Transient low shear-stress preconditioning influences long-term endothelial traction and alignment under high shear flow.

Endothelial cells (ECs) within the vascular system encounter fluid shear stress (FSS). High, laminar FSS promotes vasodilation and anti-inflammatory responses, whereas low or disturbed FSS induces dysfunction and inflammation. However, the adaptation of endothelial cells (ECs) to dynamically changing FSS patterns remains underexplored. Here, by combining traction force microscopy with a custom flow chamber, we examined human umbilical vein endothelial cells adapting their traction during transitions from short-term low shear to long-term high shear stress. We discovered that the initial low FSS elevates the traction by only half of the amount in response to direct high FSS even after flow changes to high FSS. However, in the long term under high FSS, the flow started with low FSS triggers a substantial second rise in traction for over 10 h. In contrast, the flow started directly with high FSS results in a quick traction surge followed by a huge reduction below the baseline traction in <30 min. Importantly, we find that the orientation of traction vectors is steered by initial shear exposure. Using Granger causality analysis, we show that the traction that aligns in the flow direction under direct high FSS functionally causes cell alignment toward the flow direction. However, EC traction that orients perpendicular to the flow that starts with temporary low FSS functionally causes cell orientation perpendicular to the flow. Taken together, our findings elucidate the significant influence of initial short-term low FSS on lasting changes in endothelial traction that induces EC alignment.NEW & NOTEWORTHY In our study, we uncover that preconditioning with low shear stress yields enduring impacts on endothelial cell traction and orientation, persisting even after transitioning to high-shear conditions. Using Granger causality analysis, we demonstrate a functional link between the direction of cell traction and subsequent cellular alignment across varying shear environments.

Drebrin is Required for Myosin-facilitated Actin Cytoskeletal Remodeling during Pulmonary Alveolar Development.

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

Low Shear in Short-Term Impacts Endothelial Cell Traction and Alignment in Long-Term.

Within the vascular system, endothelial cells (ECs) are exposed to fluid shear stress (FSS), a mechanical force exerted by blood flow that is critical for regulating cellular tension and maintaining vascular homeostasis. The way ECs react to FSS varies significantly; while high, laminar FSS supports vasodilation and suppresses inflammation, low or disturbed FSS can lead to endothelial dysfunction and increase the risk of cardiovascular diseases. Yet, the adaptation of ECs to dynamically varying FSS remains poorly understood. This study focuses on the dynamic responses of ECs to brief periods of low FSS, examining its impact on endothelial traction-a measure of cellular tension that plays a crucial role in how endothelial cells respond to mechanical stimuli. By integrating traction force microscopy (TFM) with a custom-built flow chamber, we analyzed how human umbilical vein endothelial cells (HUVECs) adjust their traction in response to shifts from low to high shear stress. We discovered that initial exposure to low FSS prompts a marked increase in traction force, which continues to rise over 10 hours before slowly decreasing. In contrast, immediate exposure to high FSS causes a quick spike in traction followed by a swift reduction, revealing distinct patterns of traction behavior under different shear conditions. Importantly, the direction of traction forces and the resulting cellular alignment under these conditions indicate that the initial shear experience dictates long-term endothelial behavior. Our findings shed light on the critical influence of short-lived low-shear stress experiences in shaping endothelial function, indicating that early exposure to low FSS results in enduring changes in endothelial contractility and alignment, with significant consequences for vascular health and the development of cardiovascular diseases.

Nascent adhesions shorten the period of lamellipodium protrusion through the Brownian ratchet mechanism.

Directional cell migration is driven by the conversion of oscillating edge motion into lasting periods of leading edge protrusion. Actin polymerization against the membrane and adhesions control edge motion, but the exact mechanisms that determine protrusion period remain elusive. We addressed this by developing a computational model in which polymerization of actin filaments against a deformable membrane and variable adhesion dynamics support edge motion. Consistent with previous reports, our model showed that actin polymerization and adhesion lifetime power protrusion velocity. However, increasing adhesion lifetime decreased the protrusion period. Measurements of adhesion lifetime and edge motion in migrating cells confirmed that adhesion lifetime is associated with and promotes protrusion velocity, but decreased duration. Our model showed that adhesions' control of protrusion persistence originates from the Brownian ratchet mechanism for actin filament polymerization. With longer adhesion lifetime or increased-adhesion density, the proportion of actin filaments tethered to the substrate increased, maintaining filaments against the cell membrane. The reduced filament-membrane distance generated pushing force for high edge velocity, but limited further polymerization needed for protrusion duration. We propose a mechanism for cell edge protrusion in which adhesion strength regulates actin filament polymerization to control the periods of leading edge protrusion.

Black dots: High-yield traction force microscopy reveals structural factors contributing to platelet forces.

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed 'black dots') that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces. STATEMENT OF SIGNIFICANCE: Cells produce contractile forces during division, migration, or wound healing. Measuring cellular forces provides insight into their health, behavior, and function. We developed a technique that calculates cellular forces by seeding cells onto a pattern and quantifying how much each cell displaces the pattern. This technique is capable of measuring hundreds of cells without needing to detach them. Using this technique to evaluate human platelets, we find that platelets exerting more force tend to have more spread area, are more circular in shape, and have more uniformly distributed cytoskeletal filaments. Due to our high yield of data, we were able to apply statistical techniques that revealed combinatorial effects between these factors.

Particle retracking algorithm capable of quantifying large, local matrix deformation for traction force microscopy.

Deformation measurement is a key process in traction force microscopy (TFM). Conventionally, particle image velocimetry (PIV) or correlation-based particle tracking velocimetry (cPTV) have been used for such a purpose. Using simulated bead images, we show that those methods fail to capture large displacement vectors and that it is due to a poor cross-correlation. Here, to redeem the potential large vectors, we propose a two-step deformation tracking algorithm that combines cPTV, which performs better for small displacements than PIV methods, and newly-designed retracking algorithm that exploits statistically confident vectors from the initial cPTV to guide the selection of correlation peak which are not necessarily the global maximum. As a result, the new method, named 'cPTV-Retracking', or cPTVR, was able to track more than 92% of large vectors whereas conventional methods could track 43-77% of those. Correspondingly, traction force reconstructed from cPTVR showed better recovery of large traction than the old methods. cPTVR applied on the experimental bead images has shown a better resolving power of the traction with different-sized cell-matrix adhesions than conventional methods. Altogether, cPTVR method enhances the accuracy of TFM in the case of large deformations present in soft substrates. We share this advance via our TFMPackage software.

Mechanobiology in the Comorbidities of Ehlers Danlos Syndrome.

Ehlers-Danlos Syndromes (EDSs) are a group of connective tissue disorders, characterized by skin stretchability, joint hypermobility and instability. Mechanically, various tissues from EDS patients exhibit lowered elastic modulus and lowered ultimate strength. This change in mechanics has been associated with EDS symptoms. However, recent evidence points toward a possibility that the comorbidities of EDS could be also associated with reduced tissue stiffness. In this review, we focus on mast cell activation syndrome and impaired wound healing, comorbidities associated with the classical type (cEDS) and the hypermobile type (hEDS), respectively, and discuss potential mechanobiological pathways involved in the comorbidities.

EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.

As a partner of tetraspanins, EWI2 suppresses glioblastoma, melanoma, and prostate cancer; but its role in lung cancer has not been investigated. Bioinformatics analysis reveals that EWI2 gene expression is up regulated in lung adenocarcinoma and higher expression of EWI2 mRNA may predict poorer overall survival. However, experimental analysis shows that EWI2 protein is actually downregulated constantly in the tissues of lung adenocarcinoma and lung squamous cell carcinoma. Forced expression of EWI2 in human lung adenocarcinoma cells reduces total cellular and cell surface levels of various integrins and growth factor receptors, which initiates the outside-in motogenic and mitogenic signaling. These reductions result in the decreases in 1) cell-matrix adhesion, cell movement, and cell transformation in vitro and 2) tumor growth, burden, and metastasis in vivo, and result from the increases in lysosomal trafficking and proteolytic degradation of theses membrane receptors. EWI2 elevates lysosome formation by promoting nuclear retention of TFEB, the master transcription factor driving lysosomogenesis. In conclusion, EWI2 as a lung cancer suppressor attenuates lung cancer cells in a comprehensive fashion by inhibiting both tumor growth and tumor metastasis; EWI2 as an endolysosome regulator promotes lysosome activity to enhance lysosomal degradation of growth factor receptors and integrins and then reduce their levels and functions; and EWI2 can become a promising therapeutic candidate given its accessibility at the cell surface, dual inhibition on growth factor receptors and integrins, and broad-spectrum anti-cancer activity. More importantly, our observations also provide a novel therapeutic strategy to bypass the resistance to EGFR inhibitors.

Subcellular Force Quantification of Endothelial Cells Using Silicone Pillar Arrays.

Micropillar arrays are one of the tools that quantify the mechanical forces exerted by endothelial cells and any other adherent cell types. This chapter describes the fabrication and usage of the micropillars that reports spatial distribution of traction forces by endothelial cells. The fabrication begins with making a "master" pillar substrate using photolithography, followed by softlithography of the master with polydimethylsiloxane. The pillars can then be printed with an extracellular matrix protein in specific shape using stamp-off technique. Lastly, imaging of the micropillars with cells and the analysis of the traction force using Matlab-based software are described.

High-Resolution, Highly-Integrated Traction Force Microscopy Software.

Accurate measurement of cellular traction force is critical for understanding physical interaction between cells and the extracellular matrix. Traction force microscopy (TFM) has become the most widely used tool for this purpose. While TFM has made continual progress in terms of resolution and accuracy, there have been challenges regarding obtaining user-friendly software and choosing the right values for parameters and sub-processes associated with the software. Here we provide step-by-step instructions for a MATLAB-based TFM software application equipped with multiple methods for image deformation quantification and force reconstruction, along with clarification on the computational meaning of the parameters within the software. We outline how to choose the optimal sub-methods and values for parameters for each process, depending on the characteristics of images and purpose of the analyses. The software's runtime is 20, 4, and 0.05 min by Fast BEM L1 (Boundary Element Method L1-regularization), Fast BEM L2 (L2-regularization), and FTTC (Fourier Transform Traction Cytometry), respectively, in addition to 7 min of particle-tracking velocimetry-based deformation tracking, for a single image (1280 × 960 pixel) on a standard workstation. Finally, the colocalization accuracies, in reference to a paxillin-GFP image, are compared between the three force reconstruction methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Setting up the TFM package in MATLAB Basic Protocol 2: Running the TFM package Alternate Protocol 1: Stage drift correction: Efficient subpixel registration Alternate Protocol 2: Force field calculation: FastBEM.

Pre-complexation of talin and vinculin without tension is required for efficient nascent adhesion maturation.

Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.

TRA-1-60-positive/CD45low cells found in the peripheral blood of prostate cancer patients with metastatic disease - A proof-of-concept study.

PURPOSE: Over 90% of all cancer related deaths are due to metastasis. However, current diagnostic tools can't reliably discriminate between invasive and localized cancers. PATIENTS AND METHODS: In this proof-of-concept study, we employed the embryonic stem cell marker TRA-1-60 (TRA+) to identify TRA + cells within the blood of prostate cancer patients and searched for TRA + cells in men with metastatic and localized cancers. We isolated whole peripheral blood mononuclear cells from 26 metastatic prostate cancer patients, from 13 patients with localized prostate cancer and from 17 healthy controls. Cells were stained for DAPI, CD45 and TRA + by immunofluorescence and imaged by epi-fluorescence microscopy. Imaged-based software was used both to identify TRA + cells, and to analyze CD45 levels in TRA+ and negative cells. RESULTS: We found high numbers of TRA + cells within the blood of metastatic cancer patients, whereas healthy individuals or men with localized prostate cancer showed none or very low numbers of TRA + cells. Further analysis of the CD45 levels of TRA + cells revealed a small population of TRA + cells with almost undetectable CD45 levels that were found frequently in metastatic prostate cancer patients. By excluding CD45 positive cells from the TRA + cell pool, we were able to refine the assay to be highly specific in identifying men with metastatic disease. In fact, the difference of CD45 levels between TRA+ and negative cells was a robust measure to distinguish between men with localized and metastatic prostate cancers in this small patient cohort. CONCLUSIONS: The data suggest that metastatic prostate cancer patient have significant numbers of TRA+/CD45low cells which might represent a potential tool for diagnostic assessment in the future.

The consequence of substrates of large-scale rigidity on actin network tension in adherent cells.

There is compelling evidence that substrate stiffness affects cell adhesion as well as cytoskeleton organization and contractile activity. This work was designed to study the cytoskeletal contractile activity of single cells plated on micropost substrates of different stiffness using a numerical model simulating the intracellular tension of individual cells. We allowed cells to adhere onto micropost substrates of various rigidities and used experimental traction force data to infer cell contractility using a numerical model. The model shows that higher substrate stiffness leads to an increase in intracellular tension. The strength of this model is its ability to calculate the mechanical state of each cell in accordance to its individual cytoskeletal structure. This is achieved by regenerating a numerical cytoskeleton based on microscope images of the actin network of each cell. The resulting numerical structure consequently represents pulling characteristics on its environment similar to those generated by the cell in-vivo. From actin imaging we can calculate and better understand how forces are transmitted throughout the cell.

Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1P29S Melanoma Cells.

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1P29S) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1P29S-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1P29S cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1's regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.

Vimentin fibers orient traction stress.

The intermediate filament vimentin is required for cells to transition from the epithelial state to the mesenchymal state and migrate as single cells; however, little is known about the specific role of vimentin in the regulation of mesenchymal migration. Vimentin is known to have a significantly greater ability to resist stress without breaking in vitro compared with actin or microtubules, and also to increase cell elasticity in vivo. Therefore, we hypothesized that the presence of vimentin could support the anisotropic mechanical strain of single-cell migration. To study this, we fluorescently labeled vimentin with an mEmerald tag using TALEN genome editing. We observed vimentin architecture in migrating human foreskin fibroblasts and found that network organization varied from long, linear bundles, or "fibers," to shorter fragments with a mesh-like organization. We developed image analysis tools employing steerable filtering and iterative graph matching to characterize the fibers embedded in the surrounding mesh. Vimentin fibers were aligned with fibroblast branching and migration direction. The presence of the vimentin network was correlated with 10-fold slower local actin retrograde flow rates, as well as spatial homogenization of actin-based forces transmitted to the substrate. Vimentin fibers coaligned with and were required for the anisotropic orientation of traction stresses. These results indicate that the vimentin network acts as a load-bearing superstructure capable of integrating and reorienting actin-based forces. We propose that vimentin's role in cell motility is to govern the alignment of traction stresses that permit single-cell migration.

Spatial and temporal coordination of traction forces in one-dimensional cell migration.

Migration of a fibroblast along a collagen fiber can be regarded as cell locomotion in one-dimension (1D). In this process, a cell protrudes forward, forms a new adhesion, produces traction forces, and releases its rear adhesion in order to advance itself along a path. However, how a cell coordinates its adhesion formation, traction forces, and rear release in 1D migration is unclear. Here, we studied fibroblasts migrating along a line of microposts. We found that when the front of a cell protruded onto a new micropost, the traction force produced at its front increased steadily, but did so without a temporal correlation in the force at its rear. Instead, the force at the front coordinated with a decrease in force at the micropost behind the front. A similar correlation in traction forces also occurred at the rear of a cell, where a decrease in force due to adhesion detachment corresponded to an increase in force at the micropost ahead of the rear. Analysis with a bio-chemo-mechanical model for traction forces and adhesion dynamics indicated that the observed relationship between traction forces at the front and back of a cell is possible only when cellular elasticity is lower than the elasticity of the cellular environment.

In silico CDM model sheds light on force transmission in cell from focal adhesions to nucleus.

Cell adhesion is crucial for many types of cell, conditioning differentiation, proliferation, and protein synthesis. As a mechanical process, cell adhesion involves forces exerted by the cytoskeleton and transmitted by focal adhesions to extracellular matrix. These forces constitute signals that infer specific biological responses. Therefore, analyzing mechanotransduction during cell adhesion could lead to a better understanding of the mechanobiology of adherent cells. For instance this may explain how, the shape of adherent stem cells influences their differentiation or how the stiffness of the extracellular matrix affects adhesion strength. To assess the mechanical signals involved in cell adhesion, we computed intracellular forces using the Cytoskeleton Divided Medium model in endothelial cells adherent on micropost arrays of different stiffnesses. For each cell, focal adhesion location and forces measured by micropost deflection were used as an input for the model. The cytoskeleton and the nucleoskeleton were computed as systems of multiple tensile and compressive interactions. At the end of computation, the systems respected mechanical equilibrium while exerting the exact same traction force intensities on focal adhesions as the observed cell. The results indicate that not only the level of adhesion forces, but also the shape of the cell has an influence on intracellular tension and on nucleus strain. The combination of experimental micropost technology with the present CDM model constitutes a tool able to estimate the intracellular forces.

Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient.

Traction microscopy to identify force modulation in subresolution adhesions.

We present a reconstruction algorithm that resolves cellular tractions in diffraction-limited nascent adhesions (NAs). The enabling method is the introduction of sparsity regularization to the solution of the inverse problem, which suppresses noise without underestimating traction magnitude. We show that NAs transmit a distinguishable amount of traction and that NA maturation depends on traction growth rate. A software package implementing this numerical approach is provided.

Measuring the contractile forces of human induced pluripotent stem cell-derived cardiomyocytes with arrays of microposts.

Human stem cell-derived cardiomyocytes hold promise for heart repair, disease modeling, drug screening, and for studies of developmental biology. All of these applications can be improved by assessing the contractility of cardiomyocytes at the single cell level. We have developed an in vitro platform for assessing the contractile performance of stem cell-derived cardiomyocytes that is compatible with other common endpoints such as microscopy and molecular biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were seeded onto elastomeric micropost arrays in order to characterize the contractile force, velocity, and power produced by these cells. We assessed contractile function by tracking the deflection of microposts beneath an individual hiPSC-CM with optical microscopy. Immunofluorescent staining of these cells was employed to assess their spread area, nucleation, and sarcomeric structure on the microposts. Following seeding of hiPSC-CMs onto microposts coated with fibronectin, laminin, and collagen IV, we found that hiPSC-CMs on laminin coatings demonstrated higher attachment, spread area, and contractile velocity than those seeded on fibronectin or collagen IV coatings. Under optimized conditions, hiPSC-CMs spread to an area of approximately 420 µm2, generated systolic forces of approximately 15 nN/cell, showed contraction and relaxation rates of 1.74 µm/s and 1.46 µm/s, respectively, and had a peak contraction power of 29 fW. Thus, elastomeric micropost arrays can be used to study the contractile strength and kinetics of hiPSC-CMs. This system should facilitate studies of hiPSC-CM maturation, disease modeling, and drug screens as well as fundamental studies of human cardiac contraction.