Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.     

The LINC complex is required for endothelial cell adhesion and adaptation to shear stress and cyclic stretch

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the innermost surfaces of blood vessels and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein that displaces endogenous nesprins from the nuclear envelope and disrupts nuclear–cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell–cell adhesion and barrier function, as well as altered cell–matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH–expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis.     

Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress

Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complementary techniques of photoactivation of fluorescence and fluorescence recovery after photobleaching, we have characterized endothelial actin cytoskeleton dynamics during the alignment process in response to steady laminar fluid flow and have correlated these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movement by 50%. Although movement slows, the actin filament turnover rate increases threefold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by dispersed nonconfluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells, in which cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports the notion that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returned to pre-shear stress levels but actin remodeling remained highly dynamic in many cells after alignment, suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin-remodeling response that is used for endothelial cell shape change instead of bulk cell translocation. atherosclerosis; cytoskeletal dynamics; endothelial cells; mechanotransduction     

Intermediate filaments and stress

Before we can explain why so many closely related intermediate filament genes have evolved in vertebrates, while maintaining such dramatically tissue specific expression, we need to understand their function. The best evidence for intermediate filament function comes from observing the consequences of mutation and mis-expression, primarily in human tissues. Mostly these observations suggest that intermediate filaments are important in allowing individual cells, the tissues and whole organs to cope with various types of stress, in health and disease. Exactly how they do this is unclear and many aspects of cell dysfunction have been associated with intermediate filaments to date. In particular, it is still not clear whether the non-mechanical functions now being attributed to intermediate filaments are primary functions of these structural proteins, or secondary consequences of their function to respond to mechanical stress. We discuss selected situations in which responses to stress are clearly influenced by intermediate filaments.     

Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

Role of viscoelastic properties of differentiated SH-SY5Y human neuroblastoma cells in cyclic shear stress injury

Shear stress and strain lead to neurodegeneration in vivo during head injury, glaucoma, and certain repetitive motion disorders. In vitro, shear stress and strain have been shown to lead to cell injury in a number of models using neurons and neuron-like cells. In the present study we examined the relationship between shear stress, strain, and the extent of cell injury in a cyclic shear stress induced model of cell injury using differentiated SH-SY5Y (human neuroblastoma) cells. Shear stress led to cell strain that increased with increasing stress and diminished upon cessation of shear. Strain rate during cyclic application of shear stress increased by over an order of magnitude from the first to all subsequent cycles, suggesting that the cell and/or its polymer network became more elastic upon cyclic shear stress application. To support this conclusion we measured the degree of cytoskeletal polymerization before and after exposure of cells to cyclic shear stress and found that the fraction of polymerized tubulin in the cell relative to total tubulin decreased by a factor of 2 after six cycles of shear stress. The extent of injury, as indicated by the fraction of cells with fragmented DNA, was three times higher for cyclic shear stress than for steady shear stress and may be related to the change in strain rate and/or cytoskeletal reorganization associated with cyclic stress. These findings may aid in understanding the mechanism by which neurons and neuron-like cells respond to cyclic shear stress and strain and lead to new treatments for disease or injury arising from the exposure of neurons to abnormal cyclic or repetitive stress and strain.     

The intermediate filament network in cultured human keratinocytes is remarkably extensible and resilient

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes.     

Tensile behavior of cortical bone: dependence of organic matrix material properties on bone mineral content

A porous composite model is developed to analyze the tensile mechanical properties of cortical bone. The effects of microporosity (volksman's canals, osteocyte lacunae) on the mechanical properties of bone tissue are taken into account. A simple shear lag theory, wherein tensile loads are transferred between overlapped mineral platelets by shearing of the organic matrix, is used to model the reinforcement provided by mineral platelets. It is assumed that the organic matrix is elastic in tension and elastic-perfectly plastic in shear until it fails. When organic matrix shear stresses at the ends of mineral platelets reach their yield values, the stress-strain curve of bone tissue starts to deviate from linear behavior. This is referred as the microscopic yield point. At the point where the stress-strain behavior of bone shows a sharp curvature, the organic phase reaches its shear yield stress value over the entire platelet. This is referred as the macroscopic yield point. It is assumed that after macroscopic yield, mineral platelets cannot contribute to the load bearing capacity of bone and that the mechanical behavior of cortical bone tissue is determined by the organic phase only. Bone fails when the principal stress of the organic matrix is reached. By assuming that mechanical properties of the organic matrix are dependent on bone mineral content below the macroscopic yield point, the model is used to predict the entire tensile mechanical behavior of cortical bone for different mineral contents. It is found that decreased shear yield stresses and organic matrix elastic moduli are required to explain the mechanical behavior of bones with lowered mineral contents. Under these conditions, the predicted values (elastic modulus, 0.002 yield stress and strain, and ultimate stress and strain) are within 15% of experimental data.     

Progerin-expressing endothelial cells are unable to adapt to shear stress

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a single-point mutation in the lamin A gene, resulting in a truncated and farnesylated form of lamin A. This mutant lamin A protein, known as progerin, accumulates at the periphery of the nuclear lamina, resulting in both an abnormal nuclear morphology and nuclear stiffening. Patients with HGPS experience rapid onset of atherosclerosis, with death from heart attack or stroke as teenagers. Progerin expression has been shown to cause dysfunction in both vascular smooth muscle cells and endothelial cells (ECs). In this study, we examined how progerin-expressing endothelial cells adapt to fluid shear stress, the principal mechanical force from blood flow. We compared the response to shear stress for progerin-expressing, wild-type lamin A overexpressing, and control endothelial cells to physiological levels of fluid shear stress. Additionally, we also knocked down ZMPSTE24 in endothelial cells, which results in increased farnesylation of lamin A and similar phenotypes to HGPS. Our results showed that endothelial cells either overexpressing progerin or with ZMPSTE24 knockdown were unable to adapt to shear stress, experiencing significant cell loss at a longer duration of exposure to shear stress (3 days). Endothelial cells overexpressing wild-type lamin A also exhibited similar impairments in adaptation to shear stress, including similar levels of cell loss. Quantification of nuclear morphology showed that progerin-expressing endothelial cells had similar nuclear abnormalities in both static and shear conditions. Treatment of progerin-expressing cells and ZMPSTE24 KD cells with lonafarnib and methystat, drugs previously shown to improve HGPS nuclear morphology, resulted in improvements in adaptation to shear stress. Additionally, the prealignment of cells to shear stress before progerin-expression prevented cell loss. Our results demonstrate that changes in nuclear lamins can affect the ability of endothelial cells to properly adapt to shear stress.     

Altered architecture and cell populations affect bone marrow mechanobiology in the osteoporotic human femur

Age-related increases in trabecular bone porosity, as seen in osteoporosis, not only affect the strength and stiffness, but also potentially the mechanobiological response of bone. The mechanical interaction between trabecular bone and bone marrow is one source of mechanobiological signaling, as many cell populations in marrow are mechanosensitive. However, measuring the mechanics of this interaction is difficult, due to the length scales and geometric complexity of trabecular bone. In this study, a multi-scale computational scheme incorporating high-resolution, tissue-level, fluid–structure interaction simulations with discrete cell-level models was applied to characterize the potential effects of trabecular porosity and marrow composition on marrow mechanobiology in human femoral bone. First, four tissue-level models with different volume fractions (BV/TV) were subjected to cyclic compression to determine the continuum level shear stress in the marrow. The calculated stress was applied to three detailed models incorporating individual cells and having differing adipocyte fractions. At the tissue level, compression of the bone along its principal mechanical axis induced shear stress in the marrow ranging from 2.0 to 5.6 Pa, which increased with bone volume fraction and strain rate. The shear stress was amplified at the cell level, with over 90% of non-adipocyte cells experiencing higher shear stress than the applied tissue-level stress. The maximum shear stress decreased by 20% when the adipocyte volume fraction (AVF) increased from 30%, as seen in young healthy marrow, to 45 or 60% AVF typically found in osteoporotic patients. The results suggest that increasing AVF has similar effects on the mechanobiological signaling in bone marrow as decreased volume fraction.     

Nanomechanics of Native Thick Filaments from Indirect Flight Muscles

During flight, the wings of Drosophila melanogaster beat nearly 200 times per second. The indirect flight muscle fibers that power this movement have evolved to resist the repetitive mechanical stress that results from the 5-ms wing beat cycle at a strain amplitude of 3.5%. In order to understand how this is achieved at the sarcomere level, we have analyzed the mechanical properties of native thick filaments isolated from indirect flight muscle. Single filaments adsorbed onto a solid support were manipulated in physiological buffer using an atomic force microscope. Images taken after the manipulation revealed that segments were stretched, on average, to 150%, with a maximum at 385% extension. The lateral-force-versus-displacement curve associated with each manipulation contained information about the bending and tensile properties of each filament. The bending process was dominated by shearing between myosin dimers and yielded a shear modulus between 3 and 13 MPa. Maximum tension along the stretched filaments was observed at ∼ 200% extension and varied between 8 and 17 nN. Based on current models of thick filament structure, these variations can be attributed to cross-links between myosin dimers distributed along the filament.     

Interactions between peripherin and neurofilaments in cultured cells: disruption of peripherin assembly by the NF-M and NF-H subunits.

Neurofilaments are the principal intermediate filament type expressed by neurons. They are formed by the co-assembly of three subunits: NF-L, NF-M, and NF-H. Peripherin is another intermediate filament protein expressed mostly in neurons of the peripheral nervous system. In contrast to neurofilaments, peripherin can self-assemble to establish an intermediate filament network in cultured cells. The co-expression of neurofilaments and peripherin is found mainly during development and regeneration. We used SW13 cells devoid of endogenous cytoplasmic intermediate filaments to assess the exact assembly characteristics of peripherin with each neurofilament subunit. Our results demonstrate that peripherin can assemble with NF-L. In contrast, the co-expression of peripherin with the large neurofilament subunits interferes with peripherin assembly. These results confirm the existence of interactions between peripherin and neurofilaments in physiological conditions. Moreover, they suggest that perturbations in the stoichiometry of neurofilaments can have an impact on peripherin assembly in vivo.     

Keratin filament suspensions show unique micromechanical properties.

All epithelial cells feature a prominent keratin intermediate filament (IF) network in their cytoplasm. Studies in transgenic mice and in patients with inherited epithelial fragility syndromes showed that a major function of keratin IFs is to provide mechanical support to epithelial cell sheets. Yet the micromechanical properties of keratin IFs themselves remain unknown. We used rheological methods to assess the properties of suspensions of epidermal type I and type II keratin IFs and of vimentin, a type III IF polymer. We find that both types of IFs form gels with properties akin to visco-elastic solids. With increasing deformation they display strain hardening and yield relatively rapidly. Remarkably, both types of gels recover their preshear properties upon cessation of the deformation. Repeated imposition of small deformations gives rise to a progressively stiffer gel for keratin but not vimentin IFs. The visco-elastic moduli of both gels show a weak dependence upon the frequency of the input shear stress and the concentration of the polymer, suggesting that both steric and nonsteric interactions between individual polymers contribute to the observed mechanical properties. In support of this, the length of individual polymers contributes only modestly to the properties of IF gels. Collectively these properties render IFs unique among cytoskeletal polymers and have strong implications for their function in vivo.     

Effects of cell tension on the small GTPase Rac

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.     

Modeling the tensile mechanical behavior of bone along the longitudinal direction

The tensile stress-strain behavior of bone along its longitudinal axis is modeled by using a simple shear-lag theory, wherein, stresses and strains in a unit cell consisting of an organic matrix reinforced by overlapped mineral platelets are computed. It is assumed that loads are transferred between overlapped mineral-platelets by shear in the organic matrix. The mechanical behavior of bone in which the matrix partially or completely debonds from the sides of the overlapped mineral platelets (after an ultimate interfacial shear stress value is exceeded) is modeled. It is shown that the tensile mechanical behavior of bone can be modeled only by assuming little or no debonding of the organic from the mineral. A physical phenomenon that explains the tensile behavior of bone is, after the interfacial shear stress has reached a constant value over the length of the mineral platelets, the collagen molecules/microfibrils (with the associated mineral platelets) move relative to one another. The tensile stress-strain curve of bovine bone is modeled using this model. The theory predicts the mechanical behavior of the tissue in the elastic, yield and post-yield region. The ultimate strain and strengths are not predicted in the present model.     

Calcium-Activated Proteolysis of Neurofilament Proteins in the Squid Giant Neuron

The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium-activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [gamma-32P]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220-kilodalton (NF-220) and a high-molecular-weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium-activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 110K protein is the putative side arm of the NF-220. In contrast, neither the HMW or the NF-220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium-activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium-activated proteolysis generated characteristic fragments that could be labeled with the anti-intermediate filament antibody.     

Micropatterned structural control suppresses mechanotaxis of endothelial cells

Vascular endothelial cell migration is critical in many physiological processes including wound healing and stent endothelialization. To determine how preexisting cell morphology influences cell migration under fluid shear stress, endothelial cells were preset in an elongated morphology on micropatterned substrates, and unidirectional shear stress was applied either parallel or perpendicular to the cell elongation axis. On micropatterned 20-μm lines, cells exhibited an elongated morphology with stress fibers and focal adhesion sites aligned parallel to the lines. On 115-μm lines, cell morphology varied as a function of distance from the line edge. Unidirectional shear stress caused unpatterned cells in a confluent monolayer to exhibit triphasic mechanotaxis behavior. During the first 3 h, cell migration speed increased in a direction antiparallel to the shear stress direction. Migration speed then slowed and direction became spatially heterogeneous. Starting 11-12 h after the onset of shear stress, the unpatterned cells migrated primarily in the downstream direction, and migration speed increased significantly. In contrast, mechanotaxis was suppressed after the onset of shear stress in cells on micropatterned lines during the same time period, for the cases of both parallel and perpendicular flow. The directional persistence time was much longer for cells on the micropatterned lines, and it decreased significantly after flow onset. Migration trajectories were highly correlated among micropatterned cells within a three-cell neighborhood, and shear stress disrupted this spatially correlated migration behavior. Thus, presetting structural morphology may interfere with mechanisms of sensing local physical cues, which are critical for establishing mechanotaxis in response to hemodynamic shear stress.     

Numerical investigation of the role of intercellular interactions on collective epithelial cell migration

During collective cell migration, the intercellular forces will significantly affect the collective migratory behaviors. However, the measurement of mechanical stresses exerted at cell–cell junctions is very challenging. A recent experimental observation indicated that the intercellular adhesion sites within a migrating monolayer are subjected to both normal stress exerted perpendicular to cell–cell junction surface and shear stress exerted tangent to cell–cell junction surface. In this study, an interfacial interaction model was proposed to model the intercellular interactions for the first time. The intercellular interaction model-based study of collective epithelial migration revealed that the direction of cell migration velocity has better alignment with the orientation of local principal stress at higher maximum shear stress locations in an epithelial monolayer sheet. Parametric study of the effects of adhesion strength indicated that normal adhesion strength at the cell–cell junction surface has dominated effect on local alignment between the direction of cell velocity vector and the principal stress orientation, while the shear adhesion strength has little effect, which provides compelling evidence to help explain the force transmission via cell–cell junctions between adjacent cells in collective cell motion and provides new insights into “adhesive belt” effects at cell–cell junction.     

Theoretical Predictions of the Effects of Force Transmission by Desmin on Intersarcomere Dynamics

Desmin is an intermediate filament protein in skeletal muscle that forms a meshlike network around Z-disks. A model of a muscle fiber was developed to investigate the mechanical role of desmin. A two-dimensional mesh of viscoelastic sarcomere elements was connected laterally by elastic elements representing desmin. The equations of motion for each sarcomere boundary were evaluated at quasiequilibrium to determine sarcomere stresses and strains. Simulations of passive stretch and fixed-end contractions yielded values for sarcomere misalignment and stress in wild-type and desmin null fibers. Passive sarcomere misalignment increased nonlinearly with fiber strain in both wild-type and desmin null simulations and was significantly larger without desmin. During fixed-end contraction, desmin null simulations also demonstrated greater sarcomere misalignment and reduced stress production compared with wild-type. In simulations with only a fraction of wild-type desmin present, fixed-end stress increased as a function of desmin concentration and this relationship was influenced by the cellular location of the desmin filaments. This model suggests that desmin stabilizes Z-disks and enables greater stress production by providing a mechanical tether between adjacent myofibrils and to the extracellular matrix and that the significance of the tether is a function of its location within the cell.     

Strain field in actin filament network in lamellipodia of migrating cells: Implication for network reorganization

Cell motility is spatiotemporally regulated by interactions among mechanical and biochemical factors involved in the regulation of cytoskeletal actin structure reorganization. Although the molecular mechanisms underlying cell motility have been well investigated, the contributions of mechanical factors such as strain in the network reorganization remain unclear. In this study, we have quantitatively evaluated the strain field in the actin filament network forming the lamellipodia of migrating fish keratocytes to elucidate the mechanism by which actin filament network reorganization is regulated by biomechanical factors. The results highlight the existence of a negative (compressive) strain in the lamellipodia whose direction is parallel to that of cell movement. A close correlation was found between the distributions of the strain and the actin filament density in the lamellipodia, suggesting that negative strain may be involved in filament depolymerization. Based on this result, we propose a selective depolymerization model which suggests that negative strain may couple with biomechanical factors such as ADF/cofilin to promote selective depolymerization of filaments oriented in the direction of the deformation because such filaments experience relatively higher levels of the deformation. This model, in conjunction with others, may explain the observed reduction in filament density and the reorganization of actin filament network at the back of the lamellipodia of migrating fish keratocytes. Thus, we suggest that by coupling with biochemical factors, mechanical factors are involved in the regulation of actin filament depolymerization, thereby contributing to the regulation of cell motility.