Stress Fiber Orientation Research Articles

Experimental and numerical study of the effects of fiber orientation on stress concentration of CFRE

Carbon fiber reinforced composites are broadly employed, as structural members, in many industrial applications especially in the avionic industry due to their high strength-to-weight ratio. Circular holes are commonly used in these members for fastening, bearing, reducing weight, and others. Therefore, several experimental and analytical studies were introduced to figure out the stress concentration behavior around the vicinity of the holes under different loading conditions. However, no reports are delivered to study the effect of fiber orientation on the stress concentration factor of unidirectional carbon fiber reinforced epoxy (CFRE) composites with a hole under tension. Thus, the current work is targeted to understand the relationship between stress concentration factor (SCF) and fiber orientation in CFRE orthotropic plates. The unnotched and notched composite samples, with four different fiber orientations, were experimentally investigated in a tension test. The SCF of samples was calculated using analytical and computational FE methods. The failure criterion of open-hole tensile (OHT) test samples was also analyzed. The experimental results demonstrated that there is no significant effect of fiber orientation on the normalized strength. Yet based on the stiffness matrix of isotropic materials, a pronounced effect of the fiber orientation on the SCF is detected when using analytical calculations. The present research arises the need for further investigations to find a correlation between experimental and analytical results of SCF in unidirectional CFRE with a central hole at different fiber orientations.

An Inverse Modeling Approach to Estimate Three-Dimensional Aortic Valve Interstitial Cell Stress Fiber Force Levels.

Within the aortic valve (AV) leaflet exists a population of interstitial cells (AVICs) that maintain the constituent tissues by extracellular matrix (ECM) secretion, degradation, and remodeling. AVICs can transition from a quiescent, fibroblast-like phenotype to an activated, myofibroblast phenotype in response to growth or disease. AVIC dysfunction has been implicated in AV disease processes, yet our understanding of AVIC function remains quite limited. A major characteristic of the AVIC phenotype is its contractile state, driven by contractile forces generated by the underlying stress fibers (SF). However, direct assessment of the AVIC SF contractile state and structure within physiologically mimicking three-dimensional environments remains technically challenging, as the size of single SFs are below the resolution of light microscopy. Therefore, in the present study, we developed a three-dimensional (3D) computational approach of AVICs embedded in 3D hydrogels to estimate their SF local orientations and contractile forces. One challenge with this approach is that AVICs will remodel the hydrogel, so that the gel moduli will vary spatially. We thus utilized our previous approach (Khang et al. 2023, "Estimation of Aortic Valve Interstitial Cell-Induced 3D Remodeling of Poly (Ethylene Glycol) Hydrogel Environments Using an Inverse Finite Element Approach," Acta Biomater., 160, pp. 123-133) to define local hydrogel mechanical properties. The AVIC SF model incorporated known cytosol and nucleus mechanical behaviors, with the cell membrane assumed to be perfectly bonded to the surrounding hydrogel. The AVIC SFs were first modeled as locally unidirectional hyperelastic fibers with a contractile force component. An adjoint-based inverse modeling approach was developed to estimate local SF orientation and contractile force. Substantial heterogeneity in SF force and orientations were observed, with the greatest levels of SF alignment and contractile forces occurring in AVIC protrusions. The addition of a dispersed SF orientation to the modeling approach did not substantially alter these findings. To the best of our knowledge, we report the first fully 3D computational contractile cell models which can predict locally varying stress fiber orientation and contractile force levels.

Three-dimensional analysis of hydrogel-imbedded aortic valve interstitial cell shape and its relation to contractile behavior.

Cell-shape is a conglomerate of mechanical, chemical, and biological mechanisms that reflects the cell biophysical state. In a specific application, we consider aortic valve interstitial cells (AVICs), which maintain the structure and function of aortic heart valve leaflets. Actomyosin stress fibers help determine AVIC shape and facilitate processes such as adhesion, contraction, and mechanosensing. However, detailed 3D assessment of stress fiber architecture and function is currently impractical. Herein, we assessed AVIC shape and contractile behaviors using hydrogel-based 3D traction force microscopy to intuit the orientation and behavior of AVIC stress fibers. We utilized spherical harmonics (SPHARM) to quantify AVIC geometries through three days of incubation, which demonstrated a shift from a spherical shape to forming substantial protrusions. Furthermore, we assessed changes in post-three day AVIC shape and contractile function within two testing regimes: (1) normal contractile level to relaxation (cytochalasin D), and (2) normal contractile level to hyper-contraction (endothelin-1). In both scenarios, AVICs underwent isovolumic shape changes and produced complex displacement fields within the hydrogel. AVICs were more elongated when relaxed and more spherical in hyper-contraction. Locally, AVIC protrusions contracted along their long axis and expanded in their circumferential direction, indicating predominately axially aligned stress fibers. Furthermore, the magnitude of protrusion displacements was correlated with protrusion length and approached a consistent displacement plateau at a similar critical length across all AVICs. This implied that stress fiber behavior is conserved, despite great variations in AVIC shapes. We anticipate our findings will bolster future investigations into AVIC stress fiber architecture and function. STATEMENT OF SIGNIFICANCE: Within the aortic valve there exists a population of aortic valve interstitial cells, which orchestrate the turnover, secretion, and remodeling of its extracellular matrix, maintaining tissue integrity and ultimately sustaining the proper mechanical function. Alterations in these processes are thought to underlie diseases of the aortic valve, which affect hundreds of thousands domestically and world-wide. Yet, to date, there are no non-surgical treatments for aortic heart valve disease, in part due to our limited understanding of the underlying disease processes. In the present study, we built upon our previous study to include a full 3D analysis of aortic valve interstitial cell shapes at differing contractile levels. The resulting detailed shape and deformation analysis provided insight into the underlying stress-fiber structures and mechanical behaviors.

Coordinated regulation of Cdc42ep1, actin, and septin filaments during neural crest cell migration.

The septin cytoskeleton has been demonstrated to interact with other cytoskeletal components to regulate various cellular processes, including cell migration. However, the mechanisms of how septin regulates cell migration are not fully understood. In this study, we use the highly migratory neural crest cells of frog embryos to examine the role of septin filaments in cell migration. We found that septin filaments are required for the proper migration of neural crest cells by controlling both the speed and the direction of cell migration. We further determined that septin filaments regulate these features of cell migration by interacting with actin stress fibers. In neural crest cells, septin filaments co-align with actin stress fibers, and the loss of septin filaments leads to impaired stability and contractility of actin stress fibers. In addition, we showed that a partial loss of septin filaments leads to drastic changes in the orientations of newly formed actin stress fibers, suggesting that septin filaments help maintain the persistent orientation of actin stress fibers during directed cell migration. Lastly, our study revealed that these activities of septin filaments depend on Cdc42ep1, which colocalizes with septin filaments in the center of neural crest cells. Cdc42ep1 interacts with septin filaments in a reciprocal manner, with septin filaments recruiting Cdc42ep1 to the cell center and Cdc42ep1 supporting the formation of septin filaments.

High throughput image processing software for analyzing hiPSC-CM sarcomere physiology and cardiac fibroblast activation.

Heart disease is the leading cause of death worldwide and discovering therapies that treat its underlying cause is paramount to improving survivability and patient health. Since heart disease often results in detrimental remodeling of heart tissue, high-throughput screening (HTS) using cardiomyocytes and cardiac fibroblasts is frequently used for drug discovery. A major challenge in analyzing HTS data is ensuring that analysis is both fast and minimally biased. In many forms of heart disease and remodeling, cardiomyocytes will suffer from loss of sarcomeres and sarcomere disarray, while fibroblasts will often express increased α-smooth muscle actin (α-SMA) stress fibers when activated into myofibroblasts. Two custom MATLAB HTS image processing scripts named ‘Tamarack’ and ‘Porcupine’ were developed for quantifying human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) sarcomere morphology and cardiac fibroblast activation, respectively. Both scripts use wavelet transforms to detect subcellular structures that are of interest for analyzing phenotypes at the cellular level. Tamarack enables quantification of sarcomere metrics, including sarcomere count, length, orientation, and α-actinin rich areas. Porcupine enables quantification of fibroblast activation metrics, including α-SMA stress fiber count, length, orientation, nuclei and α-SMA overlap, and F-actin and α-SMA overlap. Both scripts enable analysis of other cellular features, such as nuclei count and stain intensity/protein expression. Tamarack obtained statistically significant results when comparing control hiPSC-CMs with hiPSC-CMs subjected to siRNA knockdowns of genes of interest. Porcupine was validated by analyzing fibroblasts with increasing concentrations of TGF-β which induced increasing activation. By developing technology for analyzing cardiomyocyte and fibroblast imaged-based screens, drug candidates can be efficiently evaluated without bias on cell types that are relevant to human heart disease. This research may help expand drug candidate portfolios and improve the number of treatments available to patients worldwide.

Cell Stretcher Assay to Analyze Mechanoresponses to Cyclic Stretching.

In their natural environment, most cells and tissues are continuously exposed to cyclic mechanical strain. Sensing these stimuli by mechanosensory proteins and subsequent conversion into a variety of biological responses (referred to as mechanotransduction) are key processes for tissue homeostasis, survival, and differentiation. Perturbations of underlying signaling pathways lead to severe diseases in vivo (Urciuoli E, Peruzzi B, Int J Mol Sci 21(24). https://doi.org/10.3390/ijms21249426, (2020)). In addition, cellular mechanoresponses to cyclic stretching of an isolated single cell differ from those of a cell monolayer, network, or even three-dimensional tissue. Since these processes depend on various physical and biological parameters, the development of a precise, well-characterized, and highly reproducible but also easily tunable stretcher assay is indispensable. Here, we describe the fabrication of defined elastic substrates and their application in cyclic stretching of cultured cells in a custom-made cell stretcher device. We focus on the detailed description of the system and provide a possibility for mechanoresponse characterization, using the analysis of actin stress fiber orientation as exemplary mechanoresponse to cyclic stretching of adherent cells.

Modeling the Effect of Stress Ratio, Loading Frequency and Fiber Orientation on the Fatigue Response of Composite Materials.

Fatigue life models are widely used to predict the fatigue behavior at arbitrary cycle counts of composite structures subjected to cyclic or highly dynamic loads. However, their predictive capacity and determination of model parameters are strongly dependent on loading conditions and large experimental efforts. This research aims to develop a new model which uses a single model parameter to predict the variation trend and distribution pattern of fatigue experimental data points subjected to different stress ratios, loading frequencies and fiber orientations. Validation of the model with several sets of experimental data shows that the proposed model is capable of adequately considering the effects of stress ratio, loading frequency and fiber orientation on the fatigue behavior of composite materials and correctly predicting the variation trend of the experimental data points using only one set of model parameters regardless of stress ratios, loading frequencies and fiber orientations.

Long-term creep behavior of a short carbon fiber-reinforced PEEK at high temperature: Experimental and modeling approach

In this work, the time-dependent mechanical behavior of a short carbon fiber-reinforced PEEK is investigated at temperatures near and above the glass transition temperature and for stresses below the damage threshold. An experimental procedure including creep/recovery tests and dynamic mechanical tests was carried out by varying stress levels, temperatures and fiber orientation. The mechanical behavior is shown to be linear viscoelastic below the damage threshold. Effect of the fiber orientation on elastic and viscoelastic behavior is similar. A procedure involving the time–temperature superposition principle (TTSP) is set to assess the long-term linear viscoelastic behavior and is shown to provide reliable results. A Maxwell Generalized model is used for unidirectional identification of the model’s parameters. It is then implemented via a UMAT in Abaqus with failure criteria for viscoelastic materials from the literature for 3D calculation of industrial parts.

Autophagy in mesenchymal progenitors protects mice against bone marrow failure after severe intermittent stress

AbstractThe cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.

A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells.

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell–cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation.

How extracellular matrix contributes to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. We found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell-autonomous control of F-actin fiber orientation relies on the preceding BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a crucial organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation.

Extracellular Matrix Proteins and Substrate Stiffness Synergistically Regulate Vascular Smooth Muscle Cell Migration and Cortical Cytoskeleton Organization.

Vascular smooth muscle cell (VSMC) migration is a critical step in the progression of cardiovascular disease and aging. Migrating VSMCs encounter a highly heterogeneous environment with the varying extracellular matrix (ECM) composition due to the differential synthesis of collagen and fibronectin (FN) in different regions and greatly changing stiffness, ranging from the soft necrotic core of plaques to hard calcifications within blood vessel walls. In this study, we demonstrate an application of a two-dimensional (2D) model consisting of an elastically tunable polyacrylamide gel of varying stiffness and ECM protein coating to study VSMC migration. This model mimics the in vivo microenvironment that VSMCs experience within a blood vessel wall, which may help identify potential therapeutic targets for the treatment of atherosclerosis. We found that substrate stiffness had differential effects on VSMC migration on type 1 collagen (COL1) and FN-coated substrates. VSMCs on COL1-coated substrates showed significantly diminished migration distance on stiffer substrates, while on FN-coated substrates VSMCs had significantly increased migration distance. In addition, cortical stress fiber orientation increased in VSMCs cultured on more rigid COL1-coated substrates, while decreasing on stiffer FN-coated substrates. On both proteins, a more disorganized cytoskeletal architecture was associated with faster migration. Overall, these results demonstrate that different ECM proteins can cause substrate stiffness to have differential effects on VSMC migration in the progression of cardiovascular diseases and aging.

Fatigue Life Estimation with Mean Stress Effect Compensation for Lightweight Structures-The Case of GLARE 2 Composite.

This paper describes the current state-of-the-art in fatigue life assessment for lightweight composite structures with the use of the frequency domain fatigue life calculation method. Random stationary gaussian loading signals have been generated and served in the process of fatigue calculation. The material information that is being used in the calculation process has been obtained from literature for the Glare 2 composite. The effect of nonzero mean stress and different fiber orientations have been taken into account. The calculations have been performed for two mean stress compensation models by Goodman and Gerber. The proposed procedure gives satisfying results for the high-cycle fatigue region for Goodman and an overall good comparison in both regimes for the Gerber model.

Modeling of nonlinear response in loading-unloading tests for fibrous composites under tension and compression

Loading-unloading tests on unidirectional HTS40/PA6 carbon/polyamide laminates are performed in this study to identify the nature of off-axis nonlinear deformation of fibrous composites under tension and compression. Experimental results reveal the involved residual strain (plastic strain) and hysteresis loop during loading and unloading, and their dependence on stress level, fiber orientation, and tension or compression mode. The hysteresis behavior is assumed to be induced by a component of strain, which is considered as anelastic strain. This strain is recoverable like elastic deformation but dissipates work like plastic deformation. An approach is developed to predict the tension-compression asymmetry in plastic strain and anelastic strain, as well as the dependence of asymmetry on stress level and fiber orientation. The proposed approach is a modification of a strength differential model that considers the nonlinear deformation completely as plastic strain for monotonic tests. Predicted off-axis loading-unloading responses of unidirectional laminates are compared with the experimental results. The modified model is found to be effective for adequately describing the tension-compression asymmetry not only in plastic strain but also in complex nonlinear hysteresis behavior.

TGF-β induces changes in breast cancer cell deformability

Mechanical properties of cells are shown to regulate cell behaviors leading to phenotypic changes that may aid in the development and progression of disease. In this study, we used atomic force microscopy (AFM) indentation with a spherical probe to characterize the elastic and viscoelastic properties of invasive (MDA-MB-231) and noninvasive (MCF-7) breast cancer cells treated with transforming growth factor-β (TGF-β). We also used confocal fluorescence imaging to investigate the sub-membrane cytoskeletal structure of the cells. Results showed significant alterations in moduli of both cell types after 24 h TGF-β treatment which had a context dependent response; moduli for MDA-MB-231 decreased whereas MCF-7 demonstrated stiffening response. Stress relaxation tests showed increased fluid-like nature of MDA-MB-231 following TGF-β treatment and lower fluidity for MCF-7 cells. We also observed significant alterations in the expression and orientation of actin stress fibers with TGF-β treatment which correlated with the changes in cell mechanics. Less invasive MCF-7 cells had a delayed overall increase in cell deformability after 48 h exposure to TGF-β; a similar trend was observed for MDA-MB cells. These changes may be important to facilitate migration, for instance, during metastasis of cancer cells through submicron sized spaces.

A model for cellular mechanotransduction and contractility at finite strain

AbstractIn this work we introduce a theoretical and computational modeling framework for the contractile response of single cells triggered by external mechanical stimuli. The structural response due to the formation and dissociation of stress fibers is modeled following isotropic anisotropic contractile phases with an orientation that evolves with time and strain. The passive and active structural components are postulated to act in parallel, and the re‐orientation process drives the anisotropic phase of stress fiber orientation to align with the direction of the maximum principal stretch. A reduced form of the Hai‐Murphy model is used to follow kinetics of myosin states considering the combined effect of “latch”‐ and “cross”‐bridge states. The introduction of distinct isotropic and anisotropic activation allows modeling of the contractile intensity of each phase. Tractions on the cell surface initiate bio‐chemical signaling through the RhoA pathway, which in turn controls both myosin contraction and F‐actin polymerization. A signaling model is introduced to effectively connect intracellular events with the tractions on the cell surface. The overall model is defined by a free energy density function that couples the deformation and the activation, and associated equilibrium and kinetic models for evolution. Features of the model are highlighted via implementation in a finite element model and application to benchmark problems. The model captures the dynamic contractile responses of cells and stress fiber re‐alignment under complex load histories. For example, physiologically relevant scenario such as relaxation of cells to their initial state upon removal of applied loads can be simulated.

The Mechanical Contribution of Vimentin to Cellular Stress Generation.

Contractile stress generation by adherent cells is largely determined by the interplay of forces within their cytoskeleton. It is known that actin stress fibers, connected to focal adhesions, provide contractile stress generation, while microtubules and intermediate filaments provide cells compressive stiffness. Recent studies have shown the importance of the interplay between the stress fibers and the intermediate filament vimentin. Therefore, the effect of the interplay between the stress fibers and vimentin on stress generation was quantified in this study. We hypothesized that net stress generation comprises the stress fiber contraction combined with the vimentin resistance. We expected an increased net stress in vimentin knockout (VimKO) mouse embryonic fibroblasts (MEFs) compared to their wild-type (vimentin wild-type (VimWT)) counterparts, due to the decreased resistance against stress fiber contractility. To test this, the net stress generation by VimKO and VimWT MEFs was determined using the thin film method combined with sample-specific finite element modeling. Additionally, focal adhesion and stress fiber organization were examined via immunofluorescent staining. Net stress generation of VimKO MEFs was three-fold higher compared to VimWT MEFs. No differences in focal adhesion size or stress fiber organization and orientation were found between the two cell types. This suggests that the increased net stress generation in VimKO MEFs was caused by the absence of the resistance that vimentin provides against stress fiber contraction. Taken together, these data suggest that vimentin resists the stress fiber contractility, as hypothesized, thus indicating the importance of vimentin in regulating cellular stress generation by adherent cells.

Gaussian Curvature Directs Stress Fiber Orientation and Cell Migration

We show that substrates with nonzero Gaussian curvature influence the organization of stress fibers and direct the migration of cells. To study the role of Gaussian curvature, we developed a sphere-with-skirt surface in which a positive Gaussian curvature spherical cap is seamlessly surrounded by a negative Gaussian curvature draping skirt, both with principal radii similar to cell-length scales. We find significant reconfiguration of two subpopulations of stress fibers when fibroblasts are exposed to these curvatures. Apical stress fibers in cells on skirts align in the radial direction and avoid bending by forming chords across the concave gap, whereas basal stress fibers bend along the convex direction. Cell migration is also strongly influenced by the Gaussian curvature. Real-time imaging shows that cells migrating on skirts repolarize to establish a leading edge in the azimuthal direction. Thereafter, they migrate in that direction. This behavior is notably different from migration on planar surfaces, in which cells typically migrate in the same direction as the apical stress fiber orientation. Thus, this platform reveals that nonzero Gaussian curvature not only affects the positioning of cells and alignment of stress fiber subpopulations but also directs migration in a manner fundamentally distinct from that of migration on planar surfaces.