Modeling actin filament reorganization in endothelial cells subjected to cyclic stretch

Hemodynamic forces affect endothelial cell morphology and function. In particular, circumferential cyclic stretch of blood vessels, due to pressure changes during the cardiac cycle, is known to affect the endothelial cell shape, mediating the alignment of the cells in the direction perpendicular to stretch. This change in cell shape proceeds a drastic reorganization at the internal level. The cellular scaffolding, mainly composed of actin filaments, reorganize in the direction which later becomes the cell’s long axis. How this external mechanical stimulus is ’sensed’ and transduced into the cell is still unknown. Here, we develop a mathematical model depicting the dynamics of actin filaments, and the influence of the cyclic stretch of the substratum based on the experimental evidence that external stimuli may be transduced inside the cell via transmembrane proteins which are coupled with actin filaments on the cytoplasmic side. Based on this view, we investigate two approaches describing the formulation of the transduction mechanisms involving the coupling between filaments and the membrane proteins. As a result, we find that the mechanical stimulus could cause the experimentally observed reorganization of the entire cytoskeleton simply by altering the dynamics of the filaments connected with the integral membrane proteins, as described in our model. Comparison of our results with previous studies of cytoskeletal dynamics reveals that the cytoskeleton, which, in the absence of the effect of stretch would maintain its isotropic distribution, slowly aligns with the precise direction set by the external stimulus. It is found that even a feeble stimulus, coupled with a strong internal dynamics, is sufficient to align actin filaments perpendicular to the direction of stretch.     

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     

Substrate deformation determines actin cytoskeleton reorganization: A mathematical modeling and experimental study

A mathematical model has been developed to define the relationship between the actin cytoskeleton reorganization of a cell and substrate deformation acting on the cell. The model is based on the following major assumptions: (a) normal substrate strain, not the shear substrate strain, determines the actin cytoskeleton reorganization; (b) the normal substrate strain is transmitted to individual actin filaments; (c) each actin filament has a basal strain energy (BSE) when the cell adheres to the substrate without stretching; and (d) the actin filaments undergo disassembly when their strain energies are decreased to zero or increased to twice their BSEs. The resulting model predicts that the actin filaments are formed in the direction where their BSEs are minimally altered. This direction is therefore the one without normal substrate strain. The prediction was confirmed by experiments conducted on both fibroblasts and endothelial cells. The present model may be relevant for understanding better the effects of mechanical stimuli on the cells.     

Biorheological views of endothelial cell responses to mechanical stimuli

Vascular endothelial cells are located at the innermost layer of the blood vessel wall and are always exposed to three different mechanical forces: shear stress due to blood flow, hydrostatic pressure due to blood pressure and cyclic stretch due to vessel deformation. It is well known that endothelial cells respond to these mechanical forces and change their shapes, cytoskeletal structures and functions. In this review, we would like to mainly focus on the effects of shear stress and hydrostatic pressure on endothelial cell morphology. After applying fluid shear stress, cultured endothelial cells show marked elongation and orientation in the flow direction. In addition, thick stress fibers of actin filaments appear and align along the cell long axis. Thus, endothelial cell morphology is closely related to the cytoskeletal structure. Further, the dynamic course of the morphological changes is shown and the related events such as changes in mechanical stiffness and functions are also summarized. When endothelial cells were exposed to hydrostatic pressure, they exhibited a marked elongation and orientation in a random direction, together with development of centrally located, thick stress fibers. Pressured endothelial cells also exhibited a multilayered structure with less expression of VE-cadherin unlike under control conditions. Simultaneous loading of hydrostatic pressure and shear stress inhibited endothelial cell multilayering and induced elongation and orientation of endothelial cells with well-developed VE-cadherin in a monolayer, which suggests that for a better understanding of vascular endothelial cell responses one has to take into consideration the combination of the different mechanical forces such as exist under in vivo mechanical conditions.     

Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm² for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.     

Controlling cytoskeleton structure by phosphoinositide-protein interactions: phosphoinositide binding protein domains and effects of lipid packing

Cell movement and resistance to mechanical forces are largely governed by the cytoskeleton, a three-dimensional network of protein filaments that form viscoelastic networks within the cytoplasm. The cytoskeleton underlying the plasma membrane of most cells is rich in actin filaments whose assembly and disassembly are regulated by actin binding proteins that are stimulated or inhibited by signals received and transmitted at the membrane/cytoplasm interface. Inositol phospholipids, or phosphoinositides, are potent regulators of many actin binding proteins, and changes in the phosphorylation of specific phosphoinositide species or in their spatial localization are associated with cytoskeletal remodeling in vitro. This review will focus on recent studies directed at defining the structural features of phosphoinositide binding sites in actin binding proteins and on the influence of the physical state of phosphoinositides on their ability to interact with their target proteins.     

Flow-induced focal adhesion remodeling mediated by local cytoskeletal stresses and reorganization

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm² for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35-40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.     

Leukotrienes and tyrosine phosphorylation mediate stretching-induced actin cytoskeletal remodeling in endothelial cells

We studied actin cytoskeletal remodeling and the role of leukotrienes and tyrosine phosphorylation in the response of endothelial cells to different types of cyclic mechanical stretching. Human aortic endothelial cells were grown on deformable silicone membranes subjected to either cyclic one-directional (strip) stretching (10%, 0.5 Hz), or biaxial stretching. After 1 min of either type of stretching, actin cytoskeletons of the stretched cells were already disrupted. After stretching for 10 and 30 min, the percentage of the stretched cells that had disrupted actin cytoskeletons were significantly increased, compared with control cells without stretching. Also, at these two time points, biaxial stretching consistently produced higher frequencies of actin cytoskeleton disruption. At 3 h, strip stretching caused the formation of stress fiber bundles, which were oriented nearly perpendicular to the stretching direction. With biaxial stretching, however, actin cytoskeletons in many stretched cells were remodeled into three-dimensional actin structures protruding outside the substrate plane, within which cyclic stretching was applied. In both stretching conditions, actin filaments were formed in the direction without substrate deformation. Moreover, substantially inhibiting either leukotriene production with nordihydroguaiaretic acid or tyrosine phosphorylation with tyrphostin A25 did not block the actin cytoskeletal remodeling. However, inhibiting both leukotriene production and tyrosine phosphorylation completely blocked the actin cytoskeletal remodeling. Thus, the study showed that the remodeling of actin cytoskeletons of the stretched endothelial cells include rapid disruption first and then re-formation. The resulting pattern of the actin cytoskeleton after remodeling depends on the type of cyclic stretching applied, but under either type of cyclic stretching, the actin filaments are formed in the direction without substrate deformation. Finally, leukotrienes and tyrosine phosphorylation are necessary for actin cytoskeletal remodeling of the endothelial cells in response to mechanical stretching.     

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.     

Short-Term Shear Stress Induces Rapid Actin Dynamics in Living Endothelial Cells

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm²). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm² followed by 15 dyn/cm² 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.     

A kinematic model coupling stress fiber dynamics with JNK activation in response to matrix stretching

The role of the actin cytoskeleton in regulating mechanotransduction in response to external forces is complex and incompletely understood. Here, we develop a mathematical model coupling the dynamic disassembly and reassembly of actin stress fibers and associated focal adhesions to the activation of c-jun N-terminal kinase (JNK) in cells attached to deformable matrices. The model is based on the assumptions that stress fibers are pre-extended to a preferred level under static conditions and that perturbations from this preferred level destabilize the stress fibers. The subsequent reassembly of fibers upregulates the rate of JNK activation as a result of the formation of new integrin bonds within the associated focal adhesions. Numerical solutions of the model equations predict that different patterns of matrix stretch result in distinct temporal patterns in JNK activation that compare well with published experimental results. In the case of cyclic uniaxial stretching, stretch-induced JNK activation slowly subsides as stress fibers gradually reorient perpendicular to the stretch direction. In contrast, JNK activation is chronically elevated in response to cyclic equibiaxial stretch. A step change in either uniaxial or equibiaxial stretch results in a short, transient upregulation in JNK that quickly returns to the basal level as overly stretched stress fibers disassemble and are replaced by fibers assembled at the preferred level of stretch. In summary, the model describes a mechanism by which the dynamic properties of the actin cytoskeleton allow cells to adapt to applied forces through turnover and reorganization to modulate intracellular signaling.     

Cytoskeletal mechanics during Shigella invasion and dissemination in epithelial cells

The actin cytoskeleton is key to the barrier function of epithelial cells, by permitting the establishment and maintenance of cell–cell junctions and cell adhesion to the basal matrix. Actin exists under monomeric and polymerized filamentous form and its polymerization following activation of nucleation promoting factors generates pushing forces, required to propel intracellular microorganisms in the host cell cytosol or for the formation of cell extensions that engulf bacteria. Actin filaments can associate with adhesion receptors at the plasma membrane via cytoskeletal linkers. Membrane anchored to actin filaments are then subjected to the retrograde flow that may pull membrane‐bound bacteria inside the cell. To induce its internalization by normally non‐phagocytic cells, bacteria need to establish adhesive contacts and trick the cell into apply pulling forces, and/or to generate protrusive forces that deform the membrane surrounding its contact site. In this review, we will focus on recent findings on actin cytoskeleton reorganization within epithelial cells during invasion and cell‐to‐cell spreading by the enteroinvasive pathogen Shigella, the causative agent of bacillary dysentery.     

Real-time observation of flow-induced cytoskeletal stress in living cells

The mechanical stress due to shear flow has profound effects on cell proliferation, transport, gene expression, and apoptosis. The mechanisms for flow sensing and transduction are unclear, but it is postulated that fluid flow pulls upon the apical surface, and the resulting stress is eventually transmitted through the cytoskeleton to adhesion plaques on the basal surface. Here we report a direct observation of this flow-induced stress in the cytoskeleton in living cells using a parallel plate microfluidic chip with a fluorescence resonance energy transfer (FRET)-based mechanical stress sensor in actinin. The sensing cassette was genetically inserted into the cytoskeletal host protein and transfected into Madin-Darby canine kidney cells. A shear stress of 10 dyn/cm(2) resulted in a rapid increase in the FRET ratio indicating a decrease in stress across actinin with flow. The effect was reversible, and cells were able to respond to repeated stimulation and showed adaptive changes in the cytoskeleton. Flow-induced Ca(2+) elevation did not affect the response, suggesting that flow-induced changes in actinin stress are insensitive to intracellular Ca(2+) level. The reduction in FRET ratio suggests actin filaments are under normal compression in the presence of flow shear stress due to changes in cell shape, and/or actinin is not in series with actin. Treatment with cytochalasin-D that disrupts F-actin reduced prestress and the response to flow. The FRET/flow method is capable of resolving changes of stress in multiple proteins with optical spatial resolution and time resolution >1 Hz. This promises to provide insight into the force distribution and transduction in all cells.     

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.     

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.     

Interplay between Cytoskeletal Stresses and Cell Adaptation under Chronic Flow

Using stress sensitive FRET sensors we have measured cytoskeletal stresses in α-actinin and the associated reorganization of the actin cytoskeleton in cells subjected to chronic shear stress. We show that long-term shear stress reduces the average actinin stress and this effect is reversible with removal of flow. The flow-induced changes in cytoskeletal stresses are found to be dynamic, involving a transient decrease in stress (phase-I), a short-term increase (3–6 min) (Phase-II), followed by a longer-term decrease that reaches a minimum in ∼20 min (Phase-III), before saturating. These changes are accompanied by reorganization of the actin cytoskeleton from parallel F-actin bundles to peripheral bundles. Blocking mechanosensitive ion channels (MSCs) with Gd³⁺ and GsMTx4 (a specific inhibitor) eliminated the changes in cytoskeletal stress and the corresponding actin reorganization, indicating that Ca²⁺ permeable MSCs participate in the signaling cascades. This study shows that shear stress induced cell adaptation is mediated via MSCs.     

Effect of LIMK2 RNAi on reorganization of the actin cytoskeleton in osteoblasts induced by fluid shear stress

The biomechanical characteristics of bone tissue and its cells under mechanical stress are significant for bone biomechanics research, but the mechanism of mechanotransduction is still unknown. It has been established that the actin cytoskeleton of osteoblasts plays an important role in this process. However, the structure of the actin cytoskeleton is reorganized when loaded with mechanical stress, which results in changes in cell stiffness. These phenomena suggest that an actin-cytoskeleton-induced feedback regulation mechanism may be involved in the mechanotransduction of osteoblasts, but this has not yet been proven. The aim of this study was to explore the role of LIMK2 in the reorganization of the actin cytoskeleton induced by fluid shear stress in osteoblasts by using RNA interference. Balb/c mouse primary osteoblasts were divided into four groups. Cells in Groups 1 and 3 were transfected with negative control RNA, while cells in Groups 2 and 4 were transfected with a specific siRNA designed to silence the LIMK2 gene. Twenty-four hours after transfection, cells in Groups 1 and 2 were loaded with fluid shear stress at 12 dyne/cm2 while cells in Groups 3 and 4 were not. Compared with Group 1, the mean fluorescence density of the actin cytoskeleton in the other three groups was 28.9%, 45.7%, and 33.0%, respectively. These results indicate that LIMK2 plays an important role in the reorganization of the actin cytoskeleton induced by fluid shear stress.     

Subcellular Localization of RhoA and Ezrin at Membrane Ruffles of Human Endothelial Cells: Differential Role of Collagen and Fibronectin

Endothelial cells and the regulation of their migration are of prime importance in many physiological and pathological processes such as angiogenesis. RhoA, an important Rho family member known to trigger actin reorganization, has been shown to mediate the formation of focal adhesions and stress fibers in quiescent fibroblasts. However, recent studies have emphasized its functional diversity and its implication in migration or metastatic processes in different cell types other than fibroblasts. Its role in endothelial cells is little known. In this study, we were interested by analyzing in human endothelial cells the subcellular redistribution of endogenous RhoA and the reorganization of cytoskeletal actin induced by two important extracellular matrix proteins, collagen and fibronectin. This paper shows a translocation of RhoA and its association with cortical actin in focal contact domains at membrane ruffles and at lamellipodia of spread or migrating endothelial cells, in the absence of any soluble mitogen stimulation. Furthermore, RhoA was found colocalized with ezrin, a member of the ERM family proteins newly described as important membrane-actin cytoskeleton linkers, at early membrane ruffles of endothelial cells spread on collagen but not on fibronectin. The present study points out that extracellular matrix, depending on the nature of its components, may promote distinct assemblies of focal contact constitutive proteins and strongly suggests that endothelial RhoA, like Rac1, may be an important mediator of matrix signaling pathway regulating endothelial cell adhesiveness and motility, independently of growth factor stimulation.     

Specificity of endothelial cell reorientation in response to cyclic mechanical stretching

Evidence suggests that cellular responses to mechanical stimuli depend specifically on the type of stimuli imposed. For example, when subjected to fluid shear stress, endothelial cells align along the flow direction. In contrast, in response to cyclic stretching, cells align away from the stretching direction. However, a few aspects of this cell alignment response remain to be clarified: (1) Is the cell alignment due to actual cell reorientation or selective cell detachment? (2) Does the resulting cell alignment represent a response of the cells to elongation or shortening, or both? (3) Does the cell alignment depend on the stretching magnitude or rate, or both? Finally, the role of the actin cytoskeleton and microtubules in the cell alignment response remains unclear. To address these questions, we grew human aortic endothelial cells on deformable silicone membranes and subjected them to three types of cyclic stretching: simple elongation, pure uniaxial stretching and equi-biaxial stretching. Examination of the same cells before and after stretching revealed that they reoriented. Cells subjected to either simple elongation or pure uniaxial stretching reoriented specifically toward the direction of minimal substrate deformation, even though the directions for the two types of stretching differed by only about 20°. At comparable stretching durations, the extent of cell reorientation was more closely related to the stretching magnitude than the stretching rate. The actin cytoskeleton of the endothelial cell subjected to either type of stretching was reorganized into parallel arrays of actin filaments (i.e., stress fibers) aligned in the direction of the minimal substrate deformation. Furthermore, in response to equi-biaxial stretching, the actin cytoskeleton was remodeled into a “tent-like” structure oriented out of the membrane plane—again towards the direction of the minimal substrate deformation. Finally, abolishing microtubules prevented neither the formation of stress fibers nor cell reorientation. Thus, endothelial cells respond very specifically to the type of deformation imposed upon them.