Integrin-Generated Forces Lead to Streptavidin-Biotin Unbinding in Cellular Adhesions

The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ∼10⁶ - 10⁸ times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods.     

Integrin-Generated Forces Lead to Streptavidin-Biotin Unbinding in Cellular Adhesions

The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ∼10⁶ - 10⁸ times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods.     

Talin B is required for force transmission in morphogenesis of Dictyostelium

Talin plays a key role in the assembly and stabilisation of focal adhesions, but whether it is directly involved in force transmission during morphogenesis remains to be elucidated. We show that the traction force of Dictyostelium cells mutant for one of its two talin genes talB is considerably smaller than that of wild-type cells, both in isolation and within tissues undergoing morphogenetic movement. The motility of mutant cells in tightly packed tissues in vivo or under strong resistance conditions in vitro was lower than that of wild-type cells, but their motility under low external force conditions was not impaired, indicating inefficient transmission of force in mutant cells. Antibody staining revealed that the talB gene product (talin B) exists as small units subjacent to the cell membrane at adhesion sites without forming large focal adhesion-like assemblies. The total amount of talin B on the cell membrane was larger in prestalk cells, which exert larger force than prespore cells during morphogenesis. We conclude that talin B is involved in force transmission between the cytoskeleton and cell exterior.     

Traction forces exerted through N-cadherin contacts

BACKGROUND INFORMATION: Mechanical forces play an important role in the organization, growth and function of living tissues. The ability of cells to transduce mechanical signals is governed by two types of microscale structures: focal adhesions, which link cells to the extracellular matrix, and adherens junctions, which link adjacent cells through cadherins. Although many studies have examined forces induced by focal adhesions, there is little known about the role of adherens junctions in force-regulation processes. The present study focuses on the determination of force transduction through cadherins at a single cell level. RESULTS: We characterized for the first time the distribution of forces developed by the cell through cadherin contacts. A N-cadherin (neural cadherin)-Fc chimaera, which mimicks the cell adhesion molecule N-cadherin, was immobilized on a muFSA (micro-force sensor array), comprising a dense array of vertical elastomer pillars, which were used both as a cell culture support for N-cadherin-expressing C2 myogenic cells and as detectors for force mapping. We coated the top of the pillars on which cells adhere and recruit adhesion complexes and F-actin. Individual pillar bending allowed the measurement of forces that mainly developed at the cell edge and directed toward their centre. Similar force distribution and amplitude were detected with an unrelated cell line of neuronal origin. Further comparison with forces applied by cells on pillars coated with fibronectin indicates that mechanical stresses transduced through both types of adhesions were comparable in distribution, orientation and amplitude. CONCLUSIONS: These results present a versatile method to measure and map forces exerted by cell-cell adhesion complexes. They show that cells transduce mechanical stress through cadherin contacts which are of the same order as magnitude of those previously characterized for focal adhesions. Altogether, they emphasize the mechanotransduction role of cytoskeleton-linked adhesion receptors of the cadherin family in tissue cohesion and reshaping.     

Mechanotransduction at cadherin-mediated adhesions

Cell-to-cell junctions are crucial mechanical and signaling hubs that connect cells within tissues and probe the mechanics of the surrounding environment. Although the capacity of cell-to-extracellular-matrix (ECM) adhesions to sense matrix mechanics and proportionally modify cell functions is well established, cell-cell adhesions only recently emerged as a new class of force sensors. This finding exposes new pathways through which force can instruct cell functions. This review highlights recent findings, which demonstrate that protein complexes associated with classical cadherins, the principal architectural proteins at cell-cell junctions in all soft tissues, are mechanosensors. We further discuss the current understanding of the rudiments of a cadherin-based mechanosensing and transduction pathway, which is distinct from the force sensing machinery of cell-ECM adhesions.     

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy

The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered–integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin–substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.     

Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2 ~ 5 microm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of alpha(5)- and beta(3)-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800 ~ 1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to alpha(5)beta(1)- and alpha(v)beta(3)-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.     

Force-induced adsorption and anisotropic growth of focal adhesions

Focal adhesions are micrometer-sized protein aggregates that connect actin stress fibers to the extracellular matrix, a network of macromolecules surrounding tissue cells. The actin fibers are under tension due to actin-myosin contractility. Recent measurements have shown that as the actin force is increased, these adhesions grow in size and in the direction of the force. This is in contrast to the growth of condensed domains of surface-adsorbed molecules in which the dynamics are isotropic. We predict these force-sensitive, anisotropic dynamics of focal adhesions from a model for the adsorption of proteins from the cytoplasm to the adhesion site. Our theory couples the mechanical forces and elasticity to the adsorption dynamics via force-induced conformational changes of molecular-sized mechanosensors located in the focal adhesion. We predict the velocity of both the front and back of the adhesion as a function of the applied force. In addition, our results show that the relative motion of the front and back of the adhesion is asymmetric and in different ranges of forces, the adhesion can either shrink or grow in the direction of the force.     

Spatiotemporal constraints on the force-dependent growth of focal adhesions

Focal adhesions (FAs) are the predominant mechanism by which cells mechanically couple to and exert traction forces on their extracellular matrix (ECM). It is widely presumed that FA size is modulated by force to mediate changes in adhesion strength at different levels of cellular tension. However, previous studies seeking correlations between force and FA morphology have yielded variable and often conflicting results. Here we show that a strong correlation between adhesion size and traction force exists only during the initial stages of myosin-mediated adhesion maturation and growth. For mature adhesions, no correlation between traction stress and size is observed. Rather, the tension that is sustained at mature adhesions is more strongly influenced by proximity to the cell edge, with peripheral adhesions transmitting higher tension than adhesions near the cell center. Finally, we show that mature adhesions can withstand sixfold increases in tension without changes in size. Thus, although a strong correlation between adhesion size and mechanical tension is observed during the initial stages of myosin-mediated adhesion maturation, no correlation is observed in mature, elongated adhesions. This work places spatiotemporal constraints on the force-dependent growth of adhesions and provides insight into the mechanical regulation of cell-ECM adhesion.     

Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.     

Mast cell dynamics and involvement in the development of peritoneal adhesions in the rat

Mast cells have been implicated in the ethiopathology of post-operative peritoneal adhesions. However an evaluation of their role in this condition is missing. Adhesions were induced in rats using small intestinal scraping. These rats or rats injected ip with either Stem Cell Factor (SCF) or nedocromil sodium or compound 48/80 (day 0-20) were sacrificed for grading of peritoneal adhesions, for evaluating mast cells and inflammatory cells in adhesions and peritoneal lavage (histochemical staining) and for histamine content (peritoneal lavage, radioenzymatic assay) on days 1-21. Mast cell sonicate was added to intestinal fibroblast and their proliferation was assessed (cell counting). All the rats developed adhesions (day 1) and after 3 days the adhesion score remained constant. Early adhesions were avascular and made of fibrinous exudate containing many mast cells. Thereafter adhesions became denser, and the number of stainable mast cells decreased and then stabilized. On the first few days, inflammatory cells in the peritoneal lavage increased while mast cells and histamine content were significantly reduced indicating their activation. Injection of SCF for 1 week slightly increased peritoneal adhesion formation while nedocromil sodium reduced their development. Compound 48/80 had no significant influence. Addition of mast cell sonicate to normal intestine or to peritoneal adhesion fibroblasts resulted in a significant increase of fibroblast proliferation. In conclusion, mast cell presence correlated with the establishment of peritoneal adhesions, and their pharmacological modulation influenced adhesion formation. In vitro mast cell induced fibroplasia. Therefore, mast cells have a profibrogenic role in this model of peritoneal adhesions.     

Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/Wrch‐1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N‐ and C‐terminal domains. The N‐terminal extension promotes binding to SH3 (Src homology 3)‐domain‐containing adaptors, whereas the C‐terminal extension mediates membrane targeting through palmitoylation of its non‐conventional CAAX box. RhoU/Wrch‐1 possesses transforming activity, which is negatively regulated by its N‐terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c‐Src‐expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N‐terminal extension and the palmitoylation site were dispensable, whereas the C‐terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked‐down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C‐terminal domains are critical for these functions.     

Adhesion of T lymphocytes to human endothelial cells is regulated by the LFA-1 membrane molecule

Human T lymphocyte adhesion to human endothelial cells is the initial event in T cell migration to areas of extravascular inflammation. The molecular basis for T cell-endothelial cell adhesion was investigated using two different cell-cell adhesion assays: a) a fluorescein cell-cell adhesion assay using nonadherent endothelial cells and fluorescein-labeled T lymphocytes, and b) a radionuclide cell-cell adhesion assay using adherent endothelial cells and 51Cr-labelled T cells. Both assay systems demonstrated comparable quantitative assessment of cell-cell adhesions. The assays were performed at 22 degrees C and adhesions were maximal at 30 min. The results of these adhesion assays confirmed previous reports that T cells adhere to endothelial cells. In addition, we have shown that T cells adhere only marginally to foreskin fibroblasts or bone marrow derived fibroblasts. T cell-endothelial cell adhesions were significantly stronger than either monocytes or B lymphoblastoid cells adhesion to endothelial cells. To demonstrate the molecular mechanisms involved in regulating T cell-endothelial cell adhesions, a panel of function-associated monoclonal antibodies (MAb) were tested for their ability to inhibit T cell adhesion. MAb reactive with the leukocyte surface glycoprotein LFA-1 significantly inhibited T cell-endothelial cell adhesions in both assay systems. In contrast, MAb directed at other surface antigens did not inhibit T cell adhesion. The involvement of the LFA-1 glycoprotein in T lymphocyte adhesion to endothelial cells suggest that the LFA-1 molecule may be important in the regulation of leukocyte interactions.     

Zyxin Is a Transforming Growth Factor-β (TGF-β)/Smad3 Target Gene That Regulates Lung Cancer Cell Motility via Integrin α5β1

Although TGF-β acts as a tumor suppressor in normal tissues and in early carcinogenesis, these tumor suppressor effects are lost in advanced malignancies. Single cell migration and epithelial-mesenchymal transition (EMT), both of which are regulated by TGF-β, are critical steps in mediating cancer progression. Here, we sought to identify novel direct targets of TGF-β signaling in lung cancer cells and have indentified the zyxin gene as a target of Smad3-mediated TGF-β1 signaling. Zyxin concentrates at focal adhesions and along the actin cytoskeleton; as such, we hypothesized that cytoskeletal organization, motility, and EMT in response to TGF-β1 might be regulated by zyxin expression. We show that TGF-β1 treatment of lung cancer cells caused rapid phospho-Smad3-dependent expression of zyxin. Zyxin expression was critical for the formation and integrity of cell adherens junctions. Silencing of zyxin decreased expression of the focal adhesion protein vasodilator-activated phospho-protein (VASP), although the formation and morphology of focal adhesions remained unchanged. Zyxin-depleted cells displayed significantly increased integrin α5β1 levels, accompanied by enhanced adhesion to fibronectin and acquisition of a mesenchymal phenotype in response to TGF-β1. Zyxin silencing led to elevated integrin α5β1-dependent single cell motility. Importantly, these features are mirrored in the K-ras-driven mouse model of lung cancer. Here, lung tumors revealed decreased levels of both zyxin and phospho-Smad3 when compared with normal tissues. Our data thus demonstrate that zyxin is a novel functional target and effector of TGF-β signaling in lung cancer. By regulating cell-cell junctions, integrin α5β1 expression, and cell-extracellular matrix adhesion, zyxin may regulate cancer cell motility and EMT during lung cancer development and progression.     

Distinct Roles of Frontal and Rear Cell-Substrate Adhesions in Fibroblast Migration

Cell migration involves complex physical and chemical interactions with the substrate. To probe the mechanical interactions under different regions of migrating 3T3 fibroblasts, we have disrupted cell-substrate adhesions by local application of the GRGDTP peptide, while imaging stress distribution on the substrate with traction force microscopy. Both spontaneous and GRGDTP-induced detachment of the trailing edge caused extensive cell shortening, without changing the overall level of traction forces or the direction of migration. In contrast, disruption of frontal adhesions caused dramatic, global loss of traction forces before any significant shortening of the cell. Although traction forces and cell migration recovered within 10-20 min of transient frontal treatment, persistent treatment with GRGDTP caused the cell to develop traction forces elsewhere and reorient toward a new direction. We conclude that contractile forces of a fibroblast are transmitted to the substrate through two distinct types of adhesions. Leading edge adhesions are unique in their ability to transmit active propulsive forces. Their functions cannot be transferred directly to existing adhesions upon detachment. Trailing end adhesions create passive resistance during cell migration and readily redistribute their loads upon detachment. Our results indicate the distinct nature of mechanical interactions at the leading versus trailing edges, which together generate the mechanical interactions for fibroblast migration.     

原子力显微镜研究粘附分子与细胞膜上整合素分子的相互作用

目的：研究肝癌细胞弹性变化对其表达的整合素分子与配体分子相互作用的影响。方法：以壳聚糖/ 聚丙烯酰胺水凝胶作为 可变基底材料,并将人肝肿瘤细胞（HepG2）接种到不同软硬度壳聚糖/ 聚丙烯酰胺水凝胶基底上,利用原子力显微镜力与距离模 式定量测定不同软硬基底上生长的HepG2 肝瘤细胞膜表面整合素分子与层粘连蛋白分子之间相互作用力。结果：功能化的原子 力显微镜探针与不同软硬基底上生长的细胞所产生的粘附情况不相同,细胞生长在培养皿的为对照组;细胞生长在硬度为1000 Pa 壳聚糖/聚丙烯酰胺水凝胶基底上的为实验组,表达在HepG2 肝瘤细胞膜上的alpha-6-beta-1 整合素与其配体层粘连蛋白相互作用力 的大小分别为19± 7 pN和38.85± 19.7 pN。结论：基底软硬度会影响细胞整合素与配体分子间的相互作用。     

Vinculin在细胞响应力学刺激中的作用

Vinculin是一种细胞骨架蛋白兼粘着斑组成蛋白,主要分布于细胞 细胞连接处及细胞 细胞外基质(extracellular matrix, ECM)粘着斑部位．Vinculin通过与多种粘着斑蛋白、细胞骨架蛋白及细胞骨架F-肌动蛋白相结合并相互作用,参与细胞的力 化学信号转导,在细胞粘附、伸展、运动、增殖、存活等过程中起重要作用．本文结合本课题组研究工作,在介绍vinculin分子结构的基础上,对其在细胞力 化学信号转导中的作用做一综述．     

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.     

Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

ABSTRACT

Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.     

Quantitative measurement of changes in adhesion force involving focal adhesion kinase during cell attachment, spread, and migration

Focal adhesion kinase (FAK) is a critical protein for the regulation of integrin-mediated cellular functions and it can enhance cell motility in Madin-Darby canine kidney (MDCK) cells by hepatocyte growth factor (HGF) induction. We utilized optical trapping and cytodetachment techniques to measure the adhesion force between pico-Newton and nano-Newton (nN) for quantitatively investigating the effects of FAK on adhesion force during initial binding (5 s), beginning of spreading (30 min), spreadout (12 h), and migration (induced by HGF) in MDCK cells with overexpressed FAK (FAK-WT), FAK-related non-kinase (FRNK), as well as normal control cells. Optical tweezers was used to measure the initial binding force between a trapped cell and glass coverslide or between a trapped bead and a seeded cell. In cytodetachment, the commercial atomic force microscope probe with an appropriate spring constant was used as a cyto-detacher to evaluate the change of adhesion force between different FAK expression levels of cells in spreading, spreadout, and migrating status. The results demonstrated that FAK-WT significantly increased the adhesion forces as compared to FRNK cells throughout all the different stages of cell adhesion. For cells in HGF-induced migration, the adhesion force decreased to almost the same level (approximately 600 nN) regardless of FAK levels indicating that FAK facilitates cells to undergo migration by reducing the adhesion force. Our results suggest FAK plays a role of enhancing cell adhesive ability in the binding and spreading, but an appropriate level of adhesion force is required for HGF-induced cell migration.