Time-resolved Force Research Articles

Unveiling dissociation mechanisms and binding patterns in the UHRF1-DPPA3 complex via multi-replica molecular dynamics simulations.

Ubiquitin-like with PHD and RING finger domain containing protein 1 (UHRF1) is responsible for preserving the stability of genomic methylation through the recruitment of DNA methyltransferase 1 (DNMT1). However, the interaction between Developmental pluripotency associated 3 (DPPA3) and the pre-PHD-PHD (PPHD) domain of UHRF1 hinders the nuclear localization of UHRF1. This disruption has implications for potential cancer treatment strategies. Drugs that mimic the binding pattern between DPPA3 and PPHD could offer a promising approach to cancer treatment. Our study reveals that DPPA3 undergoes dissociation from the C-terminal through three different modes of helix unfolding. Furthermore, we have identified key residue pairs involved in this dissociation process and potential drug-targeting residues. These findings offer valuable insights into the dissociation mechanism of DPPA3 from PPHD and have the potential to inform the design of novel drugs targeting UHRF1 for cancer therapy. To comprehend the dissociation process and binding patterns of PPHD-DPPA3, we employed enhanced sampling techniques, including steered molecular dynamics (SMD) and conventional molecular dynamics (cMD). Additionally, we utilized self-organizing maps (SOM) and time-resolved force distribution analysis (TRFDA) methodologies. The Gromacs software was used for performing molecular dynamics simulations, and the AMBER FF14SB force field was applied to the protein.

Insights into the Dissociation Process and Binding Pattern of the BRCT7/8-PHF8 Complex.

DNA topoisomerase 2-binding protein 1 (Topbp1) plays a crucial role in activating the ataxia-telangiectasia mutated and rad3-related (ATR) complex to initiate DNA damage repair responses. For this process to occur, it is necessary for PHF8 to dissociate from Topbp1. Topbp1 binds to the acidic patch sequence (APS) of PHF8 through its C-terminal BRCT7/8 domain, and disrupting this interaction could be a promising strategy for cancer treatment. To investigate the dissociation process and binding pattern of BRCT7/8-PHF8, we employed enhanced sampling techniques, such as steered molecular dynamics (SMD) simulations and accelerated molecular dynamics (aMD) simulations, along with self-organizing maps (SOM) and time-resolved force distribution analysis (TRFDA) methodologies. Our results demonstrate that the dissociation of PHF8 from BRCT7/8 starts from the N-terminus, leading to the unfolding of the N-terminal helix. Additionally, we identified critical residues that play a pivotal role in this dissociation process. These findings provide valuable insights into the disassociation of PHF8 from BRCT7/8, which could potentially guide the development of novel drugs targeting Topbp1 for cancer therapy.

Nanoactuator for Neuronal Optoporation.

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.

Nanoscale visualization of fast carrier dynamics in organic thin-film transistors by time-resolved electrostatic force microscopy

Fast carrier dynamics in organic thin-film transistors (OTFTs) was investigated by time-resolved electrostatic force microscopy (tr-EFM). We found that the carrier diffusion in the OTFTs proceeded in two stages: fast diffusion and slow diffusion. By applying the instantaneous frequency method to EFM, the temporal evolution of the spatial distribution of fast carriers in the channel region of the OTFTs, which took place on the timescale of several hundreds of nanoseconds, was evaluated. The inhomogeneous distribution of the local decay time constant showed that the carrier diffusion of the OTFTs was limited by the grain boundaries between each crystalline region. The quantitative capability of the method was verified by comparing the values of the carrier mobility estimated by the tr-EFM measurement and a numerical simulation. The mobility estimated from the experiment and the simulation showed good agreement, showing the possibility of the tr-EFM to evaluate the time evolution of dynamic phenomena in semiconductor devices.

High Dynamic Range Probing of Single-Molecule Mechanical Force Transitions at Cell-Matrix Adhesion Bonds by a Plasmonic Tension Nanosensor.

Mechanical signals in animal tissues are complex and rapidly changed, and how the force transduction emerges from the single-cell adhesion bonds remains unclear. DNA-based molecular tension sensors (MTS), albeit successful in cellular force probing, were restricted by their detection range and temporal resolution. Here, we introduced a plasmonic tension nanosensor (PTNS) to make straight progress toward these shortcomings. Contrary to the fluorescence-based MTS that only has specific force response thresholds, PTNS enabled the continuous and reversible force measurement from 1.1 to 48 pN with millisecond temporal resolution. We used the PTNS to visualize the high dynamic range single-molecule force transitions at cell-matrix adhesions during adhesion formation and migration. Time-resolved force traces revealed that the lifetime and duration of stepwise force transitions of molecular clutches are strongly modulated by the traction force through filamentous actin. The force probing technique is sensitive, fast, and robust and constitutes a potential tool for single-molecule and single-cell biophysics.

Effect of methanol and photoinduced surface oxygen vacancies on the charge carrier dynamics in TiO2

The migration of holes in metal-oxide semiconductors such as TiO2 plays a vital role in (photo)catalytic applications. The dynamics of charge carriers under operation conditions can be influenced by both methanol addition and photoinduced surface oxygen vacancies (PI-SOVs). Nevertheless, the existing knowledge of the effect of methanol as a function of PI-SOVs solely concentrates on the chemical reduction process. For this reason, the fundamental understanding of the time-dependent charge carrier-vacancy interactions in the presence of methanol is impaired. Here, we conducted time-resolved atomic force microscopy measurements to quantitatively disclose the effect of methanol adsorption on the dynamics of hole migration in TiO2. Our results show that time constants associated with the migration of charge carriers significantly change due to methanol adsorption. Moreover, the energy landscape of the hole migration barrier was dominated and lowered by PI-SOVs. Our findings contribute to the physics of charge carrier dynamics by enabling the engineering of charge carrier-vacancy interactions.

Time-resolved force microscopy using the delay-time modulation method

We developed a time-resolved force microscopy technique by integrating atomic force microscopy using a tuning-fork-type cantilever with the delay time modulation method for optical pump-probe light. We successfully measured the dynamics of surface recombination and diffusion of photoexcited carriers in bulk WSe2, which is challenging owing to the effect of the tunneling current in time-resolved scanning tunneling microscopy. The obtained results were comprehensively explained with the model based on the dipole-dipole interaction induced by photo illumination.

Time-resolved electrostatic force microscopy under base-bias-level control

We propose a base-bias-level control method, in which the contact potential difference is always compensated in a similar way to Kelvin probe force microscopy, applicable to time-resolved electrostatic force microscopy using the pump–probe method. We experimentally acquired temporal waveforms of the electrostatic force signal on two SiO2/n-type Si samples, one of which was as-grown and the other was treated by forming gas annealing. Consequently, the effectiveness of the proposed method was confirmed.

Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane

Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface composition, and molecular cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane physical properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-negative breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of ‘raft’-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is observed over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concentration. Our findings suggest the biophysical characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.

Experimental mitigation of large-amplitude transverse gusts via closed-loop pitch control

In this work, we experimentally demonstrate the utility of unsteady potential flow models in developing closed-loop control strategies for mitigating the lift transients on a wing experiencing large-amplitude transverse gusts. The developed closed-loop controller mitigates lift for gusts of various strengths and directions and for wings with pre- and post-stall angles of attack. Time-resolved force and flow field measurements are used to discover the salient flow physics during these encounters and illustrate how closed-loop actuation mitigates their lift transients.

Understanding the evolution of lithium dendrites at Li6.25Al0.25La3Zr2O12 grain boundaries via operando microscopy techniques

The growth of lithium dendrites in inorganic solid electrolytes is an essential drawback that hinders the development of reliable all-solid-state lithium metal batteries. Generally, ex situ post mortem measurements of battery components show the presence of lithium dendrites at the grain boundaries of the solid electrolyte. However, the role of grain boundaries in the nucleation and dendritic growth of metallic lithium is not yet fully understood. Here, to shed light on these crucial aspects, we report the use of operando Kelvin probe force microscopy measurements to map locally time-dependent electric potential changes in the Li6.25Al0.25La3Zr2O12 garnet-type solid electrolyte. We find that the Galvani potential drops at grain boundaries near the lithium metal electrode during plating as a response to the preferential accumulation of electrons. Time-resolved electrostatic force microscopy measurements and quantitative analyses of lithium metal formed at the grain boundaries under electron beam irradiation support this finding. Based on these results, we propose a mechanistic model to explain the preferential growth of lithium dendrites at grain boundaries and their penetration in inorganic solid electrolytes.

Aerodynamics and fluid–structure interaction of an airfoil with actively controlled flexible leeward surface

Piezoelectric macro-fibre composite (MFC) actuators are employed onto the flexible leeward surface of an airfoil for active control. Time-resolved aerodynamic forces, membrane deformations and flow fields are synchronously measured at low Reynolds number (Re = 6 × 104). Mean aerodynamics show that the actively controlled airfoil can achieve lift-enhancement and drag-reduction simultaneously in the angle of attack range of 10° ≤ α ≤ 14°, where the rigid airfoil encounters stall. The maximum increments of lift and lift-to-drag ratio are 27.1 % and 126 % at the reduced actuation frequency of ${f^ + } = 3.52$ . The unsteady coupling features are further analysed at α = 12°, where the maximum lift-enhancement occurs. It is newly discovered that the membrane vibrations and flow fields are locked into half of the actuation frequency when ${f^ + } > 3$ . The shift of the dominant vibration mode from bending to inclining is the reason for the novel ‘half-frequency lock-in’ phenomenon. To the fluid–structure interaction, there are three characteristic frequencies for the actively controlled airfoil: $S{t_1} = 0.5{f^ + }$ , $S{t_2} = {f^ + }$ , and $S{t_3} = 1.5{f^ + }$ . Here, St1 and its harmonics (St2, St3) are coupled with the natural frequencies of the leading-edge shear layer, resulting in the generation of multi-scale flow structures and suppression of flow separation. The lift presents comparable dominant frequencies between St1 and St3, which means the instantaneous lift is determined by the flow structures of St1 and St3. The local membrane bulge and dent affect the instantaneous swirl strength of flow structures near the maximum vibration amplitude location, which is the main reason for the variation of instantaneous lift.

A Robust Neural Network for Extracting Dynamics from Electrostatic Force Microscopy Data.

Advances in scanning probe microscopy (SPM) methods such as time-resolved electrostatic force microscopy (trEFM) now permit the mapping of fast local dynamic processes with high resolution in both space and time, but such methods can be time-consuming to analyze and calibrate. Here, we design and train a regression neural network (NN) that accelerates and simplifies the extraction of local dynamics from SPM data directly in a cantilever-independent manner, allowing the network to process data taken with different cantilevers. We validate the NN's ability to recover local dynamics with a fidelity equal to or surpassing conventional, more time-consuming, calibrations using both simulated and real microscopy data. We apply this method to extract accurate photoinduced carrier dynamics on n = 1 butylammonium lead iodide, a halide perovskite semiconductor film that is of interest for applications in both solar photovoltaics and quantum light sources. Finally, we use SHapley Additive exPlanations to evaluate the robustness of the trained model, confirm its cantilever-independence, and explore which parts of the trEFM signal are important to the network.

Recognizing the Binding Pattern and Dissociation Pathways of the p300 Taz2-p53 TAD2 Complex.

The dynamic association and dissociation between proteins are the basis of cellular signal transduction. This process becomes much more complicated if one or both interaction partners are intrinsically disordered because intrinsically disordered proteins can undergo disorder-to-order transitions upon binding to their partners. p53, a transcription factor with disordered regions, plays significant roles in many cellular signaling pathways. It is critical to understand the binding/unbinding mechanism involving these disordered regions of p53 at the residue level to reveal how p53 performs its biological functions. Here, we studied the dissociation process of the intrinsically disordered N-terminal transactivation domain 2 (TAD2) of p53 and the transcriptional adaptor zinc-binding 2 (Taz2) domain of transcriptional coactivator p300 using a combination of classical molecular dynamics, steered molecular dynamics, self-organizing maps, and time-resolved force distribution analysis (TRFDA). We observed two different dissociation pathways with different probabilities. One dissociation pathway starts from the TAD2 N-terminus and propagates to the α-helix and finally the C-terminus. The other dissociation pathway is in the opposite order. Subsequent TRFDA results reveal that key residues in TAD2 play critical roles. Besides the residues in agreement with previous experimental results, we also highlighted some other residues that play important roles in the disassociation process. In the dissociation process, non-native interactions were formed to partially compensate for the energy loss due to the breaking of surrounding native interactions. Moreover, our statistical analysis results of other experimentally determined complex structures involving either Taz2 or TAD2 suggest that the binding of the Taz2-TAD2 complex is mainly governed by the binding site of Taz2, which includes three main binding regions. Therefore, the complexes involving Taz2 may follow similar binding/unbinding behaviors, which could be studied together to generate common principles.

In Situ Observation of Dicalcium Phosphate Monohydrate Formation and Phase Transformation.

Calcium orthophosphates (CaPs), as important minerals in biomineralization and biomedicine, have attracted wide attention. Dicalcium phosphate monohydrate (DCPM, CaHPO4·H2O), the recently discovered crystalline CaP phase, has a higher metastability than dihydrate (DCPD, CaHPO4·2H2O) and anhydrate (DCPA, CaHPO4), which may lead to many potential applications in functional biomaterial development. However, the preparation of large-sized DCPM and the underlying mechanisms of its formation and phase evolution remain unclear. Herein, for the first time, we propose a method to prepare micrometer-sized DCPM under an acidic water-methanol mixture and using in situ time-resolved atomic force microscopy further explore its crystallization via dissolution of an acidic amorphous calcium phosphate. In support of the potential role of DCPM as the biomaterial, we demonstrate that DCPM can quickly evolve into more stable octacalcium phosphate in a near-physiological solution. This work provides a mechanistic understanding of the formation and phase transformation of DCPM, which may serve as a basis for subsequent synthesis and application of DCPM as functional biomaterials.

Regularities between kinematic and aerodynamic characteristics of flexible membrane wing

The kinematic characteristics of flexible membrane wing have vital influences on its aerodynamic characteristics. To deeply explore the regularities between them, time-resolved aerodynamic forces and deformations at different aeroelastic parameters and angles of attack (α) were measured synchronously by wind tunnel experiments. The membrane motion can be mainly divided into two states at α > 0° with various lift-enhancement regularities: Deformed-Steady State (DSS) at pre-stall, and Dynamic Balance State (DBS) at around stall and post-stall. Besides, the mean camber, maximum vibration amplitude, and lift coefficient almost reach their maxima simultaneously within the DBS region. By introducing momentum coefficient Cμ of membrane vibration, positive correlation among amplitude, momentum and lift is successfully established, and the lift-enhancement mechanism of membrane vibration is revealed. Moreover, it is newly and surprisingly found that at different vibration modes, the maximum vibration amplitude and root mean square of vibration velocity present positive and linear correlation with different slopes, and their chordwise locations are basically consistent. Therefore, novel ideas for active control of flexible wing are proposed: by controlling the vibration amplitude, frequency, and mode, while selecting the specific chordwise locations for intensive excitation, Cμ can be efficiently increased. Ultimately, the aerodynamic performance will be improved.

Challenges and opportunities in measuring time-resolved force chain evolution in 3D granular materials

Granular materials are found throughout nature and industry: in landslides, avalanches, and river beds, and also in pharmaceutics, food, and mineral processing. Many behaviors of these materials, including the ways in which they pack, deform, flow, and transmit energy, can be fully understood only in the context of inter-particle forces. However, we lack techniques for measuring 3D inter-particle force evolution at subsecond timescales due to technological limitations. Measurements of 3D force chain evolution at subsecond timescales would help validate and extend theories and models that explicitly or implicitly consider force chain dynamics in their predictions. Here, we discuss open challenges associated with force chain evolution on these timescales, challenges limiting such measurements, and possible routes for overcoming these challenges in the coming decade.

Mode of action of virulence factors of intracellular pathogens studies with time-resolved and high-resolution atomic force microscopy

An essential part of the innate immune defenses is the phagocytosis of invading pathogenic microbes by professional phagocytes and their subsequent digestion within the phagosome. It is often forgotten that not all pathogens surrender without a fight—in particular, some species succeed in surviving inside the phagosome. These intracellular pathogens have evolved various individual strategies to counteract the killing mechanisms of the host cell, many of which have been poorly understood.

Wavelet transform based method of measuring multi-frequency electrostatic force microscopy dynamic process

Electrostatic force microscopy (EFM) has high sensitivity and lateral resolution, and it is widely used to measure the electrostatic properties of new energy materials. The time-resolved electrostatic force microscope technology is used to measure the dynamic electrical properties of materials, pump detection method commonly used in this technology has problems such as complex equipment, high cost, and uncertainty in the measurement. In this work the method of directly measuring the time domain is adopted. This method reduces the complexity of measurement. By using the multi-frequency or high-frequency excitation method, the simultaneous measurement of multiple EFM parameters and the improvement of time resolution can be achieved, reaching a time resolution of microseconds, and by applying wavelet transform to the tip signal obtained by the measurement the dynamic electrical properties of the materials can be extracted. Applying this technology to simulation experiments, it is possible to measure the dynamic potential changes and the characteristic time parameter of ion movement in the microsecond-level electrical dynamic process of the simulated battery materials.

Force measurements near a natural frequency of a measurement system using inverse filters

Accurate time-resolved force measurements for complex experimental systems are important for minimizing erroneous and misleading data. These measurements become difficult when a natural frequency of the system is in or near the expected frequency domain of the time-varying force being applied. In the cases where it is not possible to avoid this occurrence, the experimenter typically abandons the setup. This work presents an inverse filter method to compensate for the dynamic response of the measurement system. A two degree-of-freedom measurement system is used to obtain force measurements with dominant forcing frequencies above and below the first natural frequency of the system. The results show that inverse filtering can be used along with digital low pass filters to correct amplification and phase shift due to the dynamic response of the measurement system to within ±4.0 of total forcing amplitude and ±5.0∘. A simple cam follower mechanism is proposed as a method of low-frequency dynamic testing.