Cytoskeleton

Actin-bundle organization is essential for vascular smooth muscle cell mechanics and is implicated in actin-related diseases. However, it remains unclear how cell stretching affects intracellular actin bundles when actin polymerization is impaired. Here, we performed live imaging of Latrunculin A-treated A7r5 vascular smooth muscle cells in a stretch chamber. GFP--actinin imaging showed that Latrunculin A reduced actin-bundle coverage while periodicity was maintained. Subsequent mechanical stretch disrupted both actin-bundle coverage and periodicity. We constructed a stochastic filament bundle model in which actin filament length, actin crosslinking protein dynamics, external stretch, and myosin-driven contractile shortening determine bundle connectivity. The model generated non-spanning, collapse, and persistent states based on spanning connectivity before and after stretch, shaped by filament length and applied strain. A reduced model further showed that these states are governed by a balance between connectivity formation and stretch-induced loss. Together, our results suggest that reduced actin polymerization destabilizes intracellular actin-bundle organization under mechanical stretch, providing a mechanism linking actin polymerization defects to mechanical fragility in vascular smooth muscle cells.

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

Missense mutations in the TPM2 gene encoding skeletal muscle tropomyosin Tpm2.2 cause congenital myopathies associated with hyper- and hypocontractile phenotypes. Mutation-dependent defects in thin filament stability and length maintenance may contribute to sarcomere dysfunction. To address this possibility, four disease-associated substitutions in Tpm2.2 were analyzed: hypercontractile D20H and E181K, and hypocontractile E41K and N202K. Recombinant proteins were examined in vitro for their effects on actin filament polymerization, stability, and cofilin-2-dependent filament length regulation in the absence and presence of troponin (+Ca2+). Wild-type Tpm2.2 inhibited spontaneous actin polymerization and reduced polymerization cooperativity in the presence of cofilin-2. Hypercontractile substitutions D20H and E181K further decreased the polymerization rate, whereas hypocontractile variants had little effect. Under ATP-driven actomyosin interactions, E41K and N202K stabilized filaments, resulting in increased filament length, but this effect was abolished by troponin. All variants slightly decreased cofilin-2 affinity for F-actin without affecting cooperativity. Troponin prevented displacement of Tpm2.2 from the filament at increasing cofilin-2 occupancy, indicating concomitant binding of all proteins to the thin filament, consistent with a structural model based on high-resolution F-actin-Tpm-Tn and cofilactin structures.Tpm2.2-N202K inhibited cofilin-2-dependent depolymerization, whereas Tpm2.2-E181K increased susceptibility to depolymerization. Although cofilin-2 induced filament severing in all cases, the Tpm2.2-Tn complex protected filaments from disassembly. These findings support a model in which the Tpm2.2-Tn complex forms a cooperative regulatory strand that constrains filament dynamics and transmits structural perturbations along the filament. Disease-causing substitutions differentially alter filament length and stability, potentially contributing to the pathogenesis of myopathies.

A hallmark of damaged skeletal muscle fibers is displaced myonuclei that are no longer peripherally positioned. Displaced myonuclei are dogmatically thought to be derived exclusively from muscle stem cell (satellite cell) fusion. Using a surgical resection muscle injury model and in vivo recombination-independent resident myonuclear labeling, we detail the prevalence, time course, and origin of displaced myonuclei in response to a non-chemically-mediated muscle trauma. We found that: 1) non-satellite cell-derived (resident) displaced myonuclei emerge seven days after surgical injury in similar proportion to exogenous (satellite cell-derived) displaced myonuclei in intact muscle fibers, with a biased prevalence in myosin heavy chain IIB muscle fibers, 2) muscle fibers with multiple ([≥]2) displaced resident myonuclei was an unexpected but noteworthy feature of muscle fibers 7 days after injury, 3) embryonic myosin-expressing fibers at seven days post-surgery expectedly contain predominantly satellite-cell derived displaced myonuclei, but a subset have displaced resident myonuclei, and 4) satellite cell numbers in intact muscle do not increase until 7 days post-surgery. These data may help inform whether to target satellite cell-initiated processes, myonuclear-initiated processes, or both to facilitate muscle fiber injury repair. This information could lead to more effective therapeutic strategies for treating muscle trauma.

Increased mechanical loading induces skeletal muscle growth and, at the ultrastructural level, promotes myofibrillogenesis and the radial growth of myofibrils. However, the mechanisms regulating these ultrastructural adaptations are not known. Here, we sought to determine whether the mechanistic target of rapamycin complex 1 (mTORC1) regulates these processes. To accomplish this, muscle-specific, tamoxifen-inducible raptor knockout (iRAmKO) mice were used to inhibit signaling through mTORC1, and growth was induced with a model of chronic mechanical overload (MOV). Using a next-generation fluorescence imaging pipeline for ultrastructural analyses, we found that mTORC1 is a critical regulator of the myofibrillogenesis and radial growth of myofibrils that occur in response to MOV. Together with other recent advances in the field, we propose a model in which mTORC1 acts as a gatekeeper that permits the retention, rather than the synthesis, of proteins that drive the ultrastructural adaptations.

GFAP is a type III intermediate filament primarily found within astrocytes and is known to maintain proper cell structure and mechanical strength. Mutations in GFAP are implicated in the pathology of Alexander disease, a neurodegenerative disease characterized by cytoplasmic inclusions of protein, known as Rosenthal fibers. GFAP has a typical type III intermediate filament domain structure, consisting of a highly conserved alpha-helical rod domain bracketed by an intrinsically disordered N-terminal head and C-terminal tail domains. While the general domain organization of monomeric GFAP and the assembly process for higher order quaternary structures are known, we lack an atomic resolution mechanistic understanding of GFAP assembly into mature filaments. Understanding the structure of GFAP filaments and how mutations disrupt this structure will provide vital information into how mutations produce Alexander disease pathology. As a first step towards a mechanistic description, we characterized GFAP wild type tetrameric and filamentous assemblies using solid state NMR and compared the results to those obtained from an assembly-deficient GFAP mutant. For wild-type GFAP, we observe surprisingly uniform rigid alpha helical structure and can spectroscopically resolve highly mobile intrinsically disordered regions in the filament assemblies. Wild type tetramers show increased mobility, likely arising from the head and tail domains. Mutation of the highly conserved cysteine at position 294 to serine results in an inability to form full-length filament assemblies. We show that the rigid regions of the C294S mutant assemblies largely remain structurally consistent with wild type tetrameric assemblies but differ from wild-type filament assemblies. There is an increase in highly mobile regions for the C294S mutant relative to the wild-type. Our results provide a foundation for developing solid state NMR approaches to characterize intermediate filament assembly mechanisms and the interfering effect of disease mutations.

Cell size in a proliferating cell population generally varies over a limited range ([~]2-4-fold). Within such populations, organelle content increases with cell size maintaining a relatively constant organelle density (amount per cell volume). However, cells of different types can differ greatly in cell size as well as in organelle composition. In such cases, it is often unclear to what degree, if any, the differences in organelle composition are due to the difference in cell size. In principle, this issue could be resolved by examining situations where a proliferating population of cells of the same cell type exhibit much greater size variation. Here we characterize how organelle content scales with cell volume in the polymorphic fungus, A. pullulans, whose proliferating cells span a [~]100-fold size range. We find that mitochondria and ER content increases in proportion to cell volume, while this is not the case for vacuoles and peroxisomes. Thus, organelle composition is affected by cell size in this system.

Sarcomeres, the basic repeating unit of striated muscle, are joined together by crosslinked actin filaments found at the boundaries of muscle sarcomeres, termed Z-discs. Z-discs play a key role in cardiac signalling and disease, however, the arrangement and function of many of the proteins present in the Z-disc remain to be understood. Here, we determined the organisation of 3 key proteins, ZASP, [a]-Actinin-2 and the Z1Z2 epitope of titin, located within the Z-disc. We fluorescently labelled these proteins in cardiac myofibrils using Adhirons specific to each protein and used interferometric photoactivated localization microscopy (iPALM) to obtain the 3D position of these proteins to a high precision (<10nm in x,y,z). We then used PERPL (Pattern Extraction from Relative Positions of Localisations) to analyse patterns in the relative positions of the proteins and reveal their underlying organisation. This analysis revealed that ZASP and [a]-Actinin-2 have a similar repeating organisation, but that the organisation of Z1Z2 is different.

Mechanical properties of epithelial tissues play essential roles in morphogenesis and physiological function. In this study, we analytically derived the in-plane bulk modulus, shear modulus, and Poissons ratio of a three-dimensional cell vertex model of epithelial monolayers. We showed that the model can robustly reproduce a near-zero in-plane Poissons ratio, a mechanical feature reported in cultured epithelial tissues. Numerical simulations further confirmed that the theoretically predicted Poissons ratio accurately describes the response of the model under finite, biologically relevant strains. In addition, the model exhibits not only morphological bistability between squamous-like and columnar-like states, but also mechanical bistability characterized by distinct elastic responses. Together, these results provide a minimal three-dimensional framework that links cell-scale mechanical interactions and epithelial morphology to tissue-scale elastic properties.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Cell migration is fundamental to various biological processes, including morphogenesis, wound healing, and cancer metastasis. Durotaxis--directed migration of cells in response to spatial variations in stiffness--has been extensively studied using engineered substrates with prescribed stiffness. However, recent work has increasingly shifted toward understanding cell migration in fibrous matrices that can be actively remodeled by the actomyosin contractility, as commonly observed in tumor and epithelial cells. Despite these advances, a theoretical framework explaining how cells structurally remodel their surrounding matrix to promote their own durotaxis, and which cellular forces govern this behavior, remains elusive. To address this gap, we developed a biomechanical model in which polarized cells contract and migrate over a fibrous matrix. Using this model, we first confirmed that cells on an externally strained matrix preferentially migrate along the direction of applied strain. Then, we investigated how cells autonomously remodel the matrix to create stiffness patterns favorable for durotaxis. In the presence of intercellular adhesion, cells acted collectively to stiffen the matrix, after which a small subset of cells escaped the main population and migrated outward. This behavior is reminiscent of intravasation during cancer metastasis, where cohesive cell clusters generate local matrix remodeling that facilitates the departure of more motile subpopulations. These results illustrate how matrix stiffening driven by cell cohesion and contractility regulates durotactic behavior and provide mechanistic insight into collective invasion processes relevant to cancer metastasis.

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

Chromosome segregation fidelity during meiosis is critical for genome integrity, with aneuploidy causing infertility, miscarriages, and congenital anomalies. In the oocytes of many species, spindle assembly occurs in the absence of centrosomes that normally function as microtubule-organizing centers at the poles. Such acentrosomal spindles are believed to pose significant challenges for accurate chromosome segregation compared to centrosomal organized spindles. Previous work in Drosophila has shown that the chromosomal passenger complex (CPC) is required for acentrosomal spindle assembly. We found that heterochromatin protein-1 (HP1) plays a critical role in regulating CPC localization and spindle assembly. Furthermore, HP1 moves to the microtubules, where it has roles in building a functional spindle and interacts with the CPC to regulate chromosome biorientation. These results indicate that spindle assembly is mediated by multiple interactions between the CPC, HP1, and the chromosomes, and provide insights into the mechanisms that restricts spindle assembly to the chromosomes in Drosophila oocytes.

Idiopathic scoliosis is a common spinal disorder characterized by progressive three-dimensional curvature of unknown cause. Although biomechanical imbalance has long been proposed to contribute to scoliosis, the early physiological states that precede curvature onset remain poorly understood. Here, we investigated this problem using zebrafish uts2r3 mutants, which develop fully penetrant juvenile-onset spinal curvature following disruption of urotensin signaling. Transcriptomic analysis before curvature revealed altered expression of muscle-associated genes, suggesting that Uts2r3 influences axial muscle development or function. However, immunofluorescence, birefringence imaging, and quantitative analysis of myotome morphology showed that mutants lack overt muscle architectural defects or dystrophic pathology. By contrast, direct measurements of isolated larval trunks revealed pre-curvature biomechanical abnormalities: namely, uts2r3 mutants generated reduced active force following electrical stimulation while also exhibiting increased passive resistance to stretch. These findings identify urotensin signaling as a regulator of axial tissue biomechanics during growth and suggest that scoliosis-like curvature can arise from an early imbalance between active force generation and passive tissue stiffness. SignificanceSpinal curvature is common, but the biological events that cause the spine to bend during growth remain poorly understood. Animal models, especially zebrafish, make it possible to study these events before curvature begins. Zebrafish lacking urotensin signaling develop spinal curves that arise during juvenile growth, similar to idiopathic scoliosis in humans. Here, we demonstrate that zebrafish lacking the urotensin pathway receptor Uts2r3 develop an abnormal biomechanical state prior to curve onset. Their axial tissues generate less active force when contracting and, at the same time, show increased passive resistance to stretch--an unexpected combination that reveals a distinct pre-curvature biomechanical state. These findings suggest that spinal curvature can arise from an early imbalance in tissue mechanics during growth and identify urotensin signaling as a pathway that helps preserve spinal morphology through a biomechanical mechanism.

Myosin 3A (MYO3A) is an unconventional myosin involved in the formation and maintenance of hair-cell stereocilia of the sensory epithelia in the inner ear. The kinase domain has been implicated in phosphoregulation of MYO3A activity through intermolecular autophosphorylation. Previous studies using mass spectrometry identified two potential phosphorylation sites in the motor domain. To investigate the regulatory roles of these sites, we generated glutamic acid point mutations in our mchr-MYO3A{Delta}K construct to mimic phosphorylation and assayed the constructs for their ability to tip-localize and influence filopodial density via transfection into COS7 cells. The phosphomimic constructs were less able to generate filopodia when compared to wildtype constructs. To gain a better understanding of the phosphoregulation of MYO3A, we transfected COS7 cells with mchr-MYO3A{Delta}K in combination with GFP-tagged full-length MYO3A (GFP-MYO3AFL), or GFP attached to just the kinase domain of MYO3A (GFP-MYO3AKIN). Coexpression of mchr-MYO3A{Delta}K with either construct resulted in decreased mchr-MYO3A levels at the tips of filopodia and fewer filopodia at the edge of the cell, compared to cells expressing mchr-MYO3A{Delta}K alone. This implies that the kinase domain does not require motor activity to contribute to phosphoregulation of MYO3A, and that MYO3A phosphoregulation may be influencing filopodia initiation. Informatic analyses and structural predictions suggest that the two phosphorylation sites in the motor domain inhibit actin/MYO3A interactions. Taken together, these analyses link MYO3A phosphorylation with the regulation of its ability to create actin protrusions such as filopodia and stereocilia.

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

Skeletal muscle fibers are among the largest cells in the body and rely on the spatial distribution of numerous nuclei to maintain intracellular function across vast cytoplasmic volumes. How nuclear organization adapts when nuclear number is reduced remains unclear. Here, we analyzed three-dimensional myonuclear positioning during postnatal development in a mouse model with impaired myonuclear accretion. Across more than 1,000 fibers and nearly 15,000 nuclei, reduced myonuclear number led to increased spatial regularity, including more even longitudinal spacing, lower variability in inter-nuclear distances, and the emergence of a preferred inter-nuclear spacing. These changes persist after accounting for fiber size and nuclear density, indicating an active reorganization rather than a passive geometric consequence. Mapping myonuclei onto the fiber surface further revealed more uniform and locally ordered two-dimensional organization, although this surface pattern was largely explained by the improved longitudinal spacing. Together, these results show that muscle fibers do not simply tolerate fewer nuclei, but actively adapt myonuclear positioning in a manner that promotes efficient cytoplasmic coverage across large cellular dimensions.

The microtubule and actin cytoskeletons form dynamic, interconnected networks that are critical for eukaryotic cell function. These networks govern intracellular organization, cargo transport, cell migration, and tissue morphogenesis. Microtubules and actin filaments are regulated by diverse binding proteins that control many aspects of their function. However, identifying cytoskeletal-interacting proteins has been challenging due to the transient and weak nature of many interactions and the disruption of native architecture by conventional biochemical approaches. These limitations suggest that numerous physiologically relevant cytoskeletal regulators remain undiscovered. Identifying these factors requires novel and sensitive methodologies that can capture cytoskeletal interactions under native cellular conditions. Here, we present MT-ID and Act-ID, powerful proximity-labeling tools for identifying microtubule and actin-interacting proteins, respectively. MT-ID employs the microtubule-binding domain of MAP7 (EMTB) fused to TurboID, a highly active promiscuous biotin ligase. Act-ID utilizes the actin-binding domain of ITPKA (F-tractin) similarly fused to TurboID. We validate both approaches by successfully identifying numerous known cytoskeletal regulators and discovering potentially novel interacting proteins. Functional characterization reveals that LIMCH1 is a previously unrecognized microtubule-associated protein whose depletion increases microtubule density. Additionally, we identify FBXO30 as a novel actin-interacting protein, with its loss promoting increased focal adhesion formation. MT-ID and Act-ID will be useful not only to identify cytoskeletal interacting proteins but also to define changes to the cytoskeletal interactome when cells are exposed to changing physiological conditions.

The Ezrin, Radixin, and Moesin (ERM) family of proteins anchors the actin cytoskeleton to the plasma membrane for the purpose of either stabilizing or altering cell shape. In Caenorhabditis elegans, ERM-1, is essential for cell polarity, signaling, intestine development, and larval viability. Interestingly, ERM-1 proteins are produced by erm-1 mRNA transcripts that concentrate at the plasma membrane in embryos. The localization of erm-1 mRNA to the plasma membrane occurs in a 3UTR-independent, translation-dependent manner, directed by the PH-subdomain within ERM-1s N-terminal FERM domain. This has led to the model that erm-1 mRNA, its associated ribosome, and its emerging nascent peptide are all transported together to the plasma membrane as a complex. Here, we characterize the transport mechanism. Using a microscopy approach, we observed that the localizations of erm-1 mRNA and ERM-1 protein to the plasma membrane were disrupted by nocodazole treatment, illustrating a microtubule role. Furthermore, erm-1 mRNA and ERM-1 protein localized to the plasma membrane independently of myosin and dynein motors, but dependent on the kinesin bmk-1 (bmk-1), a plus-end-directed, Kinesin-5 family motor protein. Loss of bmk-1 did not reduce the total number of erm-1 mRNA molecules in the cell, arguing against a diffusion- and protection-based mechanism of mRNA localization. Together, these findings suggest that erm-1 mRNA is localized via an active transport pathway mediated by a plus-end-directed kinesin adapter. Interestingly, loss of bmk-1 led to diffuse localization of ERM-1 protein along the plasma membrane and reduced ERM-1 protein levels at the site of abscission, the midbody, and the midbody remnant. This suggests that ERM-1 local translation at the plasma membrane is critical for its proteins ultimate spatial patterning in the cell.

Take-off is a fast and energy-efficient strategy for bipedal animals, such as birds, to achieve rapid movement; however, how muscle physiology scales to govern this universal behavior remains unresolved. Research in other species physiologies is not readily applicable. As a result, important questions, whether theropod dinosaurs such as Tyrannosaurus rex were capable of jumping, remain unanswered. In this article, we coupled Lagrangian dynamics with Hills muscle equations and developed new experimental methods to quantify joint rotational stiffness and damping, thereby enabling a systematic description of lower-limb mechanics. The approach establishes a novel kinetic framework that links muscle contractile properties to lower-limb performance without invoking control optimization. Animal observations and tabletop mechanisms validate the framework. The mechanics model reveals that the take-off time of about 0.1 s across body masses of 0.003 to 90 kg is achievable, as heavier birds generate proportionally higher reaction forces. Additionally, Tyrannosaurus rex should be capable of jumping, based on the available physiology data. Beyond evolutionary insights, our framework provides a new methodology for analyzing the mechanical properties of biological joints and informing the design of scalable bio-inspired robots.