Cell Traction Research Articles

Modulation of the gene expression of annulus fibrosus-derived stem cells using poly(ether carbonate urethane)urea scaffolds of tunable elasticity

Annulus fibrosus (AF) injuries commonly lead to substantial deterioration of the intervertebral disc (IVD). While tissue engineering has recently evolved into a promising approach for AF regeneration, it remains challenging due to the cellular, biochemical, and mechanical heterogeneity of AF tissue. In this study, we explored the use of AF-derived stem cells (AFSCs) to achieve diversified differentiation of cells for AF tissue engineering. Since the differentiation of stem cells relies significantly on the elasticity of the substrate, we synthesized a series of biodegradable poly(ether carbonate urethane)urea (PECUU) materials whose elasticity approximated that of native AF tissue. When AFSCs were cultured on electrospun PECUU fibrous scaffolds, the gene expression of collagen-I in the cells increased with the elasticity of scaffold material, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. At the protein level, the content of collagen-I gradually increased with substrate elasticity, while collagen-II and GAG contents decreased. In addition, the cell traction forces (CTFs) of AFSCs gradually decreased with scaffold elasticity. Such substrate elasticity-dependent changes of AFSCs were similar to the gradual transition in the genetic, biochemical, and biomechanical characteristics of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that AFSCs, depending on the substrate elasticity, have strong tendencies to differentiate into various types of AF-like cells, thereby providing a solid foundation for the tissue engineering applications of AFSCs. Statement of significanceRepairing the annulus fibrosus (AF) of intervertebral disc (IVD) is critical for the treatment of disc degeneration disease, but remains challenging due to the significant heterogeneity of AF tissue. Previously, we have identified rabbit AF-derived stem cells (AFSCs), which are AF tissue-specific and hold promise for AF regeneration. In this study, we synthesized a series of poly(ether carbonate urethane)ureas of various elasticity (or stiffness) and explored the potential of induced differentiation of AFSCs using electrospun PECUU scaffolds. This work has, for the first time, found that AFSCs are able to present different gene expression patterns simply as a result of the elasticity of scaffold material. Therefore, our findings will help supplement current knowledge of AF tissue regeneration and may benefit a diversified readership from scientific, engineering, and clinical settings whose work involves the biology and tissue engineering of IVD.

Interfacing Inorganic Nanowire Arrays and Living Cells for Cellular Function Analysis.

Inorganic nanowires are among the most attractive functional materials, which have emerged in the past two decades. They have demonstrated applications in information technology and energy conversion, but their utility in biological or biomedical research remains relatively under-explored. Although nanowire-based sensors have been frequently reported for biomolecular detection, interfacing nanowire arrays and living mammalian cells for the direct analysis of cellular functions is a very recent endeavor. Cell-penetrating nanowires enabled effective delivery of biomolecules, electrical and optical stimulation and recording of intracellular signals over a long period of time. Non-penetrating, high-density nanowire arrays display rich interactions between the nanostructured substrate and the micro/nanoscale features of cell surfaces. Such interactions enable efficient capture of rare cells including circulating tumor cells and trafficking leukocytes from complex biospecimens. It also serves as a platform for probing cell traction force and neuronal guidance. The most recent advances in the field that exploits nanowire arrays (both penetrating and non-penetrating) to perform rapid analysis of cellular functions potentially for disease diagnosis and monitoring are reviewed.

Bio-inspired micropatterned hydrogel to direct and deconstruct hierarchical processing of geometry-force signals by human mesenchymal stem cells during smooth muscle cell differentiation

Micropatterned biomaterial-based hydrogel platforms allow the recapitulation of in vivo-like microstructural and biochemical features that are critical physiological regulators of stem cell development. Herein, we report the use of muscle mimicking geometries patterned on polyacrylamide hydrogels as an effective strategy to induce smooth muscle cell (SMC) differentiation of human mesenchymal stem cells (hMSCs). hMSCs were systemically coerced to elongate with varying aspect ratios (AR) (that is, 1:1, 5:1, 10:1 and 15:1) at a fixed projection area of ~7000 μm2. The results showed engineered cellular anisotropy with an intermediate AR 5:1 and AR 10:1, promoting the expression of alpha smooth muscle actin (α-SMA) and enhancement of contractile output. Further mechanistic studies indicated that a threshold cell traction force of ~3.5 μN was required for SMC differentiation. Beyond the critical cytoskeleton tension, hMSCs respond to higher intracellular architectural cues such as the stress fiber (SF) alignment, SF subtype expression and diphosphorylated myosin regulatory light-chain activity to promote the expression and incorporation of α-SMA to the SF scaffold. These findings underscore the importance of exploiting biomimetic geometrical cues as an effective strategy to guide hMSC differentiation and are expected to guide the rational design of advanced tissue-engineered vascular grafts. Muscle-mimicking geometries patterned on hydrogel facilitate the acquisition of a smooth-muscle-cell-like phenotype from human stem cells. Adult mesenchymal stem cells (MSCs) are a valuable source of cells for regenerative medicine. Now, researchers in Singapore and China have examined the complex interdependence of engineered cell shape and human transforming growth factor beta-1 treatment to direct smooth muscle cells differentiation of MSCs. They discovered that geometries resembling those of human smooth muscle cells provide the optimal shape for the expression and recruitment of the mechano-sensitive protein α-smooth muscle actin to the filamentous actin cytoskeleton. Based on these results, the researchers proposed an elegant model to describe the decision-making process that human MSCs take during differentiation into smooth muscle cells. The findings highlight the importance of using biomimetic cues for guiding differentiation of human MSCs. Inspired by the intrinsic morphology of smooth muscle cells (SMCs), a micropatterned hydrogel is developed to direct and define the boundary conditions for efficient SMC differentiation of human mesenchymal stem cells (hMSCs). The results show that in conjunction with TGF-β1 treatment, muscle-mimicking shapes with intermediate aspect ratios ranging from 5:1 to 10:1 exert the strongest pro-SMC differentiation effects in a structural–contractile force-dependent manner. These findings are expected to provide critical insights and design rules for vascular-related engineered tissue grafts.

June 2015: This Month in JoVE - Celebrating JoVE's 100th Issue

Here's a look at what's coming up in the June 2015 issue of JoVE: The Journal of Visualized Experiments. This month marks the release of JoVE's 100th issue, and we now have 10 scientific sections: Biology, Neuroscience, Immunology & Infection, Medicine, Bioengineering, Engineering, Chemistry, Behavior, Environment, and Developmental Biology. In the field of Developmental Biology, sometimes you'll come across the term coat-which might sound like a pet's coat color, or maybe a manicure, or maybe the leather under-armor worn by 17th Century cavalry. But a buffy coat is a product of centrifuging whole blood, and refers to the cloudy layer containing white blood cells, which forms between the red blood cells and the plasma. Meraviglia et al. collect the buffy coats and turn peripheral blood mononuclear cells (PBMNCs) into induced pluripotent stem cells (iPSCs). Instead of inducing pluripotency as usual with viral vectors, our authors reprogram the cells using non-integrating plasmids. The resulting iPSC colonies show normal morphology, stable karyotypes, and significant expression of pluripotency markers. As far as we know, this is the first report of deriving virus-free iPSCs from frozen buffy coats, which are commonly stored in biobanks, and could become a valuable source of iPSCs for regenerative medicine. In JoVE Bioengineering, we know that cells can behave differently depending on the stiffness of the extracellular matrix, which is measured in kiloPascals (kPA). Metastatic cancer cells, in particular, show higher traction stress than non-metastatic cells, so scientists examine tumor cell invasion and metastasis using traction force assays. However, traction data are lacking for primary tumor cells. Therefore, Yakut Ali et al. extract primary cells from human tumors and culture them on hydrogel substrates for traction cytometry. This technique may lead to the use of biophysical parameters, such as tumor cell stiffness and traction, in cancer prognostics. In JoVE Behavior, we feature protocols that deal with cognition and learning in children. In the experimental paradigm by Sargent et al., a mobile hangs above an infant, and a light-emitting diode (LED) is secured to the infant's leg. When it reaches a certain height, the LED activates the mobile. The infants discover that the mobile will spin and play music if they kick their legs up. A motion capture system records these movements so the learning process can be quantified. Also in JoVE Behavior, children with speech impairments may benefit from a speech-generating device (SGD), which produces an electronic voice using a synthesizer. Some devices have dynamic interfaces, so users can access new words by changing levels. Cognitive factors can affect the ability to navigate through these levels, so Robillard et al. present a detailed protocol for assessing how cognitive factors contribute to navigational abilities. This could, in turn, guide clinicians to select the appropriate speech-generating device for a patient. You've just had a sneak peek of the June 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Collective Matrix Remodeling by Isolated Cells: Unionizing Home Improvement Do-It-Yourselfers

How do isolated cells reach out collectively to remodel the environment in which they reside? We have known for some time that in certain processes, such as wound-healing, cells appear to act together, contacting one another through bands of remodeled collagen stretching over distances that are very long compared to the size of a cell (Fig. 1, left panel). We also know that fibroblasts act locally, synthesizing and remodeling extracellular matrix (ECM) proteins in response to signals that are only partially understood (Fig. 1, right panel).

Biomechanistic Study of Smooth Muscle Cell Sheet during Circumferential Alignment in Circular Micropatterns.

Engineered functional blood vessels play an important role in tissue regeneration and wound healing, but they remain far from clinical practices because of the complex three-layer capillary histology. In general, the reconstruction of tunica media from vascular smooth muscle cells (SMCs) that mediate vasoconstriction and vasodilation of circulatory system is critical for engineering functional blood vessels. To date, the mechanotransduction during circumferential alignment of SMC layer in tunica media remains to be elucidated. In this study, concentric microwalls fabricated on a polydimethylsiloxane (PDMS) substrate were developed for cell traction force microscopy (CTFM) of SMC layer. First, the cell shape, density, and alignment orientation of SMCs during circumferential cell sheet formation were measured during 7-day cell culture. Second, a collective CTFM assay was carried out to measure the traction forces exerted by the confluent cell layer at various stages of circumferential alignment. It was found that the mechanotransduction of SMC layer grown between the circular microwalls was significantly altered compared to the SMC layer grown on a flat substrate. The results indicated the presence of an optimal region for formation of circumferentially aligned SMC layer verified by both quantitative analysis and biomechanical study. Moreover, the distributions of traction forces and von Mises stresses during cell alignment were affected by the cellular mechanotransduction from the extracellular physical constraints and cell-cell interactions. The immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR) analyses of actin filaments further revealed the relation between the monolayer-generated traction forces and the direction of aligned actin filaments.

Role of Cytoskeletal Tension in the Induction of Cardiomyogenic Differentiation in Micropatterned Human Mesenchymal Stem Cell.

The role of biophysical induction methods such as cell micropatterning in stem cell differentiation has been well documented previously. However, the underlying mechanistic linkage of the engineered cell shape to directed lineage commitment remains poorly understood. Here, it is reported that micropatterning plays an important role in regulating the optimal cytoskeletal tension development in human mesenchymal stem cell (hMSC) via cell mechanotransduction pathways to induce cardiomyogenic differentiation. Cells are grown on fibronectin strip patterns to control cell polarization and morphology. These patterned cells eventually show directed commitment toward the myocardial lineage. The cell's mechanical properties (cell stiffness and cell traction forces) are observed to be very different for cells that have committed to the myocardial lineage when compared with that of control. These committed cells have mechanical properties that are significantly lower indicating a correlation between the micropatterning-induced differentiation and actomyosin-generated cytoskeletal tension within patterned cells. To study this correlation, patterned cells are treated with RhoA pathway inhibitor. Severely down-regulated cardiomyogenic marker expression is observed in those treated patterned cells, thus emphasizing the direct dependence of hMSCs differentiation fate on the cytoskeletal tension.

Periodic traction in migrating large amoeba of Physarum polycephalum.

The slime mould Physarum polycephalum is a giant multinucleated cell exhibiting well-known Ca(2+)-dependent actomyosin contractions of its vein network driving the so-called cytoplasmic shuttle streaming. Its actomyosin network forms both a filamentous cortical layer and large fibrils. In order to understand the role of each structure in the locomotory activity, we performed birefringence observations and traction force microscopy on excised fragments of Physarum. After several hours, these microplasmodia adopt three main morphologies: flat motile amoeba, chain types with round contractile heads connected by tubes and motile hybrid types. Each type exhibits oscillations with a period of about 1.5 min of cell area, traction forces and fibril activity (retardance) when fibrils are present. The amoeboid types show only peripheral forces while the chain types present a never-reported force pattern with contractile rings far from the cell boundary under the spherical heads. Forces are mostly transmitted where the actomyosin cortical layer anchors to the substratum, but fibrils maintain highly invaginated structures and contribute to forces by increasing the length of the anchorage line. Microplasmodia are motile only when there is an asymmetry in the shape and/or the force distribution.

Micropillar displacements by cell traction forces are mechanically correlated with nuclear dynamics

Cells sense physical cues at the level of focal adhesions and transduce them to the nucleus by biochemical and mechanical pathways. While the molecular intermediates in the mechanical links have been well studied, their dynamic coupling is poorly understood. In this study, fibroblast cells were adhered to micropillar arrays to probe correlations in the physical coupling between focal adhesions and nucleus. For this, we used novel imaging setup to simultaneously visualize micropillar deflections and EGFP labeled chromatin structure at high spatial and temporal resolution. We observed that micropillar deflections, depending on their relative positions, were positively or negatively correlated to nuclear and heterochromatin movements. Our results measuring the time scales between micropillar deflections and nucleus centroid displacement are suggestive of a strong elastic coupling that mediates differential force transmission to the nucleus.

Three-dimensional numerical model of cell morphology during migration in multi-signaling substrates.

Cell Migration associated with cell shape changes are of central importance in many biological processes ranging from morphogenesis to metastatic cancer cells. Cell movement is a result of cyclic changes of cell morphology due to effective forces on cell body, leading to periodic fluctuations of the cell length and cell membrane area. It is well-known that the cell can be guided by different effective stimuli such as mechanotaxis, thermotaxis, chemotaxis and/or electrotaxis. Regulation of intracellular mechanics and cell’s physical interaction with its substrate rely on control of cell shape during cell migration. In this notion, it is essential to understand how each natural or external stimulus may affect the cell behavior. Therefore, a three-dimensional (3D) computational model is here developed to analyze a free mode of cell shape changes during migration in a multi-signaling micro-environment. This model is based on previous models that are presented by the same authors to study cell migration with a constant spherical cell shape in a multi-signaling substrates and mechanotaxis effect on cell morphology. Using the finite element discrete methodology, the cell is represented by a group of finite elements. The cell motion is modeled by equilibrium of effective forces on cell body such as traction, protrusion, electrostatic and drag forces, where the cell traction force is a function of the cell internal deformations. To study cell behavior in the presence of different stimuli, the model has been employed in different numerical cases. Our findings, which are qualitatively consistent with well-known related experimental observations, indicate that adding a new stimulus to the cell substrate pushes the cell to migrate more directionally in more elongated form towards the more effective stimuli. For instance, the presence of thermotaxis, chemotaxis and electrotaxis can further move the cell centroid towards the corresponding stimulus, respectively, diminishing the mechanotaxis effect. Besides, the stronger stimulus imposes a greater cell elongation and more cell membrane area. The present model not only provides new insights into cell morphology in a multi-signaling micro-environment but also enables us to investigate in more precise way the cell migration in the presence of different stimuli.

Gene expression modulation in TGF-β3-mediated rabbit bone marrow stem cells using electrospun scaffolds of various stiffness.

Tissue engineering has recently evolved into a promising approach for annulus fibrosus (AF) regeneration. However, selection of an ideal cell source, which can be readily differentiated into AF cells of various regions, remains challenging because of the heterogeneity of AF tissue. In this study, we set out to explore the feasibility of using transforming growth factor-β3-mediated bone marrow stem cells (tBMSCs) for AF tissue engineering. Since the differentiation of stem cells significantly relies on the stiffness of substrate, we fabricated nanofibrous scaffolds from a series of biodegradable poly(ether carbonate urethane)-urea (PECUU) materials whose elastic modulus approximated that of native AF tissue. We cultured tBMSCs on PECUU scaffolds and compared their gene expression profile to AF-derived stem cells (AFSCs), the newly identified AF tissue-specific stem cells. As predicted, the expression of collagen-I in both tBMSCs and AFSCs increased with scaffold stiffness, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. Interestingly, the expression of collagen-I, collagen-II and aggrecan genes in tBMSCs on PECUU scaffolds were consistently higher than those in AFSCs regardless of scaffold stiffness. In addition, the cell traction forces (CTFs) of both tBMSCs and AFSCs gradually decreased with scaffold stiffness, which is similar to the CTF change of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that tBMSCs had strong tendency to differentiate into various types of AF cells and presented gene expression profiles similar to AFSCs, thereby establishing a rationale for the use of tBMSCs in AF tissue engineering.

COX-2 dependent regulation of mechanotransduction in human breast cancer cells

The ability of living cells to exert physical forces upon their surrounding is a necessary prerequisite for diverse biological processes, such as local cellular migrations in wound healing to metastatic-invasion of cancer. How forces are coopted in metastasis has remained unclear, however, because the mechanical interplay between cancer cells and the various stromal components has not been experimentally accessible. Current dogma implicates inflammation in these mechanical processes. Using Fourier transform traction microscopy, we measured the force-generating capacity of human breast cancer cells occupying a spectrum of invasiveness as well as basal and inducible COX-2 expression (MCF-7<SUM-149<MDA-MB-231). Compared with non-invasive MCF-7 and moderately-invasive SUM-149, poorly-differentiated MDA-MB-231 cells showed increased cellular dispersion on collagen matrix that was accompanied by emergent distribution of contractile stresses at the interface between the adherent cell and its substrate, defined herein as the traction field. In metastatic MDA-MB-231 cells, the local tractions were precisely tuned to the surrounding matrix rigidity in a physiologic range with the concomitant expression of mechanosensitive integrin β1. These discrete responses at the single-cell resolution were correlated with PGE2 secretion and were ablated by shRNA-mediated knockdown of COX-2. Both COX-2-silenced and COX-2-expressing cells expressed EP2 and EP4 receptors, but not EP1 and EP3. Exogenous addition of PGE2 increased cell tractions and stiffened the underlying cytoskeletal network. To our knowledge this is the first report linking the expression of COX-2 with mechanotransduction of human breast cancer cells, and the regulation of COX-2-PGE2-EP signaling with physical properties of the tumor microenvironment. Drug treatments aimed at reducing this mechanical interplay may have therapeutic potential in the treatment of breast cancer.

Matrix Confinement Plays a Pivotal Role in Regulating Neutrophil-generated Tractions, Speed, and Integrin Utilization

Neutrophils are capable of switching from integrin-dependent motility on two-dimensional substrata to integrin-independent motion following entry into the confined three-dimensional matrix of an afflicted tissue. However, whether integrins still maintain a regulatory role for cell traction generation and cell locomotion under the physical confinement of the three-dimensional matrix is unknown, and this is challenging to deduce from motility studies alone. Using three-dimensional traction force microscopy and a double hydrogel sandwich system, we determined the three-dimensional spatiotemporal traction forces of motile neutrophils at unprecedented resolution and show, for the first time, that entry into a highly confined space (2.5D) is a sufficient trigger to convert to integrin-independent migration. We find that integrins exert a significant regulatory role in determining the magnitude and spatial distribution of tractions and cell speed on confined cells. We also find that 90% of neutrophil tractions are in the out-of-plane axis, and this may be a fundamental element of neutrophil traction force generation.

Measurement systems for cell adhesive forces.

Cell adhesion to the extracellular matrix (ECM) involves integrin receptor-ligand binding and clustering to form focal adhesion (FA) complexes, which mechanically link the cell's cytoskeleton to the ECM and regulate fundamental cell signaling pathways. Although elucidation of the biochemical events in cell-matrix adhesive interactions is rapidly advancing, recent studies show that the forces underlying cell-matrix adhesive interactions are also critical to cell responses. Therefore, multiple measurement systems have been developed to quantify the spatial and temporal dynamics of cell adhesive forces, and these systems have identified how mechanical events influence cell phenotype and FA structure-function relationships under physiological and pathological settings. This review focuses on the development, methodology, and applications of measurement systems for probing (a) cell adhesion strength and (b) 2D and 3D cell traction forces.

Vinculin-dependent actin bundling regulates cell migration and traction forces.

Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been limited. In the present study, we found mutation (R1049E) of the vinculin tail impairs its ability to bind F-actin, stimulate actin polymerization, and bundle F-actin in vitro. Further, mutant (R1049E) vinculin expressing cells are altered in cell migration, which is accompanied by changes in cell adhesion, cell spreading and cell generation of traction forces, providing direct evidence for the critical role of vinculin in mechanotransduction at adhesion sites. Lastly, we discuss the viability of models detailing the F-actin-binding surface on vinculin in the context of our mutational analysis.

Tracking traction force changes of single cells on the liquid crystal surface.

Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration.

708 細胞牽引力の測定のためのシリコーン製マイクロチャンバーの開発(バイオエンジニアリングII+機械力学・計測制御VII)

708 細胞牽引力の測定のためのシリコーン製マイクロチャンバーの開発(バイオエンジニアリングII+機械力学・計測制御VII)

Forces Behind Cell Adhesion and Migration in Microgravity

Cells sense and respond to their environment according to many factors, including gravity. Changes in the gravitational field during space exploration may alter cellular interactions. Our understanding of the fundamental mechanisms by which gravitational forces ultimately affect cell function, however, is limited. Based on our prior observations, human mesenchymal stem cells (hMSCs) adopt a more rounded morphology during simulated microgravity (clinorotation). We hypothesize that microgravity affects the cell-substrate forces, which in turn affects cell adhesion and motility. Therefore, we investigated the correlation between traction forces, spreading and chemotaxis. As an extension to our previously reported “clinochip” device, we developed a lab-on-a-chip device suitable for implementing traction force microscopy during clinorotation. The device contains a channel coated with an array of fluorescent beads embedded in a polyacrylamide substrate that can be processed to calculate cell-substrate traction forces. For our studies, we investigated both hMSCs and osteosarcoma cancer cells (143-B), because they represent highly regulated and deregulated cell states in the osteogenic lineage, respectively. Clinorotation speeds of 0, 30, and 75 rpm were examined, and cell shape, adhesion area, traction forces, and chemotactic migration were measured. Interestingly, results indicate that hMSCs exhibit a dose-dependent response to clinorotation speed based on a shift in the population distribution of cell shape and adhesion area, while osteosarcoma cells do not. These results suggest that a deregulated cell phenotype may possess distinct mechanosensing characteristics, which may be related to our measures of cell adhesion, traction and chemotaxis. Our results are among the first efforts to directly measure the physical interplay between the cell and its substrate during simulated microgravity. This will allow us to gain a deeper understanding of the cellular mechanisms that lead to tissue-level changes, such as atrophy and reduced bone mineral density, observed in astronauts.

Finite Element Modeling of Cell Traction

Cell traction forces generated via the actomyosin interaction within the cell play a critical role in many biological and pathological processes including angiogenesis, wound healing, embryogenesis, metastasis, tumor invasion, and inflammation. In the case of tumor invasion, several studies have reported a correlation between cellular traction and invasion potential; however, these results are inconsistent. In their study, Kraning-Rush et al. reported a positive correlation between cellular traction as measured by total force and metastasis potential. On the other hand, Koch et al. reported a negative correlation between tumor cell invasion and cellular traction as measured by strain energy. We hypothesized that this ambiguity could be a result of overlooked effects of other parameters on measured cellular traction. Using finite element modeling of cell traction, we explored the effect of multiple morphological and mechanical parameters on the measured cellular traction using both strain energy and cellular traction forces as the model outputs. Our results demonstrate a clear dependence of strain energy on substrate stiffness, cell polarity, and contact area, whereas, total force was independent of any of the tested parameters. In addition, our results predict the ability of cells to alter the apparent stiffness of their environment by changing their focal adhesion size.

Three-Dimensional Traction Forces Exerted by Filopodia and Membrane Protrusions Drive Neutrophil Invasion

The infiltration of circulating neutrophils into the vascular wall is an early step leading to inflammation and atherogenesis, and requires transendothelial migration and invasion through the basement membrane. While much research has been dedicated to the identification of the signaling cascade involved in neutrophil recruitment, less is known about how neutrophils generate the three-dimensional (3-D) forces and shape changes required for invasion. To address this issue, differentiated HL-60 neutrophil-like cells are plated on a reconstituted basement membrane made of Matrigel and incorporated with chemoattractant fMLP. The cells subsequently adhere and invade into the 3-D Matrigel. Using 3-D Fourier traction force microscopy, we measure the evolution of cell shape and traction stresses in this 3-D invasion model. Our results show that several filopodia-like cell extensions at cell edge lead the way and exert pulling forces, which contributes to generate a large invasive protrusion that burrows into the Matrigel by generating pushing forces. We find that the total pulling force from all the filopodia structures balances the pushing force exerted by the invasive structure, revealing a coordination between morphodynamic changes and 3-D traction stresses during cell invasion. Furthermore, since the number of filopodia exceeds the number of invasive protrusions, this force balance causes the pushing stresses created at the invasive site to be much higher than the pulling stresses exerted by the filopodia. Thus, the 3-D traction forces exerted by filipodia play an important role in regulating protrusion dynamics of invading neutrophils in 3-D microenvironments, and are mechanistically coordinated to enhance invasion.