During metaphase of mitosis, the oscillation and pulsation of sister kinetochores are present in many spindle systems. In the spindle of higher eukaryotes the microtubule poleward flux is present. To understand the physical mechanism of the kinetochore oscillation and pulsation in the spindle with the presence of the microtubule poleward flux, here we present a minimal model, where only two spindle poles, two kinetochores and two microtubules are required, with one microtubule connecting one spindle pole and one kinetochore while the other microtubule connecting the other spindle pole and the other kinetochore. The depolymerase activity by kinesin-13 motors at the microtubule minus end and the net polymerization at the microtubule plus end are considered, generating the microtubule poleward flux. With the model, we show that the stochastic depolymerase activities at the minus end can result in the large-scale kinetochore oscillation and pulsation, with the numerical results being consistent with the available experimental results.

Accurate chromosome segregation requires efficient corrections of erroneous kinetochore-microtubule attachments during metaphase. However, the detailed mechanisms of how the erroneous attachments can be corrected in the metaphase spindle with the presence of microtubule poleward flux are unclear. To explore the mechanisms and understand the roles the flux plays in the error correction, here we study numerically the correction of various erroneous (merotelic, syntelic, and monotelic) attachments in the metaphase spindle exhibiting the flux. We show that with the effect of kinase Aurora B activity, the erroneous attachments can be corrected efficiently. In contrast, without the effect of Aurora B activity the erroneous attachments cannot be corrected efficiently. More interestingly, we find that an optimum rate of the microtubule poleward flux or an optimum amplitude of the kinetochore oscillation is present, which can result in both the efficient error correction and high mitotic fidelity.

Abstract Myosin-V is homodimeric motor protein that can step processively on actin filaments toward the barbed end, performing the biological function of the intracellular cargo transport. Here, a model is presented for the chemomechanical coupling of myosin-V homodimer, which is consistent with the available high-speed atomic force microscopic data showing that at high ADP concentrations nearly all neck domains of the leading head are in the straight conformation and the proportion of the leading neck domains in the sharply bent conformation increases with the decrease of ADP concentration. With the model, the dynamics of the myosin-V homodimer versus load on a large-sized bead attached to its coiled-coil stalk and that versus load on the bead attached to one of its two heads is studied theoretically, reproducing quantitatively the available single-molecule optical trapping data. Predicted results are also provided.

Ncd is the founding member of the kinesin-14 family, which can move on microtubules toward the minus end in a nonprocessive manner by hydrolyzing ATP molecules. It was experimentally observed that while multiple anchored Ncd motors can drive the gliding of a microtubule with high efficiency, the full-length Ncd motors can drive the sliding of one microtubule relative to the antiparallel one with a much lower efficiency. However, a quantitative explanation of these experimental results is not available. The mechanism of how the microtubule sliding by the full-length Ncd motors has much lower efficiency than the microtubule gliding by the anchored Ncd motors is unclear. Here, we use both theoretical analysis and numerical simulation to study microtubule gliding and sliding by the Ncd motors, explaining quantitatively the available experimental data. The studies show that the competition between the motor's stalk rotation and its tail diffusion results in the much lower efficiency of the microtubule sliding than that of the microtubule gliding. This mechanism of the lower efficiency of microtubule sliding by the Ncd motors is different from the previously proposed mechanism of the nonefficient microtubule gliding by kinesin-1 motors anchored to the slippery surface.

Abstract How the metaphase spindle in higher eukaryotes maintains stability in the presence of poleward microtubule (MT) flux is a confusing issue. Here, we present a model for the spindle by incorporating augmin-mediated MT connections and MT crosslinking by NuMA proteins. On the basis of the model, we study computationally the dynamics of the spindle for the wild-type case and for the case with depletion, inhibition, or overexpression of various associated proteins such as kinesin-8 KIF18A, CLASP, NuMA, kinesin-4 KIF4A, augmin complex, kinesin-13 MCAK, kinesin-13 KIF2A, and kinesin-5, as well as with addition of the MT-targeting agent BAL27862. The numerical results are consistent with the available experimental data. We explain the mechanism of the stability of the spindle, namely, the mechanism of how each MT maintains a constant length, how antiparallel MT overlaps maintain their constant lengths, how each MT maintains on average a fixed position relative to another one despite having different flux rates, and how the spindle length is kept constant. The mechanisms of different associated proteins regulating differentially the MT flux rate, antiparallel MT overlap length, spindle length, and interkinetochore distance are explained. Moreover, our model explains well various other puzzling experimental results such as those showing that the normal MT flux was still present despite MT minus ends being detached from the spindle poles and the MT depolymerization at spindle poles being inhibited.

Kinesin-1 protein is a biological molecular motor that can step processively along microtubules toward the plus end (the forward direction) and can also step backward occasionally under small backward loads. Puzzlingly, recent high-temporal-resolution optical trapping data showed that under large backward loads, besides the conventional slow backward steps and detachment with dwell times (> 3 ms) dependent sensitively on the load and ATP concentration, the fast backward steps and detachment with dwell times (sub-milliseconds) independent on the load and ATP concentration are also present. While the slow backward steps and detachment can be understood easily, the origin of the fast backward steps and detachment is elusive. The explanation of the origin is critical to the mechanochemical coupling mechanism of the motor. Here, based on our proposed stepping pathway, we study numerically the dwell times and fractions of the forward steps, slow backward steps, slow detachment, fast backward steps and fast detachment under the backward loads in a wide range. The numerical results explain well the optical trapping data. The physical origin of the fast backward steps and detachment is explained.

Necrosis, long considered an uncontrolled and passive process, is now known to involve active cellular regulation. While significant research has focused on biochemical pathways of necrosis, the physical changes within the nucleus, particularly chromatin dynamics, remain unknown. By combining the single-particle tracking of telomeres and particle image velocimetry of global chromatin, we characterize the spatiotemporal evolution of chromatin dynamics during necrosis. We reveal a distinct biphasic pattern of chromatin motion with an initial deceleration followed by a late acceleration, accompanied by a transient increase and a subsequent decrease in intranuclear spatial heterogeneity. Through systematic perturbation, we establish a stage-specific regulatory model: the early deceleration of chromatin is driven by mechanical restraint from the cytoskeletal network, while the late acceleration results from the combined effects of nuclear swelling and DNA fragmentation. Our findings highlight necrosis as a programmed process, uncovering a previously unrecognized layer of cytoskeleton-mediated mechanical regulation in cell death.

During metaphase, the spindle stabilizes chromosomes and maintains its size despite continuous microtubule poleward flux. To investigate the mechanism of the spindle stability and how the poleward flux regulates the spindle size, we establish a minimal spindle model that incorporates kinetochores, microtubules, spindle poles, and microtubule sliding proteins such as kinesin-5, microtubule depolymerizing proteins such as kinesin-13, and microtubule crosslinking proteins such as NuMA. We find that the poleward flux stabilizes the spindle by regulating the spindle length and the length of antiparallel microtubule overlaps to achieve equal rates of microtubule sliding, plus-end polymerization, and minus-end depolymerization. We reveal the underlying mechanism of how the poleward flux rate scales linearly with the spindle length and microtubule overlap length in small cells and how microtubule nucleation affects spindle dynamics in large cells.

Cell death is a fundamental biological process with different modes including apoptosis and necrosis. In contrast to programmed apoptosis, necrosis was previously considered disordered and passive, but it is now being realized to be under regulation by certain biological pathways. However, the intracellular dynamics that coordinates with cellular structure changes during necrosis remains unknown, limiting our understanding of the principles of necrosis. Here, we characterized the spatiotemporal intracellular diffusion dynamics in cells undergoing necrosis, using three-dimensional single-particle tracking of quantum dots. We found temporally increased diffusion rates in necrotic cells and spatially enhanced diffusion heterogeneity in the cell periphery, which could be attributed to the reduced molecular crowding resulting from cell swelling and peripheral blebbing, respectively. Moreover, the three-dimensional intracellular diffusion transits from strong anisotropy to nearly isotropy, suggesting a remodeling of the cytoarchitecture that relieves the axial constraint on intracellular diffusion during necrosis. Our results reveal the remarkable alterations of intracellular diffusion dynamics and biophysical properties in necrosis, providing insight into the well-organized nonequilibrium necrotic cell death from a biophysical perspective.

In eukaryotic cell division, a series of events are organized to produce two daughter cells. The spindle elongation in anaphase B is essential for providing enough space to maintain cell size and distribute sister chromatids properly, which is associated with microtubules and microtubule-associated proteins such as kinesin-5 Eg5 and the Ase1-related protein, PRC1. The available experimental data indicated that after the start of anaphase B more PRC1 proteins can bind to the antiparallel microtubule pairs in the spindle but the excess amount of PRC1 proteins can lead to the failure of cell division, indicating that PRC1 proteins can regulate the spindle elongation in a concentration-dependent manner. However, the underlying mechanism of the PRC1 proteins regulating the spindle elongation has not been explained up to now. Here, we use a simplified model, where only the two important participants (kinesin-5 Eg5 motors and PRC1 proteins) are considered, to study the spindle elongation during anaphase B. We first show that only in the appropriate range of the PRC1 concentration can the spindle elongation complete properly. Furthermore, we explore the underlying mechanism of PRC1 as a regulator for spindle elongation.

The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription’s (FACT’s) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT’s function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.

Histone H2B mono-ubiquitination at lysine 120 (ubH2B) has been found to regulate transcriptional elongation by collaborating with the histone chaperone FACT (Facilitates Chromatin Transcription) and plays essential roles in chromatin-based transcriptional processes. However, the mechanism of how ubH2B directly collaborates with FACT at the nucleosome level still remains elusive. In this study, we demonstrate that ubH2B impairs the mechanical stability of the nucleosome and helps to recruit FACT by enhancing the binding of FACT on the nucleosome. FACT prefers to bind and deposit H2A-ubH2B dimers to form an intact nucleosome. Strikingly, the preferable binding of FACT on ubH2B-nucleosome greatly enhances nucleosome stability and maintains its integrity. The stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as revealed by genome-wide and time-course ChIP-qPCR analyses. Our findings provide mechanistic insights of how ubH2B directly collaborates with FACT to regulate nucleosome dynamics for gene transcription.

Cell migration plays important roles in many biological processes, but how migrating cells orchestrate intracellular molecules and subcellular structures to regulate their speed and direction is still not clear. Here, by characterizing the intracellular diffusion and the three-dimensional lamellipodium structures of fish keratocyte cells, we observe a strong positive correlation between the intracellular diffusion and cell migration speed and, more importantly, discover a switching of cell migration modes with reversible intracellular diffusion variation and lamellipodium structure deformation. Distinct from the normal fast mode, cells migrating in the newly-found slow mode have a deformed lamellipodium with swollen-up front and thinned-down rear, reduced intracellular diffusion and compartmentalized macromolecule distribution in the lamellipodium. Furthermore, in turning cells, both lamellipodium structure and intracellular diffusion dynamics are also changed, with left-right symmetry breaking. We propose a mechanism involving the front-localized actin polymerization and increased molecular crowding in the lamellipodium to explain how cells spatiotemporally coordinate the intracellular diffusion dynamics and the lamellipodium structure in regulating their migrations.

G-quadruplex (G4) is one of the higher-order DNA structures in guanine-rich sequences which are widely distributed across the genome. Due to their presence in oncogenic promoters and telomeres, G4 DNA structures become the novel targets in anticancer drug designs. Curaxin CBL0137, as an important candidate anticancer drug, can effectively inhibit the growth of multiple cancers. Although there is evidence that anticancer activity of curaxin is associated with its ability to bind DNA and to change the DNA topology, its therapeutic target and the underlying anti-cancer mechanism are still unclear. Here we show, for the first time, that curaxin CBL0137 induces G4 folding from anti-parallel to parallel structures, by single-molecule fluorescence resonance energy transfer technique. More importantly, we find that curaxin CBL0137 promotes G4 folding as well as stabilizes the folded G4 structures with long loops, giving a novel insight into effects of curaxin CBL0137 on DNA structures. Our work provides new ideas for the therapeutic mechanism of curaxin CBL0137 and for designs of new G4-targeting anticancer drugs.

Cell morphology and migration depend critically on the adhesions on the extracellular matrix (ECM), determined by the transmembrane protein integrins. The epithelial to mesenchymal transition (EMT) is a prominent transformation process in which adherent cells acquire a mesenchymal phenotype and a promoted migration. EMT plays important roles in embryonic development and cancer metastasis, and its hallmarks include the acquisition of front-back cell polarity and loss of cell-cell contact. However, how integrins dynamically regulate cell-ECM adhesions and cellular behaviors during EMT is still unclear. Using single-particle tracking of β1-integrins labeled with quantum dots, the temporal-spatial on-membrane dynamics of integrins in the EMT of MCF10A cells is revealed. β1-integrins exhibit significantly enhanced dynamics, which temporally behave more diffusive and less immobilized, and spatially become distributed asymmetrically with front regions being more dynamic. These dynamic alterations are shown to arise from microtubule remodeling in EMT. The results shed new light on the EMT mechanism from the cell-ECM adhesion perspective, and suggest that the enhanced integrin diffusion may represent as a new hallmark of EMT.

In eukaryote cells, cargos are often transported cooperatively by kinesin motors and nonmotor microtubule-associated proteins（MAPs）. The prior in vitro experimental data showed that the velocity of the cargo transported by kinesin motors and Ndc80（a member of MAP） proteins of truncated coiled-coil stalks decreases sensitively with the increase of the ratio of Ndc80 to motor number. However, the underlying mechanism of Ndc80 affecting sensitively the cooperative cargo transport by kinesin motors is unclear. To understand the mechanism, here we study numerically the cooperative cargo transport by kinesin motors and Ndc80 proteins. Our results showed that for the case of the motors and Ndc80 proteins with truncated short stalks, as used in the experiments, the calculated results reproduce quantitatively the prior experimental data. The mechanism of the cargo velocity decreasing sensitively with the ratio of Ndc80 to motor number is revealed. By contrast, for the case of the motors and Ndc80 proteins with full-length long stalks, the velocity of the cargo decreases slowly with the increase in the ratio of Ndc80 to kinesin number. Our results thus give an explanation of why the kinesin motors working in the cell have long stalks.

Kinesin is a molecular motor that can step processively on microtubules via the hydrolysis of ATP molecules. An important factor characterizing the processivity of the kinesin motor is its dissociation from the microtubule. Here, using all-atom molecular dynamics simulations, we studied the dissociation process of the kinesin head in weak-microtubule-binding or ADP state from tubulin on the basis of the available high-resolution structural data for the head and tubulin. By analyzing the simulated snapshots of the structure of the head-tubulin complex we provided detailed structural and dynamic information for the dissociation process. We found that the dissociation of the head along different directions relative to the tubulin exhibits very different dynamic behaviors. Moreover, the potential forms or energy landscapes of the interaction between the head and tubulin along different directions were determined. The studies have important implications for the detailed molecular mechanism of the dissociation of the kinesin motor and thus are critical to the mechanism of its processivity.

In the Large High Altitude Air Shower Observatory (LHAASO), one square kilometer array (KM2A), with 5242 electromagnetic particle detectors (EDs) and 1171 muon detectors (MDs), is designed to study ultra-high energy gamma-ray astronomy and cosmic ray physics. The remoteness and numerous detectors extremely demand a robust and automatic calibration procedure. In this paper, a self-calibration method which relies on the measurement of charged particles within the extensive air showers is proposed. The method is fully validated by Monte Carlo simulation and successfully applied in a KM2A prototype array experiment. Experimental results show that the self-calibration method can be used to determine the detector time offset constants at the sub-nanosecond level and the number density of particles collected by each ED with an accuracy of a few percents, which are adequate to meet the physical requirements of LHAASO experiment. This software calibration also offers an ideal method to realtime monitor the detector performances for next generation ground-based EAS experiments covering an area above square kilometers scale.

Intracellular transport plays an important role in maintaining the physiological functions of cells. Here, we describe a protocol for 3D single-particle tracking within living cells. We detail the use of a two-focal imaging system and the analytical steps for quantifying 3D transport dynamics. This protocol can be used to characterize the intracellular diffusion and trafficking of macromolecules, nanoparticles, and endocytic vesicles in adherent cells. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2022).

The histone chaperone FACT (FAcilitates Chromatin Transcription) plays an essential role in transcription and DNA replication by its dual functions on nucleosome assembly to maintain chromatin integrity and nucleosome disassembly to destabilize nucleosome and facilitate its accessibility simultaneously. Mono-ubiquitination at Lysine 119 of H2A (ubH2A) has been suggested to repress transcription by preventing the recruitment of FACT at early elongation process. However, up to date, how ubH2A directly affects FACT on nucleosome assembly and disassembly remains elusive. In this study, we demonstrated that the dual functions of FACT are differently regulated by ubH2A. The H2A ubiquitination does not affect FACT's chaperone function in nucleosome assembly and FACT can deposit ubH2A-H2B dimer on tetrasome to form intact nucleosome. However, ubH2A greatly restricts FACT binding on nucleosome and inhibits its activity of nucleosome disassembly. Interestingly, deubiquitination of ubH2A rescues the nucleosome disassembly function of FACT to activate gene transcription. Our findings provide mechanistic insights of how H2A ubiquitination affects FACT in breaking nucleosome and maintaining its integrity, which sheds light on the biological function of ubH2A and various FACT's activity under different chromatin states.