Cytoskeleton

Schwann cell precursors (SCPs) migrate with peripheral sensory axons from DRGs to target regions. SCP migration involves adhesions to substrata to stabilize advancing cell margins, and contractile forces that pull a SCP forward and break rear adhesions. Ncadherin on axons provides adhesion for SCPs, and ephrin-A2 signaling from axons stimulates SCP contractions to complete SCP translocation. Modulation of these adhesive and contractile forces regulates SCP migratioin during development of peripheral nerves.

The cytoskeleton is a dynamic biopolymer network whose shear rheological properties are crucial for cellular physiology and pathology. However, its mechanical behavior spans multiple spatiotemporal scales, and the coupling of dynamic remodeling and viscoelastic dissipation mechanisms poses a challenge for traditional models to comprehensively capture complex cellular responses. This study aims to establish a multiscale cytoskeletal network model that integrates the bio-chemo-mechanical properties of local linked proteins, the viscoelasticity of actin filaments, and their deformation states. Developing a boundary-modified finite element method with an incremental iterative algorithm, we demonstrated the dynamic remodeling of network and the resultant rheological properties of cytoskeleton by extending the predictive time scale to one thousand seconds. The results not only reproduced the short- and intermediate-term power-law creep behavior and long-term strain plateau response of the cytoskeletal network observed in shear rheological experiments, but also indicate that the synergy among the chemo-mechanical coupling of cross-linked proteins and the bending-to-tension transition of actin filaments govern both the network remodeling and its power-law response evolutionary, whereas the steady-state properties of actin filaments determine the long-term network behavior. Simulations of cancerous and drug effects show that cancer-induced softening and reduced filament viscosity lead to accelerated cytoskeletal responses and decreased apparent shear modulus, respectively; and drug-enhanced filament prestress, along with promoting association or inhibiting dissociation of cross-linked proteins, can effectively increase the steady-state shear modulus. These findings advance the understanding of the spatiotemporal evolution and pathological mechanisms of cellular mechanical responses and provide insights for regulating polymer network performance.

The podocyte foot process network forms the final barrier of the kidneys glomerular filtration system. Under mechanical stress this network is prone to injury in which podocytes lose connectivity to their neighbors and begin the progression toward effacement, but what governs its mechanical resilience is unknown. We show that the network is built like a lap joint: two major processes coupled through interdigitating foot processes, a configuration that behaves as a classical shear lag system, with force concentrating at the joint ends and decaying over a characteristic transfer length set by geometry and stiffness. A discrete network model reproduces the continuum shear lag solution and identifies a hierarchy among governing parameters, with cytoskeletal stiffening of the major process amplifying foot process force more potently than basement membrane stiffness. Applying the model to morphometric data from puromycin aminonucleoside nephrosis, a model of human minimal change disease and early focal segmental glomerulosclerosis, reveals a mechanical positive feedback loop: force concentration drives foot process loss, which raises force on surviving segments and accelerates further loss. This nonlinear amplification implies a threshold beyond which failure becomes self-sustaining, analogous to the critical crack length in fracture mechanics.

Myosin II generates force through the collective action of mechanically coupled motor ensembles, yet the mechanisms by which these ensembles sense changes in motor occupancy and coordinate force generation remain poorly understood. Ensemble force production may be governed by an optimal balance between effective motor occupancy and mechanical coordination rather than by motor number alone. We reconstituted cardiac myosin ensembles and systematically perturbed effective motor occupancy using the small-molecule drugs omecamtiv mecarbil (OM), which prolongs actomyosin interactions, and mavacamten (MAVA), which reduces the number of available force-generating myosin heads. Optical trapping measurements of full-length and S1 cardiac myosin ensembles revealed that force generation depended on both myosin concentration and pharmacological perturbation. Reducing myosin concentration increased force generation in the absence of drug, while OM and MAVA produced responses that varied with the initial occupancy state of the ensemble. Low concentrations of MAVA enhanced force generation under high motor occupancy but reduced force under low motor occupancy, whereas OM produced occupancy-dependent changes in both endpoint force and force dynamics. Force traces further revealed changes in the persistence and temporal coordination of force generation. These findings support a model in which cardiac myosin ensembles operate along an occupancy-coordination landscape, where maximal force generation is achieved at an intermediate level of effective motor occupancy. Our results illuminate how changes in motor occupancy are translated into coordinated ensemble mechanics and suggest that emergent mechanical feedback through the shared actin filament may enable ensembles to collectively sense and adapt to their mechanical state. Significance StatementForce generation by muscle emerges from the coordinated activity of myosin ensembles, yet the principles governing this collective behavior remain poorly understood. Using an in vitro force assay with full-length and truncated cardiac myosin, we systematically perturbed ensemble activity by varying myosin availability and pharmacologically altering the fraction of force-generating motors. We find that force production depends on an optimal balance of motor engagement rather than a simple increase or decrease in active motors, demonstrating that collective mechanical output arises from coordinated interactions within the ensemble. These findings reveal emergent design principles that govern molecular motor function and establish effective motor occupancy as a key regulator of collective force generation, providing new insight into the mechanisms underlying muscle contractility.

Integrins are adhesion proteins that diffuse along the cell membrane, bind to ligands, and cluster with each other in the early stage of cell adhesion. Integrin clustering and its specific spatial distribution play important roles in subsequent biological processes; however, the mechanisms that give rise to their characteristic spatial distribution remain poorly understood. To address this issue, we developed a cell adhesion model that incorporates cell membrane deformation and integrin dynamics. A hybrid continuous/discrete model was applied to represent membrane deformation, whereas Brownian dynamics combined with a transition state model was used to describe integrin dynamics and binding kinetics. Comparison of numerical simulations of cell adhesion to a substrate with experimental observations at the early stage of adhesion successfully reproduced the characteristic spatial distribution of integrin clusters, in which high-density clusters formed at the periphery of the region adhering to the substrate. These results suggest that the cellular-scale distribution of integrin clusters can be reproduced using only minimal elements, such as adhesion-driven membrane deformation and integrin-ligand binding. In addition, we found that the strength of integrin-ligand binding regulates the degree of clustering by changing the size of the part of the membrane that is deformed, thereby mechanically supporting the mechanical involvement of the actin cytoskeleton in integrin clustering. Furthermore, the formation and spatial distribution of integrin clusters were shown to be determined not only by the static mechanical equilibrium of membrane deformation and physical adsorption, but also by membrane spreading/deformation and the dynamic behavior of integrins. This suggests that the size and spatial distribution of integrin clusters may be controllable by modulating the speed of membrane spreading.

Motile cilia rely on highly stable axonemal microtubules reinforced by microtubule inner proteins (MIPs) that form a network within their lumen, yet the functions of individual MIPs remain poorly understood. Here, we characterize the conserved MIP sperm acrosome-associated protein 9 (SPACA9), which localizes to respiratory cilia and sperm flagella. Using in vitro reconstitution assays, we show that human SPACA9 (hSPACA9) acts as a molecular staple: it stabilizes protofilaments at growing microtubule ends, and inhibits dynamic instability. Surprisingly, these effects do not confer resistance to motor-induced lattice damage, indicating that regulation of microtubule dynamics can be uncoupled from mechanical resilience. Mechanistically, we identify an unstructured C-terminal region that is sufficient for microtubule binding and recapitulates the effects on dynamics. Together, our findings reveal functional specialization among MIPs and provide a mechanistic framework for how lumenal proteins tune distinct properties of axonemal microtubules.

One of the first genes identified to regulate the spacing of nuclei in the multinucleated myofiber was Kinesin-1. However, the mechanism by which Kinesin-1 or other kinesins regulate myonuclear spacing is not known. Critically, the myofiber lacks centrosomes, and the many myonuclei act as the primary microtubule organizing centers of the cell. Because of this unique re-structuring, we hypothesized that the kinesins that drive centrosomes apart during mitotic spindle elongation may play a similar role in spacing myonuclei. We found that the bipolar Kinesin-5 (Klp61f) and the (-)-end directed Kinesin-14 (ncd) were both necessary for myonuclear spacing at different times, with both being necessary during embryogenesis, but only ncd being necessary in the fully differentiated myofiber. To investigate the shared mechanisms during embryogenesis, we used live-imaging and found that, similar to the mitotic spindle, Klp61f acts as an accelerator for myonuclear movement, whereas ncd acts as a brake contrary to this movement. To investigate these mechanisms and test the hypothesis that this is dependent on microtubule-sliding, we used super-resolution microscopy to visualize and quantify the microtubule network in animals with disrupted Klp61f or ncd. We found that in both cases, there was a decrease in the amount of microtubule overlap between neighboring myonuclei. Furthermore, we found that disrupting ncd led to severe changes in microtubule network organization, supporting our hypotheses that microtubule-sliding is necessary to space myonuclei, and that ncd likely functions through a unique mechanism in the differentiated myofiber to maintain myonuclear spacing. Together, our data supports a model where myonuclear spacing is regulated by a counteracting force generated by different kinesins during embryonic development. Furthermore, one kinesin, ncd, is repurposed in the differentiated myofiber to dynamically crosslink microtubules, a function necessary to anchor nuclei in place. Thus, kinesin motors regulate myonuclear spacing across developmental time by leveraging opposing forces through diverse mechanisms.

The mechanical properties of the nucleus are critical for maintaining nuclear integrity and function. We previously showed that chromatin dominates short-extension mechanics whereas lamins provide long-extension strain stiffening. To distinguish the roles of lamin isoforms, micromanipulation nucleus force measurements were performed on isolated nuclei from lamin A/C (Lmna-/-) and lamin B1 (Lmnb1-/-) knockout mouse embryonic fibroblast cells. Lamin A/C knockout does not alter short-extension nuclear stiffness but is essential for strain stiffening at longer extensions. Oppositely, lamin B1 loss reduced short-extension stiffness due to facultative heterochromatin loss while long-extension strain stiffening was slightly increased. Loss of lamin A/C and B1 resulted in similar lamin-chromatin linkers effects as LBR did not change and LAP2{beta} decreased in both. A simulation model of a polymeric lamin shell with stiff lamin A/C and softer lamin B1 subunits can qualitatively recapitulate experimental measurements of lamin knockout cells. Lamin A/C knockout resulted in abnormal nuclear shape but not nuclear blebbing or rupture whereas lamin B1 knockout, similar to other perturbations that cause heterochromatin loss, resulted in increased nuclear blebbing and rupture. This work illuminates the distinct mechanical roles of lamin A/C and B1 in determining nuclear structure and integrity.

In epithelial wounds, physically enlarged cells emerge along injury edges where they interact with regular-size cells during collective migration to repair tissue continuity. Although such large cells are often interpreted as migration leaders, recent observations suggest that regular-size cells can reciprocally influence them and that mixed-size cell clusters engage in distinct rotational motion before merging into confluent sheets. How cell size, polarity and adhesive coupling jointly control distinct collective migration modes remains unclear. Because many features of collective epithelial migration, including large and regular-size cell phenotypes, are conserved between in vivo and in vitro systems, here we developed a multi-scale computational model of interacting epithelial cells in two-dimensional culture with dynamic cell-substrate adhesion, cell-cell interactions, protrusion-based polarity, and contact-induced myosin redistribution as polarity regulator. We also explicitly modeled two functionally distinct cell states, featuring regular and large sizes respectively. Simulations showed that symmetric and asymmetric myosin redistribution at cell-cell junctions can produce stable cell contacts and persistent rotational motions by cell doublets. Intriguingly, in mixed-size cell clusters, straight translation can arise both from leader-like large cell and regular-size cell-mediated motion, whereas rotation emerges when large-cell-generated torque overcomes translation while cell-cell adhesion is maintained. Thus, collective migration mode depends on the balance among myosin-driven torque, regular-size cell-mediated translation, substrate coupling, and cell-cell adhesion. These modeling results suggest that collective epithelial migration can emerge in vitro from reciprocal biomechanical interactions between distinct cell states, rather than from leader-like cell behavior alone, and that such interactions can produce a predator-prey-like pursuit-escape mode of collective migration. Our model also provides a framework for investigating the biochemical and biomechanical regulation of collective cellular migration. It can be readily extended from in vitro to in vivo context and can incorporate the effects of substrate topology and soluble signaling factor-driven chemotaxis. Author summaryWhen epithelial cells repair a wound, they move as coordinated groups rather than as isolated individuals. Cells of different sizes may contribute in distinct but connected ways. We developed a computational model to examine how a large cell and neighboring regular-size cells move together. In the model, cells attach to the underlying surface and one another, form protrusions that set their direction, and redistribute the force-generating protein myosin after contact. We found that group motion depends on a balance among surface attachment, cell-cell adhesion, movement of regular-size cells toward the large cell, and myosin-driven turning of the large cell. Depending on this balance, a mixed-size cluster can travel along a nearly straight path or rotate persistently, exhibiting pursuit-and-escape-like interactions between the large cell and surrounding regular-size cells. Rotation occurs when the large cells turning effect outweighs the translational motion driven by regular-size cells, while cell-cell adhesion keeps the cluster together. Our results show that collective migration can emerge from reciprocal mechanical interactions between cells in different states, not only from a "leader" cell acting alone. The model also provides experimentally testable predictions for how cell size, adhesion, and internal myosin distribution shape cell cluster movement.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

The lateral spacing between filaments in crosslinked cytoskeletal bundles is a critical yet poorly understood physical parameter that affects force generation, transport, and bundle architecture. We develop a biophysical model of crosslinkers and crosslinking motors on filament pairs. Motor/crosslinker binding sets the filament spacing, which in turn biases which motors/crosslinkers can bind. Crosslinking motors generate pulling forces that bring antiparallel filaments closer together, while non-motor crosslinkers with a preferred binding angle exert repulsive torques that maintain larger spacing. We demonstrate these principles in a model of the fission yeast anaphase mitotic spindle midzone, where microtubules form a square array with nearest-neighbor spacing 2-5 times smaller than the length of crosslinking proteins. Our model shows that motor-crosslinker interactions alone are sufficient to drive self-organization into this experimentally observed geometry. Furthermore, the feedback between geometry and binding creates strong indirect cooperativity, because crosslinkers establish spacing that favors binding of similar-length proteins, leading to history-dependent states that persist long after individual protein binding equilibration. This feedback mechanism in which crosslinkers control geometry and geometry controls crosslinker binding should operate in any multi-crosslinker-filament system and represents a general self-organizing principle for cytoskeletal networks.

Microtubule-dependent pronuclear migration and mitotic spindle positioning are fundamental processes during the first embryonic division in many animals. In the one-cell embryo of Caenorhabditis elegans, these events are regulated by well-characterized pulling forces acting on astral microtubules, including cortical forces mediated by the G-GPR-LIN-5 dynein complex. Although the overall framework of these dynamics is conserved, recent studies have revealed substantial interspecies variation in their regulation. Here, we investigated nuclei and mitotic spindle behaviors in one-cell embryos of Caenorhabditis inopinata, the closest known relative of C. elegans, using live-cell imaging and functional perturbation. We found that C. inopinata embryos exhibit altered pronuclear migration, reduced anaphase spindle oscillations, and slower centrosome diffusion during telophase compared with C. elegans. These differences suggest weaker cortical pulling forces. Functional analyses using RNA interference showed that GPR retains its essential role in force generation, whereas the contribution of the microtubule depolymerizing kinesin KLP-7 is reduced in C. inopinata. Our results point to evolutionary changes in microtubule-regulated spindle dynamics, and provide insight into how conserved cellular processes can diversify through subtle changes in their underlying mechanisms.

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

Many adherent eukaryotic cells exhibit amoeboid locomotion, where traction stress exerted on the substrate is essential for movement. In this study, we investigated the spatiotemporal development of these forces in Amoeba proteus to clarify the mechanical dynamics underlying bleb-driven migration. By performing a multipole analysis of the stress distribution, we characterized the spatiotemporal patterns exhibited by motile cells. Furthermore, we tracked the behavior of individual localized peak structures within these profiles, which are thought to correspond to focal contact sites. These analyses revealed that the front-back asymmetry in the traction distribution correlates with the direction of migration. We also found that A. proteus exhibits a periodic pattern in which inward-directed stresses are alternately strengthened and weakened at the cell poles. Crucially, we identified a distinctive feature not observed in other cell types: the generation of large lateral traction forces at the cell center. Together, these results highlight both the universality and diversity of the biophysical mechanisms driving amoeboid locomotion.

The development and physiology of neurons rely on their microtubule organization, which is characterized by plus-end-out oriented microtubules in the axon and a mix of plus-end-out and plus-end-in oriented microtubules in dendrites. This orientational pattern is established early in neuronal development and is tightly linked to axon-dendrite differentiation. Even though multiple potentially relevant mechanisms have been proposed, fundamental questions remain: How does the microtubule organization in neurons emerge, and how does a neuron develop a single axon and multiple dendrites? Here, we address these questions at two distinct, complementary levels: at a higher level by proposing a conceptual framework, in which we classify mechanisms into three categories based on how they contribute to the microtubule organization: orientational bias, parallel amplification, and polarization; at a lower level we build a biophysical model that incorporates multiple mechanisms of microtubule dynamics in a neuron, from which, using analytical calculations and simulations, we derive insights into the emergence of microtubule organization in developing neurons. We show that geometrical effects alone can confer a bias in microtubule orientation. Parallel amplification then enhances the resulting polarity. Coupling multiple neurites to a common cell body that serves as a shared reservoir of resources allows for a polarization mechanism that ensures that the microtubule organization of one neurite becomes axonal while all others are dendritic. This framework unifies diverse molecular observations and yields experimentally testable predictions about microtubule self-organization in early neuronal development. Author summaryNeurons communicate through long protrusions called neurites, which are of two types: dendrites, which receive signals, and axons, which send signals. Their development relies primarily on microtubules, which are polar filaments with two distinct ends, known as the plus and minus ends. Microtubules self-organize into functional architectures that are significantly different between axons and dendrites. In axons, all microtubules point their plus end away from the cell body, whereas in dendrites, they point either towards the cell body or have mixed orientations depending on the species. This orientation guides intracellular transport by motors and is closely linked to whether a neurite develops into an axon or a dendrite. Despite decades of research identifying individual mechanisms, the bigger picture behind the emergence of microtubule orientation in neurons remains unclear. Here, we construct a conceptual framework and a biophysical model to identify the principles underlying the emergence of microtubule orientation in developing neurons. Our conceptual framework provides a high-level perspective on how individual mechanisms influence microtubule organization in neurites. In our concrete biophysical model, we study a selection of mechanisms to gain specific, quantitative insight into the organizational process. We propose a minimal model of a neuron that exhibits neuronal polarization, giving rise to a single axon-like neurite and multiple dendrite-like ones, consistent with experimental observations. This in silico neuron helps to explain how neurons break symmetry during development and provides a systematic way to generate and test new hypotheses about neuronal polarity.

Epithelial sheets must maintain robust barrier function while enduring severe mechanical deformations across various physiological environments. While baseline actomyosin contractility is understood to stabilize intercellular junctions and hence cell-cell contact integrity, how cell-generated active forces interact with external physical strain to dictate contact integrity remains poorly understood. In this study, we investigated the biophysical trade-offs between actomyosin contractility and barrier resilience when Madin-Darby Canine Kidney (MDCK) cell islands are subject to large stretch. In contrast to a high concentration (50 M) of the non-muscle myosin II inhibitor blebbistatin that disrupted cell-cell contacts, we first identified a lower concentration (10 M) that maintained cell-cell contact integrity in the absence of any stretch. Such moderate inhibition of non-muscle myosin II reduced, but preserved some level of actin bundle organization. Remarkably, when challenged with a pathological 38% linear stretch using a custom-built biaxial stretching device, 10 M blebbistatin treated epithelial islands exhibited significantly fewer cell-cell contact ruptures than untreated controls, demonstrating a potent protective effect against mechanical strain. Traction force microscopy revealed diminished cell-generated strain energy by over 60% indicating a partial but significant reduction in contractility upon 10 M blebbistatin treatment. Nanoindentation measurements revealed that moderate contractility inhibition decreased the cellular Young's modulus by more than 40%. Consequently, moderate contractility inhibition safeguards epithelial junctions through a dual mechanical effect: it simultaneously reduces baseline active tensile stresses due to cell contractility and lowers the passive elastic forces generated within the softened cell island during external stretch. Our findings indicate that this systemic reduction in forces dominates over any loss of biochemical adhesion strength at cell-cell contacts. We propose that shifting the epithelium from a rigid, highly stressed continuum to a more compliant, relaxed state by moderate contractility inhibition can serve as a general biophysical mechanism to preserve barrier integrity under severe mechanical challenge.

Focal adhesions maintain force-bearing attachment between cells and the extracellular matrix but can also undergo dynamic remodeling. Their assembly and actomyosin tension are coupled through mechanochemical feedback. The processes underlying this feedback are not instantaneous and therefore involve a time delay. However, how this delayed feedback gives rise to stable adhesion maintenance or dynamic remodeling remains unclear. Here, paired time-lapse measurements of vinculin fluorescence and traction stress revealed distinct local adhesion-force dynamics, including low-fluctuation and recurrent fluctuation patterns. To examine how these patterns could arise, we formulated a minimal mechanochemical model coupling focal adhesion assembly and actomyosin force through delayed reciprocal feedback. The model exhibited stable and oscillatory modes depending on feedback strength, the balance of opposing feedback effects, and the effective feedback delay. Bistability and hysteretic switching also occurred in a subset of parameter space, and the oscillation period followed a power-law relation with the delay. These results suggest that stable adhesion maintenance and dynamic remodeling can emerge from a common mechanochemical feedback architecture.

The {beta}-cardiac myosin (MYH7) mutation E525K was first identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered myosin constructs has shown that this mutation causes hypocontractility by stabilizing the interacting heads motif (IHM) of myosin despite the mutant E525K motor head exhibiting increased ATPase activity. However, no measurements have been made in myofilaments or cardiomyocytes to determine how this mutation affects contractile function. Here, we present force and contractile kinetics measurements from induced pluripotent stem cell (iPSC)-derived cardiomyocytes engineered for heterozygous expression of E525K. Contraction of E525K single cells decreased by 65%, and isometric twitch force in engineered heart tissues (EHTs) decreased by 39%. In contrast, maximal isometric force in isolated myofibrils increased by 45%. Structural analysis revealed reduced myofibril content (13.7% decrease) and organization (increased z-disk dispersion angle) in E525K cells. We confirmed that E525K S1 myosin has higher actin affinity than WT S1 and elevated ATPase activity. However, no change was observed in the rate of ADP release. Importantly, there was no change in the rate of force development or relaxation in myofibrils, cells, or EHTs. These findings suggest that myosin crossbridge cycling is not altered under load by E525K. Decreased force generation in EHTs and shortening in cardiomyocytes arise from reduced sarcomere number and myofibrillar disorganization. Additional force deficits likely result from stabilization of the IHM, as recently reported by others. This study demonstrates the value of multi-scale analysis for determining the functional profile of cardiomyocytes containing disease-related sarcomere protein mutations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/733270v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@5f85f5org.highwire.dtl.DTLVardef@153b3b6org.highwire.dtl.DTLVardef@3b8f21org.highwire.dtl.DTLVardef@31d323_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: A Model for how the E525K mutation impacts contracting myofibrils Here we have shown that the E525K mutation impacts contraction in three ways: (1) Decreased sarcomere organization in cells and tissues drives decreased force generation. (2) Increased binding affinity of E525K myosin for actin contributes to increased force generation in isolated myofibrils. (3) Increased IHM stability. (4) The rate limiting step of loaded crossbridge cycling, ADP release, is unchanged by the E525K mutation and the rate of loaded contraction and relaxation is unchanged at all scales of contraction measured here. C_FIG

Kinesins are molecular motor proteins essential for organizing and remodeling the cytoskeleton during neuronal development and maintenance. One key regulator is kinesin family binding protein (KIFBP), which inhibits a subset of kinesins by blocking motor-microtubule interactions. Homozygous mutations in KIFBP cause Goldberg-Shprintzen Syndrome (GOSHS), a neurodevelopmental disorder characterized by intellectual disability, microcephaly, and axonal neuropathy. Although loss of KIFBP has been linked to reduced neurite length and microtubule disorganization, the specific kinesins underlying these phenotypes remain unclear. Here we use a CRISPR-Cas9 generated KIFBP knockout Neuro-2a cell line to demonstrate that KIFBP is required for neurite extension and use inducible GFP-KIFBP to define the KIFBP interactome during neuronal differentiation. Immunoprecipitation coupled with mass spectrometry identified both known and novel KIFBP-associated kinesins. Single molecule TIRF microscopy confirmed direct inhibition of a subset of kinesins that co-immunoprecipitated with KIFBP. Notably, we identified KIF5A and KIF18B as previously unrecognized regulatory targets with potential roles in neuronal development. Together, these findings establish Neuro-2a cells as a model for studying KIFBP function and provide new insight into the regulation of kinesin activity and cytoskeletal dynamics in neurons.

During cell migration, branched actin filaments nucleated by the Arp2/3 complex induce leading edge protrusions, whereas directionality of cell migration is controlled by microtubules. We showed previously that Adenomatous Polyposis Coli (APC) initiates branched actin assembly at microtubule tips, which can explain how microtubules control directional protrusion. Here, we investigate a link from APC to Arp2/3 complex activity. We show that protrusion-generating activity of APC resides in its N-terminal region containing the Armadillo Repeat Domain (ARD). Furthermore, Asef1/ARHGEF4, a Cdc42 GEF known to be activated by the ARD of APC, as well as Cdc42 itself and its effector N-WASP, an Arp2/3 complex activator, are all required for the assembly of branched actin filaments in neuronal growth cones and neurite outgrowth. As downregulation of Asef1, Cdc42 or N-WASP produces phenotypes similar to APC knockdown, we propose that microtubules regulate directional cell migration and neuron navigation by inducing local membrane protrusion through the APC - Asef1 - Cdc42 - N-WASP - Arp2/3 pathway. These findings bridge a significant gap in our knowledge of cell migration.