BACKGROUND:The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by PRKCA, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα. METHODS:We generated mice harboring a phospho-resistant PKCα (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the Prkca gene (PrkcaT497A) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of PrkcaT497A was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the PRKCAT497A mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca2+ homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with PRKCAT497A under chronic AngII (angiotensin II) stimulation. RESULTS:The T497A mutation in PKCα prevented its subsequent phosphorylation and led to PKCα protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKCα phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKCα-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca2+ transients. CONCLUSIONS:The editing of PRKCAT497A through adenine base editing represents a potential therapeutic approach for human cardiac diseases.

Anomalous, non-Gaussian diffusion is ubiquitous in living cells, but whether this statistical signature arises from static structural disorder or active cytoskeletal fluctuations remains unresolved. To decouple these factors, we used high-resolution single-particle tracking to characterize intracellular diffusion, while systematically perturbing cytoplasmic molecular crowding or cytoskeletal integrity. Our results demonstrate that the non-Gaussian, Laplace-like form of the displacement distribution is robust to cytoskeletal disruption but highly sensitive to molecular crowding. Notably, the characteristic confinement length scale, ξ, is significantly reduced by both increased crowding and the suppression of active fluctuations. This decoupling supports our proposed model of “active heterogeneous confinement”, where a static landscape defined by crowding is dynamically rescaled by activity. Our framework establishes diffusion statistics as a quantitative probe of the cell’s underlying biophysical state.

Macromolecular crowding is a fundamental physical property of the cytoplasm that governs intracellular diffusion and biochemical reactions.However,in situ quantitative characterization of intracellular dynamics and associated biophysical states in intact plant tissues remains challenging.Using 40-nm genetically encoded multimeric nanoparticles(GEMs)and single-particle tracking in Arabidopsis roots,we quantitatively map the regional heterogeneity of cytoplasmic diffusion dynamics and crowding along the root developmental axis:elongation zone cells exhibit a dense,low-mobility baseline,whereas maturation zone and root hair cells display higher mobility.These regions exhibit different sensitivities to osmotic stress.Notably,under severe ionic stress,both the diffusion coefficients and non-Gaussian parameters of the maturation zone and root hair cells converge toward the levels of the elongation zone cells,suggesting an intrinsic physical baseline for cytoplasmic crowding.This kinetic convergence in these cells is accompanied by vacuolar retraction and an increase in cytoplasmic thickness.Together,our study establishes a GEMs-based platform for in situ biophysical analysis in plant cells and uncovers a spatially-resolved physical landscape of cytoplasmic crowding and its dynamic reorganization under osmotic stress.

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.

How living cells transduce thermal perturbations into altered intracellular organization remains a fundamental biophysical question. Here, using single-particle tracking of quantum dot (QD) diffusion and endocytic vesicle transport, we show that temperature-enhanced transport is not a simple passive solvent effect; instead, heating enhances active cytoplasmic dynamics mediated by myosin-II-dependent actomyosin activity. While active fluctuations enhance random QD diffusion, they also accelerate active vesicular transport by reducing its mechanical resistance. Using thermoresponsive elastin-like polypeptide (ELPV) phase separation as a physical-chemical reporter, we demonstrate that myosin-II-dependent active fluctuations modulate phase separation. Near the transition temperature, the enhanced droplet formation after myosin-II inhibition suggests that active fluctuations oppose ELPV nucleation, whereas at higher temperatures, deeper within the two-phase regime, thermodynamic driving forces dominate. This work directly links nonequilibrium cytoplasmic mechanics with the physical chemistry of intracellular phase boundaries.

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.

The mechanical characteristics of cells and extracellular matrices-such as elasticity, surface tension and viscosity-can influence diseases such as fibrosis and tumour metastasis. Multicellular tissues have traditionally been modelled as viscoelastic materials, which overlooked the abundance of intercellular space and intercellular flow within the structure. Although intercellular flow can substantially impact development and disease progression, its role in the mechanical behaviour of tissues remains unclear. Here we show that fluid transport via the intercellular space determines the immediate mechanical response of tissues upon deformation. We directly measure the mechanical response of multicellular tissues by applying parallel plate compression via a tailored micro-mechanics platform. We find that both cultured three-dimensional cell spheroids and native mouse pancreatic islets exhibit apparent poroelastic behaviour over a timescale of up to a minute. These findings highlight the fundamental role of interstitial fluid transport in the mechanics of multicellular systems and could help identify potential physical regulators of development and diseases, as well as strategies for engineering multicellular living systems.

The dynamic behaviors of the mitotic spindle composed of antiparallel microtubules (MTs) are critical for the successful completion of cell division. Motor proteins kinesin-4 and MT cross-linking proteins PRC1 can cooperatively regulate the length of antiparallel MT overlaps. However, the physical mechanism of relative sliding of the two MTs by the two types of proteins to regulate the overlap length is unclear. Here, using both the theoretical analysis and numerical simulation, we first study the clusters at MT plus ends formed by kinesin-4s alone and those formed by kinesin-4s and PRC1s, where a kinesin-4 and a PRC1 can form an unstable complex. The forward movement of the complex is proposed to be realized via a "binding site competition" mechanism; namely, the competition of the site bound by the kinesin-4 head with the PRC1 head makes the complex move forward. Furthermore, we study MT sliding mediated by the kinesin-4s and PRC1s. The theoretical/numerical results reproduce quantitatively the published experimental results and, in particular, the puzzling results showing that the initial MT sliding velocity increases with the initial MT overlap length. The study indicates that the MT sliding by the kinesin-4s and PRC1s occurs via the "binding site competition" mechanism, which is different from the well-known "force-driving" mechanism by kinesin-5 motors, where the forces generated by the kinesin-5s drive the MT sliding.

Extracellular osmotic pressure is a key modulator of intracellular biophysical properties and cellular functions. However, its impact on the cell nucleus remains elusive, largely due to the challenges in real-time measurement of local environmental properties and reaction kinetics at specific loci within the nucleus. Here, we employ the dCas9-SunTag system to investigate the biophysical response at target DNA loci to osmotic pressure alterations. We reveal that variations in extracellular osmotic pressure modulate the efficiency of dCas9-SunTag-mediated fluorescent labelling rapidly and reversibly, with hypoosmotic condition increasing and hyperosmotic condition decreasing the number and fluorescence intensity of foci for telomeres and genes. Strikingly, osmotic pressure also regulates gene transcription with the dCas9-SunTag system, mirroring its effects on fluorescent labelling, as evidenced by changes in mRNA burst frequency. The underlying mechanism is that osmotic pressure shifts the binding-unbinding equilibrium of specific proteins to dCas9-SunTag complex by altering intranuclear crowding. These findings not only highlight the role of mechanical cues in modulating DNA-related processes within the nucleus, but also establish the dCas9-SunTag system as a sensitive probe for intranuclear crowding in response to extracellular cues, notably osmotic pressure.

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.

Tumor invasion constitutes a multifaceted process encompassing collective cellular migration and dynamic cell-fate transitions. Although these aspects have been studied separately by physicists and biologists, their spatiotemporal coupling remains unclear. To bridge this gap, we introduce a tumor-adipose assembloid model that facilitates live tracking and temporal analysis of cancer cells and adipocytes. The tumor assembloids manifest two discrete phases of morphogenic behavior, delineated by the reprogramming of adipocytes. In the initial phase, the biophysical interactions between cancer cells and adipocytes can be modeled as contact between viscoelastic drops. This interaction precedes the adipocytes' dedifferentiation and subsequent myofibrogenic reprogramming. The emergence of adipocyte-derived myofibroblasts instigates assembloid invasion through the mechanical remodeling of surrounding collagen networks. Our findings unveil a paradigm shift in understanding the evolutionary dynamics of heterotypic multicellular systems, wherein cell-fate transitions act as catalytic events that initiate serial patterns of collective morphogenesis via alterations in extracellular biophysical interactions.

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.

Membrane channel proteins (MCPs) play key roles in matter transport through cell membranes and act as major targets for vaccines and drugs. For emerging ionic liquid (IL) drugs, a rational understanding of how ILs affect the structure and transport function of MCP is crucial to their design. In this work, GPU-accelerated microsecond-long molecular dynamics simulations were employed to investigate the modulating mechanism of ILs on MCP. Interestingly, ILs prefer to insert into the lipid bilayer and channel of aquaporin-2 (AQP2) but adsorb on the entrance of voltage-gated sodium channels (Nav). Molecular trajectory and free energy analysis reflect that ILs have a minimal impact on the structure of MCPs but significantly influence MCP functions. It demonstrates that ILs can decrease the overall energy barrier for water through AQP2 by 1.88 kcal/mol, whereas that for Na+ through Nav is increased by 1.70 kcal/mol. Consequently, the permeation rates of water and Na+ can be enhanced and reduced by at least 1 order of magnitude, respectively. Furthermore, an abnormal IL gating mechanism was proposed by combining the hydrophobic nature of MCP and confined water/ion coordination effects. More importantly, we performed experiments to confirm the influence of ILs on AQP2 in human cells and found that treatment with ILs significantly accelerated the changes in cell volume in response to altered external osmotic pressure. Overall, these quantitative results will not only deepen the understanding of IL-cell interactions but may also shed light on the rational design of drugs and disease diagnosis.

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.

Liquid‒liquid phase separation (LLPS) is a ubiquitous process in which proteins, RNA, and biomolecules assemble into membrane-less compartments, playing important roles in many biological functions and diseases. The current knowledge on the biophysical and biochemical principles of LLPS is largely from in vitro studies; however, the physiological environment in living cells is complex and not at equilibrium. The characteristics of intracellular dynamics and their roles in physiological LLPS remain to be resolved. Here, by using single-particle tracking of quantum dots and dynamic monitoring of the formation of stress granules (SGs) in single cells, the spatiotemporal dynamics of intracellular transport in cells undergoing LLPS are quantified. It is shown that intracellular diffusion and active transport are both reduced. Furthermore, the formation of SG droplets contributes to increased spatial heterogeneity within the cell. More importantly, the study demonstrated that the LLPS of SGs can be regulated by intracellular dynamics in two stages: the reduced intracellular diffusion promotes SG assembly and the microtubule-associated transport facilitates SG coalescences. The work on intracellular dynamics not only improves the understanding of the mechanism of physiology phase separations occurring in nonequilibrium environments but also reveals an interplay between intracellular dynamics and LLPS.

扩散对于细胞内生物分子的运输至关重要,是各种生命过程的物理基础.由于细胞内部微环境的复杂性,其内部生物大分子的扩散呈现出异常扩散、空间异质性、各向异性等新奇动力学特性.然而,细胞内扩散动力学与胞内微环境空间维度之间的关系并不清楚.利用活细胞单分子荧光跟踪技术研究了细胞内量子点探针的扩散,定量分析了细胞内准二维扩散和丝状伪足内准一维扩散的动力学特征.研究发现相比于细胞内的准二维扩散,丝状伪足内的扩散速度更快,且扩散加速现象与丝状伪足的直径成反比,表明丝状伪足提供了一种更加有效的胞内物质运输途径.本研究不仅阐明了细胞内扩散动力学与胞内空间维度的关系,而且揭示了细胞通过亚细胞结构调控生物大分子运输的复杂机制.

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.

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).