The aim of this study was to evaluate StepAn as an automated approach for quantitative gait analysis in rodents. We sought to determine whether this video-based approach, which uses standard recordings from devices like smartphones, could sensitively detect motor deficits in a pharmacological model of Parkinsonian bradykinesia. Wistar rats received a single intraperitoneal injection of saline (control), 0.15 mg/kg haloperidol, or 0.30 mg/kg haloperidol. One hour post-injection, gait was analyzed using both the traditional manual “Footprints” test and the StepAn-based automated video analysis, which performs paw detection and stride length calculation from video recordings. Both analysis methods detected a significant reduction in average stride length in haloperidol-treated rats compared to controls (p<0.001), confirming the expected bradykinetic phenotype. No significant difference was found between the two haloperidol doses. Automated analysis provided equivalent mean values but demonstrated superior precision, evidenced by significantly lower measurement variability compared to manual scoring. We evaluated StepAn as a precise and hardware-flexible tool for quantitative gait assessment. Its ability to detect drug-induced Parkinsonian gait deficits comparable to established methods supports its use for objective locomotor analysis in preclinical research.
KRAS is a small GTPase essential for cell signaling, and the G12C mutation acts as a key oncogenic driver in multiple cancers. First-generation KRAS G12C inhibitors, such as sotorasib and adagrasib, have shown clinical efficacy, but are limited by acquired resistance due to secondary mutations. In this study, we investigated the impact of secondary mutations (Y96D, Y96S, G13D, and Q99L) on the binding efficacy of sotorasib, adagrasib, and the next-generation inhibitor (MK-1084) using molecular dynamics simulations, binding free energy calculations, and dynamic protein-ligand interaction analysis. Our study revealed that each secondary mutant variant exhibited variations in the degree of resistance to the inhibitors. Two major resistance patterns were identified: direct and indirect. Our analyses revealed that Y96 mutations directly disrupt inhibitor binding, conferring high resistance to all three inhibitors, whereas G13D and Q99L indirectly alter the binding environment by influencing other residues, resulting in variable resistance profiles. This study provides detailed molecular insights into resistance mechanisms to support the rational design of more robust KRAS G12C-targeted therapies.
Bioluminescence is an oxidation-mediated chemical reaction involving the luciferase enzyme and the luciferin substrate. To date, various artificial luciferases have been developed, and their luminescence properties have been investigated. However, very few studies have focused on the solvent environment in which these luminescent reactions occur, and the details of their influence on luminescent phenomena remain unclear. In this study, we investigated the effects of various surfactants on the activity of the luciferase picALuc. To this end, we analyzed the effects of adding diverse surfactants on the luminescence properties of the luciferase picALuc. The results showed that the addition of a surfactant enhanced the luminescence intensity and extended the duration of luminescence. Dynamic light scattering measurements and microscopic observations revealed that micelle-like particles were formed by the surfactant, which suppressed luminescence inhibition. This study provides our original method for adding surfactants to enzymatic reactions, such as those involving hydrophobic substrates.
Hyperthermal sarcomeric oscillations (HSOs) expose rapid sarcomere-level motion in living cardiomyocytes and provide a mesoscopic window between actomyosin activity and robust cellular contraction. I reanalyzed high-speed sarcomere-length recordings from five consecutive sarcomeres in each of seven neonatal rat cardiomyocytes. During HSOs, local phase relations became trackable through most of the oscillatory segment (valid fraction, 0.298 before warming and 0.956 during HSOs; paired Wilcoxon P=0.0156). Neighboring-sarcomere reconfiguration was dominated by one-link switches, in which one adjacent-pair relation changed while the other three were maintained (216/230 HSO phase transitions), and anti-phase-rich occupancy increased from 0.254 to 0.509 (P=0.0156). I then measured event-local relative internal length redistribution. For each reach-qualified one-link event, compensation reach, S, was defined as the expected sarcomere-index distance between relative shortening and relative lengthening. The same directed IAAI-to-IAII switch was accompanied by short-reach redistribution in one event (S=1.29) and cross-chain redistribution in another (S=2.88). Across 248 reach-qualified events, S increased with the pre-event number of I-type links, with a cell-fixed slope of 0.148 span units per added I-link supported by cell-blocked permutation and cell-cluster bootstrap analyses. Thus, HSO reveals a mesoscale organizing process in which a local switch in neighboring-sarcomere synchrony is linked to spatially distributed relative internal length redistribution whose reach is shaped by the pre-event phase context.
Chromatin in the eukaryotic nucleus is organized into transcriptionally active euchromatin and transcriptionally silent heterochromatin. Its fundamental unit, the nucleosome, consists of two copies each of histones H2A, H2B, H3, and H4 wrapped by ~145 base pairs of DNA, flanked by linker-DNA that binds linker-histone H1 to form a chromatosome. In euchromatin, histone N-terminal tails (N-tails) are extensively acetylated. In facultative heterochromatin, H3K27 is methylated (H3K27me) and the H2A C-terminal tail (C-tail) is ubiquitinated (H2Aub), whereas in constitutive heterochromatin, H3K9 is methylated (H3K9me) and recognized by heterochromatin protein 1 (HP1). The N-tail of the HP1α homologue is phosphorylated, enhancing its binding to H3K9me and promoting liquid-liquid phase separation (LLPS). In addition, the C-tails of the histone chaperones FACT and NAP1 mediate binding to H2A-H2B. These N-tails and C-tails are intrinsically disordered regions (IDRs), whose dynamic conformations are accessible primarily through NMR spectroscopy. NMR has revealed dynamic IDR interaction networks essential for chromatin regulation, including H3 N-tail acetylation enhanced by H4 N-tail acetylation on linker-DNA, its suppression by H1, H3K27 methylation promoted by H2Aub, H3 N-tail acetylation induced by phosphorylated FACT C-tail, the role of the NAP1 C-tail in H2A-H2B binding, and phosphorylation-dependent enhancement of HP1 N-tail binding to H3K9me and LLPS. Thus, NMR has illuminated how dynamic interactions among nuclear IDRs play central roles in establishing euchromatin and heterochromatin, providing insights into the molecular basis of nuclear function.
Developing active transport systems for micro cargo delivery is challenging because it requires overcoming the constraints imposed by low Reynolds numbers. We developed a bio-hybrid micro-swimmer, “Chlamylipo” consisting of the green alga Chlamydomonas reinhardtii, encapsulated within a giant liposome. Although internal encapsulation offers cargo protection, it necessitates a mechanism to transmit the propulsion force across a closed membrane. We demonstrated that Chlamylipo exhibited forward swimming and phototactic directional control. High-speed imaging of the membrane shape and fluid flow revealed that the driving force originated from periodic membrane deformations and was accompanied by characteristic fluid dynamics. Flow analysis showed rapid oscillations at tens of hertz corresponding to flagellar beating, superimposed on slower axial migration at approximately 4 Hz, associated with cell rotation. Corresponding flow signatures were also detected in the external fluid, indicating mechanical coupling of the lipid bilayer. Membrane domain tracking further revealed that the fluid motions inside and outside the membrane were coupled through viscous friction and membrane deformation, generating a characteristic four-vortex flow field consistent with a two-point force model. Collectively, these results suggest that membrane flow primarily reflects force transmission across the bilayer, whereas forward propulsion is primarily driven by periodic membrane deformation. This study elucidates the physical mechanism of force transmission in encapsulated swimmers, demonstrating that internal hydrodynamic power can effectively drive the motion of microscopic containers.
Cellular nuclei contain various nucleases that play major roles in gene expression and regulation. They are classified into various categories based on their three-dimensional structures and specificities. Since the discovery of ribozyme in the early 1980, one nuclease class has attracted significant attention. The cleavage reactions of these nucleases essentially require Mg2+ cations and produce a 5'-phosphate nucleotide and a 3'-nucleoside at the cleavage site of polynucleotides, similarly to the reaction of ribozyme. In particular, Drs. Thomas A. Steitz and Joan A. Steitz proposed an attractive and general model for the phosphoryl transfer reaction. This was later named the “two-metal-binding mechanism”. In accordance with recent developments in cryo-electron microscopy (cryo-EM), many structural reports published about Mg2+-dependent nucleases or spliceosomes support the “two-metal-binding mechanism” for hydrolytic schemes. However, the direct visualization of Mg2+ on electron-density maps requires high resolution analyses, regardless of X-ray crystallography or cryo-EM. Furthermore, recent biochemical studies combined with structural data support the similarity in hydrolytic mechanisms between ribonuclease HI (RNase HI) and restriction endonucleases. Since the late 2000s, molecular dynamics approaches have also appeared to support the hydrolytic mechanism of single mobile metals. This review aims to critically reconsider Mg2+-dependent hydrolytic mechanisms, with a particular focus on bacterial RNase HI.
Cardiac fibroblasts play a pivotal role in heart tissue dynamics, responding to mechanical and interfacial cues in their microenvironment. This study examines how substrate stiffness and surface condition influence the motility and morphology of chicken embryonic cardiac fibroblasts cultured on collagen-coated polydimethylsiloxane (PDMS) substrates with approximate elastic moduli of ~10 kPa and ~400 kPa, representing mechanically soft and stiff conditions, respectively. The PDMS surfaces were used without plasma or UV/ozone treatment, retaining their native hydrophobic character without surface oxidation. This absence of surface activation resulted in weak cell-substrate adhesion, leading to delayed spreading and frequent partial detachment during migration. Using time-lapse phase-contrast microscopy, we observed irregular motility cycles characterized by contraction-driven retraction, followed by partial detachment and re-adhesion. On soft substrates, fibroblasts exhibited longer motility cycles and left trailing edges upon retraction, while on stiff substrates, cells remained rounded and displayed less polarized motion. Quantitative analysis revealed that the duration of static phases and the degree of cell elongation varied with substrate stiffness but were also strongly influenced by adhesion instability due to unoxidized PDMS surfaces. These findings clarify the roles of substrate stiffness and surface adhesion in regulating fibroblast motility, and demonstrate that non-oxidized PDMS surfaces—though less adhesive—can preserve a rounded, less-spread cell morphology. Rather than promoting full spreading, such conditions may maintain fibroblasts in a mechanically less engaged or undifferentiated state. This work provides insight into how surface condition and mechanics together shape fibroblast behavior, with implications for mechanobiology and in vitro model design using non-activated biomaterials.
Spiroplasma swim by switching the handedness of their helical bodies between right- and left-handed. Helicity formation and switching can be reconstituted in an immotile minimal synthetic bacterium, JCVI-syn3B by introducing a pair of bacterial actins, MreB4 and MreB5 from Spiroplasma eriocheiris. However, the mechanism is unknown. Here, to elucidate this mechanism, we analyzed MreB behaviors optically. We tried MreB4 fluorescence labeling by protein fusion. Labeling was unsuccessful because the fusion of fluorescent proteins or peptides at 16 different positions resulted in immotile cells. These results may suggest that MreB4 has many interfaces interacting with other proteins. To obtain suggestions for roles of MreB4 and MreB5, we tried induction of individual MreBs. Induced expression of MreB4 in cells with constitutive MreB5 expression resulted in earlier onset and higher frequency of motile cells, distinct from the results of constitutive MreB4 and inducible MreB5. Next, the behavior of labeled MreB5 was analyzed by photobleaching and photoactivation, suggesting static behavior of MreB5 during cell movements. Cell treatment with A22, an MreB polymerization inhibitor caused helix deformation, movement stall, and diffusion of MreB5 fluorescence, suggesting that A22 sensitive MreB5 interaction should be involved in helix formation and motility. These results suggest that the movement is caused by conformational change of MreB5 filament induced by MreB4 without obvious replacements of MreB5 subunits.
Single-molecule imaging in live cells now plays key roles in elucidating molecular dynamics and interactions. However, the imaging time resolution has remained limited, despite its critical importance for precisely capturing molecular events in cells, in contrast with major spatial resolution advances in fluorescence microscopy. To address this issue, we developed an ultrafast camera system that achieves the highest time resolutions reported to date for single fluorescent-molecule imaging and tracking (SFM-IT), reaching fluorophore photophysics-limited (photon-limited) frame times of 33 and 100 μs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore identified. Using this system, we directly detected the hop diffusion of membrane molecules in the plasma membrane, confirming its compartmentalization. Thus, ultrafast SFM-IT has helped to elucidate the principles governing the plasma membrane organization and molecular dynamics. Building on this platform, we further established ultrafast, live-cell, two-color single-molecule localization microscopy (SMLM). This method reduced the data acquisition time by ≈30-fold relative to standard methods, while simultaneously enabling much larger view-fields, with localization precisions of 29 and 19 nm for PALM and dSTORM, respectively. Combining ultrafast SMLM with ultrafast SFM-IT revealed the mesoscopic dynamical organization of focal adhesions (FAs), termed an archipelago architecture, showing that FAs consist of ≈30-nm-diameter FA-protein islands loosely clustered into ≈300-nm-diameter functional units embedded in the compartmentalized fluid membrane (74 nm inside vs. 110 nm outside the FA). Ultrafast SFM-IT and ultrafast SMLM techniques open previously inaccessible spatiotemporal regimes for biophysical cell biology research.
The cytoskeleton, comprising intracellular filamentous structures composed of polymerized proteins, is crucial for the survival of both eukaryotes and prokaryotes. Although bacterial cytoskeletal proteins have diverged, they generally do not drive cellular motility. Spiroplasma, a genus of wall-less helical bacteria, swims by propagating a helicity-switching point (kink) along its cell axis. Unlike typical walled bacteria, whose motility depends on widespread motility machineries such as flagella and pili, Spiroplasma swimming is powered by the coordinated dynamics of five isoforms of bacterial actin MreB (SMreB1-5), which are grouped into three phylogenetic classes: SMreB1 and 4, SMreB2 and 5, and SMreB3. Despite the efforts to understand Spiroplasma swimming, its molecular mechanism remains unclear. In this review, we summarize how in vitro analyses of SMreBs have provided mechanistic insights into Spiroplasma swimming. While all SMreBs conserve the canonical actin fold, each SMreB class exhibits unique characteristics in its polymerized structures, ATPase activities, polymerization dynamics, and membrane binding. Studies of an essential SMreB subset for Spiroplasma swimming, i.e. SMreB1 and SMreB5, have revealed that SMreB1 binds to polymerized SMreB5 and disassembles it depending on the nucleotide state. These results challenge the previous model in which Spiroplasma swimming is driven by the coordinated extension and contraction of two distinct SMreB filaments. Finally, we discuss potential molecular mechanisms underlying Spiroplasma swimming and highlight key questions that must be answered to validate these models.
Proteins can aggregate to form amyloid fibrils, which are associated with a group of diseases collectively known as amyloidoses. Molecular dynamics simulations are widely used to study protein aggregation; however, it remains computationally challenging to investigate the entire aggregation process, from initial nucleation to the formation of mature amyloid fibrils, within a single simulation. In this study, we propose a Monte Carlo simulation based on a mathematical lattice model in which proteins are represented as spheres on a lattice. Motivated by the role of intramolecular β-sheet structures in accelerating aggregation, we classify protein states into three categories: monomer, intermediate possessing an intramolecular β-sheet structure, and fibril. By tuning the monomer-to-intermediate transition probability PMI and the intermediate-to-fibril transition probability PIF, both kinetics and morphology of amyloid fibril formation can be systematically controlled. When PMI is low, a lag time appears in the early stage of aggregation, whereas increasing PMI shortens the lag time. High values of both PMI and PIF lead to rapid aggregation and the formation of many short fibrils, while low values result in slower aggregation and fewer but longer fibrils. These results are consistent with experimental observations and indicate that amyloid fibril formation can be understood as a crystal growth process. This approach is expected to provide further insight into the universal mechanisms of amyloid fibril formation in homogeneous aqueous environments, as well as in heterogeneous and nonequilibrium systems.
The conformational change of a protein molecule is described by the single equilibrium constant K and the single rate constants, k (forward) and k’ (reverse). The underlying assumption is that all amino acid residues undergo state changes with perfect cooperativity. However, residue-specific measurements such as NMR often show residue-to-residue variations in these constants, indicating that proteins are not fully cooperative. Theoretical considerations based on “the consistency principle of protein folding” demonstrated that the linear free energy relationship (LFER) observed in residue-specific log k vs. log K plots is the physicochemical basis for smooth conformational changes of protein molecules. The residue-based LFER was found in many protein-related phenomena, but the structural changes have been limited to relatively small ones, such as fluctuations in the folded state and the coupled binding and folding of intrinsically disordered proteins. Here, we applied NMR to the two-state exchange equilibrium of the spectrin SH3 domain in acidic solutions and determined residue-specific equilibrium and rate constants. The data points obtained from the wild-type and its two single amino acid mutants were aligned on a single line in the log k vs. log K plot, indicating that residue-based LFER applies to large structural changes between the unfolded and folded states of the SH3 domain. The mutation-induced shifts in the distribution ranges of residue-specific equilibrium and rate constants are useful for establishing residue-based LFERs for all kinds of protein structural changes, including the refolding from a fully unfolded state, caused by the removal of a denaturant.
The structural flexibility of enzymes plays an essential role in determining their catalytic efficiency and thermal stability. Cold-adapted enzymes are typically highly flexible, resulting in high catalytic activity but low stability. Glucokinase (GK) consists of the large substrates binding domain, small catalytic domain, and hinge region that undergoes conformational changes upon substrates binding. We recently reported that the psychrophilic GK from Pseudoalteromonas sp. AS-131 (PsGK) exhibits both high catalytic efficiency and remarkable thermal stability compared to the mesophilic GK from Escherichia coli (EcGK). We also found that a disulfide bond connecting the N- and C-termini in PsGK contributes to its unusual thermal stability. However, cold adaptation mechanism of cold-adapted PsGK has remained unclear. To clarify how PsGK acquires high activity, we utilized site-directed spin labeling electron spin resonance (SDSL-ESR) spectroscopy for PsGK and EcGK in the absence and presence of substrates in the wide range of temperatures. PsGK without substrates was more flexible than EcGK. Particularly, the small domain and hinge region of PsGK were highly flexible while its large domain was relatively rigid. In contrast, EcGK showed lower entire flexibility and did not exhibit domain dependent differences. When the substrates were bound, both enzymes became more rigid, but the small domain and hinge region of PsGK was still flexible whereas its large domain was considerably rigid. These results suggest that enhancing catalytic activity requires increasing flexibility only in proper sites rather than in the entire enzyme. These findings provide insight into how cold-adapted enzymes balance activity and stability.
Dielectrophoresis (DEP) is a promising label-free technique for bioparticle manipulation, offering significant potential for diagnostic application such as isolation and trapping of cancerous CCRF-CEM cells. The efficacy of DEP trapping is critically dependent on the interaction between the cell’s dielectric properties and the spatial gradient of the squared electric field (∇|E2|), which is governed by electrode geometry. This study conducts a comparative evaluation, via finite element method (FEM) analysis, to quantify the positive dielectrophoresis (pDEP) force exerted on CCRF-CEM cells. Four distinct electrode configurations were analyzed: parallel (rectangular, triangular, cylindrical) and interdigitated. Based on a single-shell model for CCRF-CEM cells within a low-conductivity buffer, the Clausius-Mossotti factor was determined at 4.6 MHz, confirming a strong pDEP response that attracts cells to high-field regions. Results demonstrate that the pDEP force scales quadratically with applied voltage and is significantly enhanced by reducing the electrode gap. At a standardized 25 Vpp and 100 μm gap, the interdigitated configuration generated the highest maximum pDEP force, substantially exceeding the parallel rectangular, triangular, and cylindrical designs. Furthermore, the interdigitated geometry produced the most extensive and uniform high-force zones along the electrode edges, creating a superior trapping area. This comparative evaluation provides quantitative guidelines for optimizing electrode design, identifying the interdigitated configuration as the most effective for developing high-efficiency microdevices for CCRF-CEM cell trapping.
Intrinsically disordered regions (IDRs) are vital for several cellular processes. They play a significant role in liquid-liquid phase separation (LLPS). LLPS enhances reaction efficiency by locally concentrating proteins and nucleic acids. The BACH2 transcription factor contains such IDRs. Early observations hinted at the phase separation capabilities of BACH2. However, how BACH2-IDR directly participates in LLPS is not fully understood. Its precise modulation by heme binding and phosphorylation also requires further elucidation. This study experimentally demonstrated the intrinsic capability of BACH2-IDR (residues 331-520) to undergo LLPS in vitro. Microscopic observations revealed that TANK-binding kinase 1(TBK1)-mediated phosphorylation suppressed LLPS of BACH2-IDR. Conversely, the coexistence of heme enhanced LLPS, initiating LLPS at lower polyethylene glycol concentrations. Bioinformatic analyses supported these experimental observations. Tools such as FuzDrop and CIDER were used to predict LLPS propensity from sequences. Specifically, the calculations for the sequence of phosphorylation-mimic mutations showed changes in LLPS-promoting regions. Heme binding influenced TBK1-mediated phosphorylation sites. Some of these sites overlap with predicted LLPS-driving regions. Heme binding also induced substantial conformational alterations within BACH2-IDR. Consequently, we propose LLPS as a fundamental regulatory mechanism for BACH2 protein function. This process complements its known regulation through heme binding and phosphorylation.
The synthesis of mirror life, an organism composed entirely of the mirror-image counterparts of naturally occurring biomolecules, may be a topic for science fiction today but is suggested to be a reality in the future. Scientists called for strict preemptive regulation for the synthesis of mirror life due to concerns regarding biosecurity risks, such as the potential threat of mirror life to humans and non-human ecosystems. This perspective reports on the discussions held at two recent symposia in Japan, at the Japanese Society for Cell Synthesis Research (JSCSR) meeting and at the Biophysical Society of Japan (BSJ) annual meeting. Participants, including synthetic biologists, social scientists and artists, discussed the feasibility of the synthesis of mirror-image artificial self-replicating cells and the balance between the risks and benefits of this emerging technology. The discussions indicate that, while safety management is essential, a rational, evidence-based framework developed through open dialogue with society is crucial for the responsible advancement of mirror life research.
In morphogenesis, mechanical forces are the fundamental drivers of tissue deformation. To elucidate the principles governing this process, a thorough understanding of these forces is indispensable, and mathematical models are invaluable tools for this purpose. Various models have been developed, including vertex models suitable for representing epithelial cell mechanics, cell particle models for simulating the dynamics of large cell populations, and deformable cell models for describing complicated cell shapes. In these models, the motion of the objects is usually assumed to be overdamped by viscous friction. The viscous frictional forces are likely provided by liquid medium or body fluid, and they are also exerted in cell-cell and cell-extracellular matrix contacts. Furthermore, if these components, including the ECM, are in motion, the viscous frictional forces are affected by their motions. Because there seems to be little consensus on how to model the viscous frictional forces, we organize and review them in this article where we exemplified the vertex model and the particle model.
Data science methodologies can be applied to “molecular archeology.” By statistically inferring ancestral gene or protein sequences from a molecular phylogeny, researchers can recreate ancient molecules for laboratory experiments, allowing direct examination of their properties and structures. This review summarizes studies that applied this approach to investigate how whales and seals readapted to deep-sea diving through the evolutionary modification of myoglobins.
