Histone chaperones exhibit conserved functionality in nucleosome remodeling

Chromatin homeostasis mediates some of the most fundamental processes in the eukaryotic cell. In this regard, histone chaperones have emerged as major regulatory factors during DNA replication, repair, and transcription. However, the dynamic nature of these processes has severely impeded their characterization at the molecular level. Here we apply single-molecule probing by fluorescence optical tweezers to follow histone chaperone dynamics in real-time. The molecular action of SET/template-activating factor-Iβ and nucleophosmin 1, representing the two most common histone chaperone folds, were examined using both nucleosomes and isolated core histones. We show that these chaperones present binding specificity for partially dismantled nucleosomes and are able to recognize and disrupt non-native histone-DNA interactions. Furthermore, we reveal that cytochrome c inhibition of histone chaperones is coupled to chaperone accumulation on DNA-bound histones. Our single-molecule approach shows that despite the drastically different structures of these chaperones, they present conserved modes of action mediating nucleosome remodeling.

Stem–loop formation drives RNA folding in mechanical unzipping experiments

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force–distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping–rezipping process, we introduce a barrier energy landscape of the stem–loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem–loops that kinetically trap the long-lived intermediates observed in the FDC. Stem–loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

Controllable shifting, steering, and expanding of light beam based on multi-layer liquid-crystal cells

Shaping and steering of light beams is essential in many modern applications, ranging from optical tweezers, camera lenses, vision correction to 3D displays. However, current realisations require increasingly greater tunability and aim for lesser specificity for use in diverse applications. Here, we demonstrate tunable light beam control based on multi-layer liquid-crystal cells and external electric field, capable of extended beam shifting, steering, and expanding, using a combination of theory and full numerical modelling, both for liquid crystal orientations and the transmitted light. Specifically, by exploiting three different function-specific and tunable birefringent nematic layers, we show an effective liquid-crystal beam control device, capable of precise control of outgoing light propagation, with possible application in projectors or automotive headlamps.

Calculating the force-dependent unbinding rate of biological macromolecular bonds from force-ramp optical trapping assays

The non-covalent biological bonds that constitute protein–protein or protein–ligand interactions play crucial roles in many cellular functions, including mitosis, motility, and cell–cell adhesion. The effect of external force ($$F$$ F ) on the unbinding rate ($${k}_{\text{off}}\left(F\right)$$ k off F ) of macromolecular interactions is a crucial parameter to understanding the mechanisms behind these functions. Optical tweezer-based single-molecule force spectroscopy is frequently used to obtain quantitative force-dependent dissociation data on slip, catch, and ideal bonds. However, analyses of this data using dissociation time or dissociation force histograms often quantitatively compare bonds without fully characterizing their underlying biophysical properties. Additionally, the results of histogram-based analyses can depend on the rate at which force was applied during the experiment and the experiment’s sensitivity. Here, we present an analytically derived cumulative distribution function-like approach to analyzing force-dependent dissociation force spectroscopy data. We demonstrate the benefits and limitations of the technique using stochastic simulations of various bond types. We show that it can be used to obtain the detachment rate and force sensitivity of biological macromolecular bonds from force spectroscopy experiments by explicitly accounting for loading rate and noisy data. We also discuss the implications of our results on using optical tweezers to collect force-dependent dissociation data.

Red blood cell in the field of a beam of optical tweezers

We consider the effect of a tightly focused laser beam with a wavelength of 1064 nm and a power from 10 to 160 mW on red blood cells during their optical trapping with optical tweezers. It is found that the shape of a red blood cell, which alters after optical trapping, ceases to change when the trapping duration is less than 5 min and the laser beam power is less than 60 mW. At a beam power above 80 mW, the red blood cell begins to fold at a trapping duration of about 1 min, and at powers above 100-150 mW, the red blood cell membrane ruptures in 1-3 min after optical trapping. It is also found that with repeated short-term capture of a red blood cell in an optical trap, the deformation properties of the membrane change: it becomes more rigid. The obtained results are important both for understanding the mechanisms of interaction of a laser beam with red blood cells and for optimising the technique of optical experiments, especially for measuring the deformation properties of a membrane using optical tweezers.

Tweezepy: A Python package for calibrating forces in single-molecule video-tracking experiments

Single-molecule force spectroscopy (SMFS) instruments (e.g., magnetic and optical tweezers) often use video tracking to measure the three-dimensional position of micron-scale beads under an applied force. The force in these experiments is calibrated by comparing the bead trajectory to a thermal motion-based model with the drag coefficient, γ, and trap spring constant, κ, as parameters. Estimating accurate parameters is complicated by systematic biases from spectral distortions, the camera exposure time, parasitic noise, and least-squares fitting methods. However, while robust calibration methods exist that correct for these biases, they are not always used because they can be complex to implement computationally. To address this barrier, we present Tweezepy: a Python package for calibrating forces in SMFS video-tracking experiments. Tweezepy uses maximum likelihood estimation (MLE) to estimate parameters and their uncertainties from a single bead trajectory via the power spectral density (PSD) and Allan variance (AV). It is well-documented, fast, easy to use, and accounts for most common sources of biases in SMFS video-tracking experiments. Here, we provide a comprehensive overview of Tweezepy’s calibration scheme, including a review of the theory underlying thermal motion-based parameter estimates, a discussion of the PSD, AV, and MLE, and an explanation of their implementation.

FOCUS SHAPING OF CYLINDRICALLY POLARIZED VORTEX BEAMS BY A LINEAR AXICON

Focus shaping of cylindrically polarized vortex beams (CPVBs) by linear axicon is studied theoretically. Vector diffraction theory has been used to derive the expressions of the light field in the focal region. It is shown that a different intensity distribution in the focal region can be obtained by adjusting the topological charge, the polarization rotation angle and the numerical aperture maximal angle. A focal spot, a dark channel and a flat-topped shapes are formed by choosing proper values of parameters. A controllable polarization state of dark channel is obtained. The different focal region shapes may find wide applications such as material processing and optical tweezers.

Review on fractional vortex beam

As an indispensable complement to an integer vortex beam, the fractional vortex beam has unique physical properties such as radially notched intensity distribution, complex phase structure consisting of alternating charge vortex chains, and more sophisticated orbital angular momentum modulation dimension. In recent years, we have noticed that the fractional vortex beam was widely used for complex micro-particle manipulation in optical tweezers, improving communication capacity, controllable edge enhancement of image and quantum entanglement. Moreover, this has stimulated extensive research interest, including the deep digging of the phenomenon and physics based on different advanced beam sources and has led to a new research boom in micro/nano-optical devices. Here, we review the recent advances leading to theoretical models, propagation, generation, measurement, and applications of fractional vortex beams and consider the possible directions and challenges in the future.