Cytoskeleton

Tropomyosins play a central role in the regulation of the actin cytoskeleton by assembling along filaments into rope-like, head-to-tail polymers ("clusters") along filaments that regulate the binding and activity of numerous actin-binding proteins. Importantly, in cells, tropomyosins do not decorate actin filaments uniformly; some filaments are devoid of tropomyosins, whereas others recruit specific isoforms. To explore the diversity of tropomyosin-isoform binding kinetics and identify factors that control their assembly, we designed tropomyosin constructs, bearing a natural N-terminal acetylation and targeted cysteine mutations for fluorescence labelling. We analysed the binding of different tropomyosin isoforms to individual actin filaments reconstituted inside a microfluidic chamber. We show that, despite significant differences in spontaneous nucleation and elongation rates, all tested tropomyosin isoforms can coassemble within the same clusters. In addition, we identified tropomodulin and leiomodin, two pointed-end-binding proteins, to efficiently nucleate the formation of tropomyosin clusters on actin filaments, for all tested tropomyosin isoforms. Notably, while tropomodulin nucleates tropomyosin clusters from the pointed end, leiomodin does so along the entire filament.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Centrosome positioning is governed by mechanical interactions between the actin and microtubule cytoskeletons, yet the contribution of different actin architectures and their attachment to the cell membrane remains unclear. We use numerical simulations to study a centrosomal array of microtubules interacting with cortical, bulk, and centrosome-associated actin networks, including passive cross-linkers, motor proteins, and ERM-mediated coupling of actin to the cell membrane. We find that bulk and centrosome-associated actin alone are insufficient for robust centrosome centering, whereas sufficiently dense cortical actin promotes centering through steric interactions with growing microtubules. ERM-mediated attachment of cortical actin to the cell membrane enhances this effect, allowing stable centering at lower actin densities. In a two-centrosome system, microtubule interactions generate a spindle-like structure, while the organization of actin and its membrane attachment through ERM regulate centrosome separation. Antagonistic microtubule motors further limit centrosome separation and establish a finite spindle length. These results demonstrate that ERM-mediated coupling of actin to the cell membrane provides an important mechanical link between the actin cortex and microtubule-driven centrosome positioning, influencing both centrosome centering and spindle geometry.

Aims: Mature, contractile vascular smooth muscle cells (vSMC) in muscular blood vessels can de-differentiate into synthetic vSMC, while human pluripotent stem cells (hPSC) differentiate into proliferating, fetal-like vSMC that can be induced to form contractile vSMC. Given the complicated embryonic origins and conflicting views on vSMC heterogeneity, our aim was to establish how closely hPSC-vSMC of a known germ layer lineage and defined phenotype are representative of vSMC obtained from aorta (AoSMC). Methods and Results: We generated paraxial mesoderm (PM)-derived synthetic and contractile vSMC from hPSC lines and performed phenotypic and mechanical comparisons with AoSMC. Synthetic hPSC-vSMC and cultured AoSMC are proliferative, sensitive to contact inhibition and exhibit phenotype switching. Contractile hPSC-vSMC are non-proliferative, capable of phenotype switching and have elevated levels of contractile proteins (CNN1, TAGLN, ACTA2). These contractile proteins also displayed density-dependent, cell-to-cell heterogeneity in both AoSMC and hPSC-vSMC. Single-cell traction force measurements on micropost array detectors (mPADs) yielded comparable forces per micropost in the nN range across cell types. Quantitative 3D force measurements on humanized smooth muscle cell microtissues (SMTs) developed similar forces per cell. Larger forces were generated by MEKi-treated hPSC-vSMC and AoSMC versus controls. Conclusions: We have systematically studied the properties of hPSC-vSMC and performed comprehensive phenotypic and mechanical comparisons with AoSMC. These results demonstrate that synthetic hPSC-vSMC generated in vitro and cultured AoSMC derived from tissues are comparable and mechanically similar, but results with hPSC-vSMC are more reproducible.

Actin cross-linkers are essential modulators of the actin cytoskeleton, enabling structural diversity and dynamic remodeling. Among them, calcium/calmodulin-dependent protein kinase II (CaMKII) is unique in serving a dual role as a kinase and as a multivalent structural binder of actin in dendritic spines, where it plays a major role in supporting dendritic spine structure via its {beta} subunit. To understand the basis of the structural function of CaMKII, we quantified the morphology and mechanics of actin bundles formed by CaMKII and directly compared them with bundles assembled by the canonical bivalent cross-linkers -actinin and fascin. Using fluorescence microscopy, we measured contour length, straightness ratio, and persistence length across different cross-linker concentrations. Fascin generated progressively shorter, straighter, and stiffer bundles with increasing concentration, whereas -actinin bundles remained largely unchanged. In contrast, increasing CaMKII concentration reduced both bundle straightness and persistence length, revealing a unique concentration-dependent increase in bundle flexibility. To investigate the structural origins of these mechanical behaviors, we complemented the experiments with coarse-grained simulations of cross-linked actin bundles. Simulations revealed that CaMKII-generated bundles retained a substantially larger fraction of their curvature in their time-averaged configuration than bundles formed by -actinin or fascin, indicating that bundle mechanics are strongly influenced by organizational features beyond thermal bending fluctuations alone. CaMKII bundles displayed behavior consistent with additional structural complexity arising from their multivalent architecture and flexible linker domains. Together, our experimental and computational results identify CaMKII as a structurally and mechanically distinct actin cross-linker. We propose that the combination of multivalency and linker flexibility enables CaMKII to function as a molecular structural pivot, promoting flexible, adaptable actin assemblies that are mechanically compatible with the dynamic remodeling required for dendritic spine plasticity.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Trigger waves are positive feedback-mediated traveling waves that propagate without slowing down or diminishing in amplitude, enabling biochemical coordination over distances where diffusion alone would be insufficient. Apoptosis can spread as trigger waves of caspase activity, as demonstrated in Xenopus eggs and egg extracts. We measured apoptotic wave speeds in extracts at two cell cycle states and found that waves propagated more slowly in M-phase arrested cytostatic factor (CSF) extracts than in interphase extracts (21 versus 27 m min-1). Luther's equation, v=2{surd}D{kappa}, predicts wave speed from the diffusion coefficient D and the positive-feedback doubling rate {kappa}. However, D and {kappa} were comparable between the two extracts and could not explain the speed difference. This discrepancy highlighted a key limitation of the classical equation: it does not account for an activation threshold in trigger wave initiation, and in fact the threshold required to trigger apoptosis was found to differ between cell cycle states. We therefore extended Luther's equation to include such a threshold, obtaining v{propto}{surd}(AD/Uc ), where A is the reaction rate and Uc is the activation threshold. Perturbing apoptotic sensitivity confirmed the predicted threshold-speed relationship. These findings establish the activation threshold as a key determinant of trigger wave speed and provide a broadly applicable scaling law for predicting the spread of switch-like biochemical signals.

Tau is a microtubule-associated protein important for neuronal structure and function, with abnormal phosphorylation associated with neuronal dysfunction and degeneration. While tau pathology has been extensively studied in neurodegenerative diseases and traumatic brain injury, its role in spinal cord injury (SCI) remains unclear. This study investigates changes in the levels of tau isoforms and phosphorylation following a high lateral C2 hemisection (C2Hx) in adult female rats. Western blot analysis was performed on spinal cord tissue collected caudal to the lesion at 4 weeks post-injury. Low-molecular-weight (LMW) tau and high-molecular-weight, Big tau did not differ significantly between injured and control groups. In contrast, phosphorylated tau (p-tau), detected using the AT8 antibody, was significantly elevated after injury, suggesting a shift in tau functional state. Assessment of microtubule stability markers revealed increased acetylated tubulin and decreased tyrosinated tubulin after SCI, indicating a shift toward a more stable cytoskeletal state. Immunohistochemistry was performed on spinal cord sections at 6 weeks post-injury and dorsal root ganglia (DRG) at 8 weeks post-injury. These analyses demonstrated increased p-tau immunoreactivity within the ipsilateral dorsal horn of the injured spinal cord and a higher proportion of p-tau-positive neurons in DRGs from injured animals compared with controls. Together, these findings demonstrate that cervical SCI induces persistent tau phosphorylation without altering total tau levels, and that this modification extends beyond the lesion site into distal spinal regions and beyond the acute phase of injury. These alterations may influence neural plasticity and remodeling in the injured spinal cord.

Cortical positioning of oocyte meiotic spindles is required for reducing chromosome number by segregating chromosomes into polar bodies. In C. elegans, kinesin-1 transports membrane-bound organelles inward into a central ball that is hypothesized to push the meiotic spindle outward. Using focused ion beam scanning electron microscopy, we found that the percent volume occupied by membrane-bound organelles was 28% in a mature oocyte that has completed endocytosis of the yolk precursor, vitellogenin, compared to 17% in a less mature oocyte that has not yet endocytosed vitellogenin. We therefore hypothesized that blocking endocytosis of vitellogenin would reduce the size of the central ball of organelles packed inward by kinesin-1 and limit outward movement of the spindle. Depletion of vitellogenin or proteins required for endocytosis resulted in a smaller central volume of kinesin-coated organelles and defective spindle positioning. A computer simulation of a simple exclusion model faithfully recapitulated wildtype and mutant spindle position phenotypes. These results support a volume exclusion model for positioning acentriolar meiotic spindles.

Clathrin-mediated endocytosis (CME) is the primary pathway for internalization of extracellular and membrane cargo in eukaryotic cells. It is characterized by a patch of the plasma membrane invaginating and pinching off to generate a cytoplasmic vesicle. Recently, experimental evidence has begun to accumulate in support of proteins assembling into liquid-like condensates at nascent CME sites, but potential functional consequences are not understood. Given that condensate capillary forces can influence membrane bending, we constructed complementary analytical and numerical model systems to probe the mechanical influence of a protein droplet on endocytosis. Model evaluations and analysis reveal that a droplet can regulate the progression of endocytosis by stalling invagination until the droplet grows past a threshold size. After the droplet-mediated brake is released, the droplet provides a force that can drive the membrane bud toward scission. Thus, we propose a ``mechanical timer'' mechanism as a function for protein phase separation in cellular membrane trafficking.

It is important to determine whether native state dynamics drive the misfolding and oligomerization of the prion protein, which are important events in prion disease, and how they are modulated by conformational conversion. Native (N) mouse prion protein (moPrP) is known to form small (OS) and large (OL) oligomers rich in {beta}-sheet, and in this study, photoinduced electron transfer-fluorescence correlation spectroscopy (PET-FCS) has been used to characterize intramolecular dynamics within individual monomeric units in both isolated OS and OL, as well as the diffusion properties of the oligomers. It is estimated that OS and OL comprise of about 15 and 55 monomeric units, respectively. Microsecond dynamics at each of the two regions that are the 1-3 and 2-3 interfaces of native protein are distinct in N, OS and OL, although they occur on very similar timescales. Analysis of the evolution of the distribution of diffusion times, determined using the maximum entropy method, indicates heterogeneity in the oligomerization reaction. Analysis of the change in the fluctuations which occur in two different timescales in the native state ensemble shows that they are damped more at the erstwhile 1-3 interface than the erstwhile 2-3 interface. The difference in the extent of damping at the erstwhile 1-3 and 2-3 interfaces can be explained on the basis of the structural changes known to occur across each region. The changes in dynamics occur concurrently in both regions, indicating that the structural changes accompanying conformational conversion also occur simultaneously during the oligomerization of moPrP.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes/glomerular epithelial cells (GECs) induces ER stress and contributes to the pathogenesis of glomerular disease. Activation of IRE1 and consequently the unfolded protein response (UPR) and autophagy sustains proteostasis. The present study examines the role of Surf4 and collagen IV-5 (COL4A5) trafficking in podocyte proteostasis. Glomerular Surf4 protein expression increased in IRE1-replete mice with experimental focal segmental glomerulosclerosis (FSGS) compared to podocyte-IRE1 knockout mice with FSGS and control mice. Glomerular Surf4 gene expression was greater in human FSGS and membranous nephropathy compared to healthy controls. In cultured GECs, basal Surf4 expression was greater in IRE1-replete (control) compared to IRE1 knockout GECs, although ER stress (tunicamycin treatment) did not increase Surf4 despite activating the UPR. Tunicamycin enhanced autophagy (LC3-II) in an IRE1-dependent manner, and the amount of Surf4 localized in autophagosomes. In GECs, tunicamycin increased the amount and colocalization of endogenous COL4A5 or ectopically-expressed COL4A5-wild type (WT) and Surf4 in autophagosomes. Compared to COL4A5-WT, the human COL4A5-C1567R mutant (which is misfolded) showed enhanced basal colocalization with Surf4 in autophagosomes; colocalization was similar to COL4A5-WT after tunicamycin treatment. Degradation of COL4A5-C1567R was more rapid compared to COL4A5-WT and was comparable to degradation of COL4A5-WT in the presence of tunicamycin. Thus, during ER stress, subsets of Surf4 and COL4A5 are redirected from the secretory pathway to autophagy. Surf4 may help deliver misfolded ER cargo, such as COL4A5 for autosomal degradation, thereby sustaining proteostasis in podocytes.

Although MAP1LC3B/LC3B (LC3) is best known as a cytoplasmic marker of autophagosome biogenesis, a substantial pool of LC3 resides in the nucleus and shuttles dynamically between nuclear and cytoplasmic compartments, yet the composition and regulation of this nuclear interactome remain poorly defined. Using mass spectrometry-based proteomic profiling of nuclear GFP-LC3 immunoprecipitates from primary human trabecular meshwork (TM) cells, a mechanosensitive ocular cell type, we identified a reproducible nuclear LC3 interactome enriched for proteins containing LC3-interacting region (LIR) and expanded LIR (xLIR) motifs and reported nuclear localization. Among these, clathrin heavy chain (CLTC) emerged as a previously unrecognized nuclear LC3 partner that localizes to the nucleus and colocalizes with nuclear LC3 puncta. CLTC depletion reduced basal LC3-II levels, consistent with a role in autophagosome biogenesis, and markedly impaired nuclear LC3 accumulation induced by both nuclear export blockade with leptomycin B and cyclic mechanical stretch, without altering total CLTC abundance, indicating that CLTC actively promotes LC3 nuclear translocation rather than serving as a passive scaffold. Strikingly, mechanical stress-induced nuclear LC3 trafficking, but not its basal component, was selectively impaired in TM cells derived from glaucoma patients, despite comparable CLTC levels, pointing to a defect in coupling this trafficking pathway to mechanotransduction rather than in the core transport machinery itself. Together, these findings establish nuclear LC3 trafficking as an actively regulated, CLTC-dependent process linked to cytoskeletal and vesicular machinery, and implicate its dysregulation in mechanically stressed glaucomatous cells, providing a framework for understanding how autophagy intersects with nuclear homeostasis, mechanotransduction, and glaucoma pathogenesis.

Cellulose has long been viewed as the major load-bearing component in plant primary cell walls. Recent studies, however, suggest that pectin plays a significant role in cell wall mechanics despite a kPa-scaled modulus. Here we quantify the mechanical properties of centimeter-sized internodal cell walls of Chara corallina following treatments to remove calcium crosslinks or digest pectin. We also characterize the flow of water and gas through the cell wall as a function of pressure. We found that removal of calcium crosslinks did not affect wall strength and stress-relaxation behavior, yet it decreased the elastic modulus and increased permeability to water. In contrast, pectin removal had a dramatic impact on all measurements. Samples from which pectin had been removed exhibited lower strength and stiffness and had greater stress relaxation under constant strain. Pectin removal increased the permeability of the cell wall to water above that observed when calcium was removed and reduced the pressure at which gas "tunnels" through the cell wall ~ 2-fold. Our findings support the idea that pectin locally restricts the movement of cellulose microfibrils, with implications for understanding functional properties of primary cell walls in vascular plants and developing high-performance double-network materials.

Large shear deformation in cartilage can lead to tissue failure and injury, which are often precursors to diseases such as post-traumatic osteoarthritis. Under shear, cartilage strain-stiffens prior to failure, which makes stiffening an important indicator of changes to the loading of the extracellular matrix that gives rise to this tissue's properties. While a variety of stiffening mechanisms ranging from uncrimping to fiber-alignment have been explored, whether such mechanisms apply to the depth dependent shear strain stiffening in articular cartilage remains poorly understood. Here, we present experiments and theory that support the idea that the strain-stiffening behavior is caused by a mechanical phase transition from a random isotropic network whose mechanics arise primarily from bending-dominated modes to one where the mechanics arise from stretching-dominated modes. Indeed, consistent with network model predictions, we find that stress-strain data for all depths can be collapsed onto a single universal curve that diverges at a depth-dependent critical strain. These results show that increasing the local concentration of collagen, which constitutes the primary load-bearing network in cartilage, decreases the critical strain at which the strain-stiffening transition occurs, while increasing the low-strain elastic modulus. Furthermore, we demonstrate that bulk compression leads to a higher critical strain and decreased low-strain elastic modulus. Network simulations capture this rich behavior, and indicate that under compression the network collapses, which allows for larger strains before the transition from bending-dominated to stretching-dominated mechanics occurs. Collectively, these findings shed light on the roles that tissue composition and axial strain play in the mechanisms that organize how articular cartilage stiffens during the lead-up to injury.

Biofilm formation--the establishment of cellular communities within a self-secreted polymer matrix--is a behavior exhibited by almost all species of bacteria. Bacterial biofilms are well described as viscoelastic materials, with properties that grant physical advantages, such as elastic resilience and viscous adaptability. Additionally, many biofilm-forming species produce a subpopulation of highly resilient spores. In the model biofilm- and spore-forming species Bacillus subtilis, sporulation and matrix production are regulated by a common gene pathway, provoking the question of how sporulation influences biofilm viscoelastic properties. Investigating this point will facilitate the management of both salutary and deleterious biofilms, especially through manipulating their establishment and dispersion. Here, we investigate the interplay of sporulation and viscoelastic properties using rheological measurements of B. subtilis biofilms with varied matrix and spore production. We find that, to a significantly greater extent than matrix production, biofilm physical development is strongly altered by activation of sporulation through the spo0A regulation pathway. Spore-deficient strains initially establish physically robust biofilms with linear viscoelastic properties and recovery behavior distinct from spore-producing biofilms. However, spore-deficient biofilms fail to maintain their physical properties over time. In contrast, spore-producing wild-type and matrix-mutant biofilms exhibit more stable physical properties. Our results demonstrate that cell-level gene regulation is associated with macroscopic mechanical transitions in cellular communities, with sporulation granting not only a cellular-level survival advantage, but also greater community-level physical stability.

Liquid-liquid phase separation is a vital and ubiquitous principle of subcellular organization. Several actin-binding proteins have recently been shown to undergo liquid-liquid phase separation, forming micron-sized droplets that assemble actin into distinct network shapes. However, our knowledge of how these phase-separated proteins organize actin filaments at the nanoscale is rather limited. Here, we seek to address this knowledge gap by investigating synapsin-1 condensates through a combination of computational simulations and nanoscale imaging experiments. Our results show that while synapsin-1 condensates by themselves lack any special structural organization, the addition of actin filaments results in the generation of a regularly organized actin scaffold within the condensates. The organized scaffold forms both when actin and synapsin-1 are added simultaneously and when actin is added after the formation of synapsin condensates. Dissolving the condensates, by synapsin-1 removal, leaves the actin scaffold largely unaffected in both simulations and experiments, implying that this scaffold should guide droplet reformation when synapsin-1 is added back. Taken together, our findings suggest that the actin-rich component of the condensate retains a structural memory that promotes and guides the reassembly of synapsin-actin condensates. This structural memory may be important in the long-term maintenance of synaptic function in vivo.

Hydrodynamics is a strong regulator of phytoplankton cell densities and population connectivity. However, the extent to which hydrodynamics, specifically dilution and advection, dictates the internal physiological state and overall bloom dynamics remains poorly understood. We conducted an integrated study of the harmful diatom Pseudo-nitzschia australis in two neighboring coastal bays with similar abiotic conditions but contrasting bloom trajectories. Using a combination of field observations, transcriptomics, domoic acid (DA) measurements, and hydrodynamic simulations, we show that a three-fold difference in dilution rates alone explains a 25-day shift in bloom timing and an order-of-magnitude difference in cell densities. Transcriptomic profiling revealed distinct metabolic and stress-associated gene modules that synchronized with hydrodynamically-mediated nutrient depletion rate, linking residence times to cellular mortality. Furthermore, directional connectivity between the bays may explain population-level toxin heterogeneity, as advection of older cells, releasing DA outside the cells, may have decreased the proportion of intracellular DA in the destination bay. Our results illustrate that hydrodynamics do not merely modulate population transport; they structure the full physiological life cycle of a bloom, from initiation to nutrient-driven demise. This highlights the necessity of considering hydrodynamics as a primary explanation for bloom dynamics with cascading effects on bloom onset, cell density, nutrient consumption, cell physiology and mortality.

Adherent cells must continuously adapt to rapid environmental fluctuations to preserve mechanical integrity. However, conventional theoretical frameworks predominantly rely on quasi-static assumptions, thereby limiting their ability to capture the transient dynamics of cellular responses to high-rate perturbations, such as acute osmotic shocks. To address this limitation, we developed a biophysical model incorporating a Hill-type law that couples active cytoskeletal mechanics to cell-edge velocity, explaining the counterintuitive experimental observation that traction forces transiently decrease during rapid hypotonic swelling despite an increase in cell size. We further construct a kinematic-geometric phase diagram that connects classical quasi-static theories with our dynamic model. Moreover, we show that traction-force dynamics are sensitive to the loading rate and amplitude of osmotic shock. In addition, stiffness-associated simulations and supporting perturbation evidence suggest a cellular stiffness-associated recovery trend in which stiffer cells exhibit faster volume recovery driven by stronger recoil of hydrostatic pressure.

Mutations in {beta}-cardiac myosin are a common cause of inherited cardiomyopathies. The dilated cardiomyopathy E525K mutation alters both thick filament regulation and motor activity, yet the structural basis for these effects has remained unclear. Here, we combined cryo-EM and MD simulations to determine how E525K affects the conformational landscape of cardiac myosin in both its autoinhibited interacting-heads motif (IHM) and isolated myosin head states. E525K-mutated myosin exclusively adopted a single S2 conformation in IHM, in contrast to the conformational heterogeneity observed in wild-type myosin. Structural and electrostatic analyses revealed that the mutation increased positive charge density at the blocked-head (BH) - S2 (subfragment-2) interface, strengthening interactions with negatively charged residues within S2. 3D variability analysis and MD simulations of the E525K IHM demonstrated reduced S2 mobility, consistent with strengthened BH - S2 interactions, and decreased conformational flexibility, providing a structural mechanism for stabilization of the IHM state. To investigate the isolated myosin head, we determined cryo-EM structures of wild-type and E525K subfragment-1 (S1). The mutation induced local conformational rearrangements in the activation-loop and Loop 3 region, the SH3-like domain, and the essential light chain of S1, revealing structural changes that support the enhanced motor activity previously reported for isolated E525K myosin motors. These findings show that E525K stabilizes the autoinhibited IHM through electrostatic stabilization of the BH - S2 interface while inducing structural changes consistent with activation of the isolated motor domain, providing structural insights into how a single DCM mutation differentially regulates cardiac myosin S1 and IHM structure and function.