Probing Chromatin Structure with Magnetic Tweezers

Kaczmarczyk, Artur; Brouwer, Thomas B.; Pham, Chi L.L.; Dekker, Nynke H.; Noort, John van

doi:10.1007/978-1-4939-8591-3_18

Cited by 20 publications

(39 citation statements)

References 30 publications

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“…The samples were dialyzed overnight at 4 C against 200 ml of a buffer with gradually decreasing ionic strength down to 100 mM NaCl. The salt gradient was maintained by a peristaltic pump (Bio-Rad Econo Gradient Pump, flow rate 0.9 mlÁmin À1 ) 70,71 . Quality assessment of the reconstituted chromatin fibers was performed via an electrophoretic band shift assay (0.7% agarose gel in 0.2 TB).…”

Section: Methodsmentioning

confidence: 99%

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Chromatin fibers stabilize nucleosomes under torsional stress

et al. 2020

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Torsional stress generated during DNA replication and transcription has been suggested to facilitate nucleosome unwrapping and thereby the progression of polymerases. However, the propagation of twist in condensed chromatin remains yet unresolved. Here, we measure how force and torque impact chromatin fibers with a nucleosome repeat length of 167 and 197. We find that both types of fibers fold into a left-handed superhelix that can be stabilized by positive torsion. We observe that the structural changes induced by twist were reversible, indicating that chromatin has a large degree of elasticity. Our direct measurements of torque confirmed the hypothesis of chromatin fibers as a twist buffer. Using a statistical mechanicsbased torsional spring model, we extracted values of the chromatin twist modulus and the linking number per stacked nucleosome that were in good agreement with values measured here experimentally. Overall, our findings indicate that the supercoiling generated by DNAprocessing enzymes, predicted by the twin-supercoiled domain model, can be largely accommodated by the higher-order structure of chromatin.

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Section: Methodsmentioning

confidence: 99%

“…Finally, force-extension curves were measured between 0 and 50 pN. The acquired data was analyzed using LabVIEW software by fitting the statistical mechanics model presented earlier 40,71 . Extension-twist curves were analyzed with the torsional spring model (explained in Supplementary Note 3).…”

Section: Methodsmentioning

confidence: 99%

Chromatin fibers stabilize nucleosomes under torsional stress

et al. 2020

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“…1 a. The composition of chromatin fibers may vary because of the quality of reconstitution (11), disassembly (6), or reflecting naturally occurring variations (53). In these applications, we noticed that the dynamic range of the LUT method was sometimes insufficient, yielding an accuracy that depended on the bead height.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Brouwer

Hermans

Noort

2020

Biophysical Journal

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Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead's position with nanometer accuracy in three dimensions but is computationally expensive. Realtime multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 mm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.

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“…By manipulation of microbeads, single-molecule force spectroscopy techniques revealed the mechanical properties of biomolecules such as DNA or RNA with unprecedented detail [2][3][4][5]. In addition, the interactions with proteins, like the DNA compaction by histones in eukaryotic chromatin [6][7][8][9][10][11] and prokaryotic architectural proteins [12][13][14][15][16], supercoiling [17][18][19][20], and repair processes [21][22][23] were extensively studied with magnetic tweezers (MT) or optical tweezers (OT), Acoustic Force Spectroscopy (AFS) [24][25][26] or tethered particle motion (TPM) [13,27,28]. These bead manipulation techniques have also been used to quantify the mechanical properties of other biological structures, such as extracellular protein collagen [29][30][31], or even entire cells [32].…”

Section: Introductionmentioning

confidence: 99%

“…Previously, we implemented the LUT bead tracking algorithm in our MT and used it to study the various transitions of chromatin fiber unfolding [6,7,52], as schematically depicted in Fig 1a. In these applications, we noticed that the dynamic range of the LUT method was sometimes insufficient, yielding an accuracy that depended on the bead height. As the composition of chromatin fibers may vary due to the quality of reconstitution [11], disassembly [6] or reflecting naturally occurring variations [53], we found it imperative to improve both the computation speed, allowing for tracking a large number of independent tethers, and the accuracy over a dynamic range that spans up to 10 micrometer. In our hands, the empirical LUT algorithm frequently flawed due to non-perfect imaging conditions, which led to discarding a large fraction of the beads, limiting the throughput.…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

Brouwer

Hermans

Noort

2019

Preprint

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Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in arange of over 10 µm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required. SignificanceMicrobeads are often used in biophysical single-molecule manipulation experiments and accurately tracking their position in 3 dimensions is key for quantitative analysis. Holographic imaging of these beads allows for multiplexing bead tracking but image analysis can be a limiting factor. Here we present a 3D tracking algorithm based on Fast Fourier Transforms that is fast, has nanometric precision, is robust against common artifacts and is accurate over 10's of micrometers. We show its real-time application for magnetic tweezers based force spectroscopy on more than 100 chromatin fibers in parallel, but we anticipate that many other bead-based biophysical essays can benefit from this simple and robust 3 phasor algorithm.

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Probing Chromatin Structure with Magnetic Tweezers

Cited by 20 publications

References 30 publications

Chromatin fibers stabilize nucleosomes under torsional stress

Chromatin fibers stabilize nucleosomes under torsional stress

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

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