsequent cellular responses. While these approaches have given insight into specific aspects of many such proteins, subsequent validation and cross‐comparison between techniques remain difficult given significant variations in reported activation thresholds and responses for the same protein across different studies. Accurately determining mechanosensitivity responses for various proteins, however, is essential for understanding mechanotransduction and potential physiological implications, including therapeutics. This critical review provides an assessment of current and emerging approaches used for mechanosensitive ion channel and G‐Coupled Receptors (GPCRs) stimulation and measurement, with a specific focus on the ability to quantitatively measure mechanosensitive responses.

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Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy

Cited by 125 publications

References 38 publications

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

Extending the Range for Force Calibration in Magnetic Tweezers

Force versus Response: Methods for Activating and Characterizing Mechanosensitive Ion Channels and GPCRs

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