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EXTRACELLULAR MATRIX, CELL INTERACTIONS
4β1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation1Department of Cell Biology, Johns Hopkins University School of Medicine, and 2Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland
Submitted 22 March 2008 ; accepted in final form 15 May 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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4β1-Integrin plays a pivotal role in cell migration in vivo. This integrin has been shown to regulate the front-back polarity of migrating cells via localized inhibition of 4-integrin/paxillin binding by phosphorylation at the 4-integrin cytoplasmic tail. Here, we demonstrate that 4β1-integrin regulates directionally persistent cell migration via a more complex mechanism in which 4-integrin phosphorylation and paxillin binding act via both cooperative and independent pathways. We show that, in response to shear flow, 4β1-integrin binding to the CS-1 region of fibronectin was necessary and sufficient to promote directionally persistent cell migration when this integrin was ectopically expressed in CHO cells. Under shear flow, the 4β1-integrin-expressing cells formed a fan shape with broad lamellipodia at the front and retracted trailing edges at the back. This "fanning" activity was enhanced by disrupting paxillin binding alone and inhibited by disrupting phosphorylation alone or together with disrupting paxillin binding. Notably, the phosphorylation-disrupting mutation and the double mutation resulted in the formation of long trailing tails, suggesting that 4-integrin phosphorylation is required for trailing edge retraction/detachment independent of paxillin binding. Furthermore, the stable polarity and directional persistence of shear flow-stimulated cells were perturbed by the double mutation but not the single mutations alone, indicating that paxillin binding and 4-integrin phosphorylation can facilitate directionally persistent cell migration in an independent and compensatory manner. These findings provide a new insight into the mechanism by which integrins regulate directionally persistent cell migration. lamellipodia; fibronectin; shear stress
4β1-Integrin belongs to a unique subgroup of integrins that not only mediate cell-ECM adhesion but also mediate heterophilic cell-cell adhesion by binding to VCAM-1, a member of the immunoglobulin superfamily. 4β1-Integrin-mediated cell-cell adhesion is essential for placental and cardiac development (38) and plays critical roles for leukocytes and pericytes to attach to vascular endothelial cells during extravasation (2) and tumor angiogenesis (9), respectively. 4β1-Integrin also binds to an alternatively spliced CS-1 region of fibronectin, the ECM ligand of 4β1-integrin. 4β1-Integrin-mediated cell-ECM adhesion is required for neural crest cell migration (18) and the migration of progenitor cells during cardiac development (30) and blood vessel remodeling (13). In culture, 4β1-integrin is required for primary embryonic pericytes to migrate in a directionally persistent manner in response to shear force (13). When ectopically expressed in CHO cells, 4β1-integrin promotes cell motility by facilitating the formation of lamellipodia in response to scratch wounding a cell monolayer (28). The mechanism by which 4β1-integrin regulates cell migration involves molecular interactions at the cytoplasmic tail of the 4-subunit (referred to as the 4-tail). The 4-tail is phosphorylated at Ser988 (14) and binds to paxillin, a signaling adaptor protein (24). Furthermore, 4-integrin and paxillin form a ternary complex with G protein-coupled receptor kinase interactor 1 (GIT1), an ADP-ribosylation factor-GTPase-activating protein, and this complex inhibits Rac activity (26). It has been proposed that the 4-integrin-paxillin-GIT1 complex polarizes Rac activities by inhibiting Rac activation at the sides and trailing edge of the cells (26).
Fluid shear stress associated with the flow of blood in the circulatory system plays an important regulatory role in angiogenesis (23), a process that involves the migration of vascular endothelial cells and pericytes. In 4 integrin-deficient mouse embryos, pericytes fail to cover angiogenic blood vessels, and the vessels are dilated (13). In culture, wild-type pericytes migrated in a directionally persistent manner in response to laminar shear flow, and this shear flow-induced motility was abolished by the 4-integrin-null mutation (13). Furthermore, an anti-4-integrin antibody inhibited the functions of endothelial cells during tumor angiogenesis (9). As a critical physiological stimulus, shear flow has a profound effect on vascular endothelial cells and other cell types at the vessel wall and in the blood circulation. Studies on endothelial cells have revealed that shear flow modulates gene expression and promotes cytoskeletal alignment, cell polarization, and cell motility via the activation of intracellular signaling components, including Rac, FAK, and phosphatidylinositol 3-kinase (15, 16, 23, 33, 36, 39). 5β1-Integrin and vβ3-integrin, which are the major components of focal adhesions, are required for some of these shear flow-induced signaling pathways (31).
In the present study, we show that 4β1-integrin is required and sufficient for shear flow-induced directionally persistent cell migration when ectopically expressed in CHO cells. This model system allowed us to analyze the effects of mutations that disrupt phosphorylation and/or paxillin binding at the 4-tail on shear flow-induced cell motility. These experiments provide new insight into the 4β1-integrin-mediated signaling pathway that regulates shear flow-induced directionally persistent cell migration.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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4-tail, respectively, were constructed using the same strategy as that for constructing pQN4Y991AG (28), which carries the Y991A single mutation, except that pQN4Y991AG was used as the PCR template for generating the double mutation in pQN4DMG. Transfection was performed using FuGene6 transfection reagent (Roche Molecular Biochemical) following the manufacturer's instructions. The selection of stable transfectants and culturing of the cell lines have been described by Pinco et al. (28).
Integrin ligands. Rat plasma fibronectin was purchased from Calbiochem (La Jolla, CA). Plasmids pGH-F12-V120-15 (expressing FNIII12-V120-15, which contains CS-1) and pGH-F7-11 (expressing FNIII7-11,which contains RGD and the synergy region), which were derived from pGH.PL (3), were provided by Richard Hynes (Massachusetts Institute of Technology, Cambridge, MA). CS-1- and RGD-containing fragments were purified from pGH-F12-V120-15- or pGH-F7-11-expressing Escherichia coli using glutathione-Sepharose beads (Pierce).
Flow cytometry, immunoprecipitation, and immunoblot analysis.
Flow cytometry, immunoprecipitation, and immunoblot analysis were performed as described by Pinco et al. (28). For flow cytometry, an anti-4-integrin antibody (clone P1H4 from Millipore-Chemicon) and a goat anti-mouse allophycocyanin-conjugated secondary antibody (Santa Cruz Biotechnology) were used. For immunoprecipitation and immunoblot analysis, the following primary antibodies were used: mouse anti-4-integrin (clone P1H4), mouse phosphorylation (phospho)-specific anti-4-integrin (Invitrogen-Biosource), mouse anti-paxillin (BD Transduction Laboratories), and rabbit anti-green fluorescent protein (GFP; for GFP-tagged 4-integrin, Molecular Probes).
Shear flow migration assay. Cells were plated on 35-mm suspension culture dishes precoated with 5 µg/ml fibronectin or other substrates for 2 h at 37°C. After cells had been allowed to adhere for 2 h at 37°C, the dish was assembled onto a perfusion chamber. The flow channel (1.3 x 0.5 x 0.0178 cm) was formed by a cutout in a silicone-based gasket, which was sealed to the transparent polycarbonate surface of the perfusion chamber. The flow chamber assembly was mounted onto the stage of an inverted microscope (Nikon TE300 or Zeiss Axiovert 200M) equipped with phase-contrast optics and enclosed in a 37°C incubator. Cells were subjected to shear stress by continuously perfusing Leibovitz's L-15 CO2-buffered medium (Invitrogen) supplemented with 10% FBS through the chamber for 4 or 6 h. Shear stress was set to 4 dyn/cm2, which is in the range of stresses encountered in the venous circulation in vivo. Digital images of a single x10 field of view were acquired every 30 s. For each experiment, the time-lapse images were compiled into a movie at 30 frames/s. Three independent assays were performed on each cell line.
Immunofluorescence and confocal microscopy.
Cells were plated on 5 µg/ml fibronectin, stimulated with shear flow at various time periods, and fixed in 3% paraformaldehyde in PBS for 10 min at room temperature. Immunofluorescence staining was performed as described by Pinco et al. (28). The following primary antibodies were used: mouse anti-paxillin (1:50), rabbit anti-GFP (1:1,000), mouse anti-GFP (Zymed, 1:100), and rabbit anti-phospho-4-integrin [generated and prepared as described by Han et al. (14), 1:50]. Confocal images were taken using a Zeiss LSM510 confocal microscope.
Detachment assay. Suspension culture dishes (35 mm) were coated with 0.2 µg/ml of the FNIII12-V120-15 fragment of fibronectin for 1 h at 37°C, blocked with 2 mg/ml heat-inactivated BSA overnight at 4°C, assembled to the flow chamber, and mounted onto the microscope stage. Cells were injected into the chamber and allowed to settle for 1 min. Cells were then subjected to a shear stress of 4 dyn/cm2 for 1 min. Thereafter, the wall shear stress was increased stepwise to 32, 64, and 96 dyn/cm2 at 1-min intervals. At the end of each shear stress increment, cells were photographed, and the percentage of cells detached was determined. Means ± SE were calculated from the data of three independent assays for each cell line. Statistical analysis of the data was performed using ANOVA (Microsoft Excel).
Analysis of cell migration speed and directional persistence time.
The centroid position (x,y) of each cell migrating under shear was calculated at each time step (
; 4.5 min) for the entire duration of the experiment (6 h) using ImageJ software (National Institutes of Health). Cells whose migration was not contained within the image field for the duration of the experimental run, as well as the cells that divided during the course of the flow assay, were excluded from this calculation. The distance that the centroid of the cell moved over each value in two-dimensional space was then used to calculate the cell migration speed during this time period. Migration speed data for each cell at all time points were then compiled to obtain the root mean square (RMS) cell migration speed (S) for the entire duration of the shear flow experiment using the following equation:
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Values of S and time-averaged MSD calculated for each cell were then used to determine the directional persistence time (PT), which is the characteristic time in which cell movement persists in the same direction, using the following equation (7, 20, 32):
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Statistics. Data are expressed as means ± SE. Statistical significance of differences between means was determined by Student's t-test or one-way ANOVA where appropriate. If means were shown to be significantly different, multiple comparisons by pairs were performed by the Holm test.
Online data supplements. The online version of this article contains time-lapse movies that accompany Figs. 1 and 3 and additional supplemental movies. Online supplemental material is available at the American Journal of Physiology-Cell Physiology website.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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4β1-Integrin promotes leading edge lamellipodia protrusion and trailing edge detachment/retraction in response to shear flow stimulation.
We have previously shown that 4β1-integrin is required for primary pericytes to respond to shear force and migrate in a directionally persistent manner (13). To elucidate how 4β1-integrin regulates cell motility, we used a CHO cell system. CHO cells do not express 4-integrin endogenously but do express 5β1-integrin. It has previously been shown that, when 4β1-integrin is stably transfected into CHO cells, this integrin promotes the formation of broad lamellipodia in response to scratch wounding a cell monolayer (12, 28). To study the role of 4β1-integrin in directionally persistent cell migration, we tested the motile activities of 4β1-integrin-expressing CHO cells (referred to as CHO-4WT cells) when stimulating the cells with a unidirectional shear flow using a parallel plate flow chamber system (6, 21). When plated on fibronectin or a recombinant FNIII12-V120-15 fragment of fibronectin that contains the CS-1 region (referred to as CS-1), CHO-4WT cells responded to the shear flow and migrated in the direction of the flow. Many migrating cells (51 ± 7.3% and 57 ± 5.3% on fibronectin and CS-1, respectively, P > 0.5) formed broad leading edge lamellipodia in a manner similar to that seen at the edges of scratch wounds (Fig. 1B) (28). These cells also had flattened and often concave trailing edges with a distinct bright phase-contrast halo along the edges, giving the cells a fan-like shape (Fig. 1A,b and c, and movies 2 and 3 in the online data supplements). These fan-shaped cells migrated in a directionally persistent manner, resembling migrating fish epidermal cells (8). In the absence of 4β1-integrin, on the other hand, parental CHO cells (referred to as CHO cells) plated on fibronectin had only a low basal level of the "fanning" activity under shear flow (Fig. 1A,a and movie 1 in the online data supplements), as did CHO-4WT cells plated on a recombinant FNIII7-11 fragment of fibronectin that contained RGD and synergy regions (Fig. 1A,d and movie 4 in the online data supplements). The majority of these cells protruded membrane extensions transiently in random directions. The fanning activity of CHO-4WT cells on fibronectin and CS-1 depended on the shear flow. In the absence of shear flow, most CHO-4WT cells failed to form the fan shape, and their protrusive activities were similar to those of CHO cells (Fig. 1A,e and movies 5 and 6 in the online data supplements). These results indicate that binding between 4β1-integrin and CS-1 is necessary and sufficient for shear flow-induced fanning activities characterized by the formation of a broad leading edge lamellipodium and a flattened trailing edge.
Phosphorylation and paxillin binding at the 4-tail regulate shear flow-induced motile activities.
The 4-tail binds to paxillin and can be phosphorylated at Ser988. It has been previously shown that the lamellipodia-promoting activity of 4β1-integrin is negatively regulated by paxillin binding (28) and requires 4-subunit phosphorylation (12). These conclusions were derived from studies involving two point mutations in the 4-tail: Y991A, which markedly reduces paxillin binding by substituting Tyr991 to Ala, and S988A, which impairs phosphorylation by substituting Ser988 to Ala. Y991A and S988A enhance and inhibit the lamellipodia-promoting activities of 4β1-integrin, respectively (12, 28). To investigate the mechanisms by which 4β1-integrin regulates shear flow-induced cell migration, we examined the effects of these mutations using a shear flow assay. We compared CHO-4WT cells with CHO cell lines that stably express 4-integrin mutants carrying Y991A, S988A, or a Y991A/S988A double mutation (referred to as CHO-4Y991A, CHO-4S988A, and CHO-4DM, respectively). In all of these cell lines, 4-integrin was tagged with GFP, which has been shown not to interfere with the motile activities of 4β1-integrin (28). Flow cytometry analysis showed that the expression levels of 4-integrin were fairly equivalent among all cell lines (Fig. 2A). Furthermore, immunoblot analysis showed that S988A and the double mutation disrupted the phosphorylation site at Ser988 (Fig. 2B) and that Y991A and the double mutation markedly reduced the ability of 4-integrin to coimmunoprecipitate with paxillin (Fig. 2C).
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4-integrin-expressing cells were compared with CHO-4WT cells for their ability to form the fan shape when plated on fibronectin or CS-1 under shear flow. We found that shear flow-induced fanning activities were also observed among CHO-4Y991A cells plated on fibronectin (Fig. 3A, b and movie 7 in the online data supplements) or CS-1 (movie 8 in the online data supplements). The percentage of CHO-4Y991A cells with fanning activity (82 ± 1.8% and 72 ± 1.3% on fibronectin and CS-1, respectively) was significantly higher than that of CHO-4WT cells (P < 0.05; Fig. 3B). On the other hand, CHO-4S988A and CHO-4DM had only low basal levels of fanning activy under shear flow (Fig. 3A,c and d, and B and movies 9–12 in the online data supplements). Thus, fanning activity is enhanced by Y991A but inhibited by S988A and the double mutation.
Under shear flow, a significant proportion of CHO-4S988A and CHO-4DM cells had a polarized "teardrop" shape featured with a pointed or elongated trailing tail (35 ± 8.0% and 37 ± 5.8% on fibronectin and CS-1, respectively, for CHO-4S988A; and 37 ± 2.6% and 37 ± 5.0% on fibronectin and CS-1, respectively, for CHO-4DM, P > 0.5; Fig. 3A,c and d, and movies 9–12 in the online data supplements). This phenotype was rarely observed in the other cell types under the same conditions (Figs. 1B and 3B). The trailing tail phenotype of CHO-4S988A and CHO-4DM cells indicates that 4-integrin phosphorylation plays a critical role in retracting/detaching the trailing edge of shear flow-stimulated cells. This event is likely to be independent of 4-integrin/paxillin interactions because the trailing tail phenotype of the double mutation resembled that of the S988A but not Y991A mutation. Teardrop-shaped CHO-4S988A and CHO-4DM cells did form leading edge lamellipodia, but compared with the broad lamellipodia of fan-shaped CHO-4WT and CHO-4Y991A cells, the lamellipodia protruded by teardrop-shaped cells were narrower and often had irregular shapes. Therefore, 4-integrin phosphorylation is not essential for lamellipodia protrusion, but it does somehow contribute to this event, again in a manner dominant over 4-integrin/paxillin interactions.
4β1-Integrin promotes directionally persistent cell migration under shear flow.
Although both CHO-4S988A and CHO-4DM cells had the same capacity to form a teardrop shape, they did not migrate in the same manner under shear flow. Teardrop-shaped CHO-4S988A cells appeared to migrate in a directionally persistent manner similar to fan-shaped CHO-4WT and CHO-4Y991A cells, whereas teardrop-shaped CHO-4DM cells had less stable front-back polarity (white arrows in Fig. 3A,d) and appeared to be less directionally persistent. To quantify directional persistence, we calculated the directional persistence time (PT), which was the characteristic time that a cell moved in the same direction. We calculated the mean PT for each cell type plated on fibronectin in the absence or presence of shear flow (Fig. 4A). In the absence of shear flow, mean PTs of all cell types were <6 min. Under shear flow, mean PTs for CHO (23 ± 3.5 min) and CHO-4DM cells (25 ± 3.8 min) were similar (P > 0.5); both had an increased mean PT compared with the same cell types without shear flow, consistent with these cells having some directionally persistent movement under shear flow. However, these mean PT values were much lower than those for CHO-4WT (99 ± 25 min), CHO-4Y991A (183 ± 32 min), and CHO-4S988A (75 ± 11 min) cells under shear flow. These data show that 4β1-integrin drastically enhances directionally persistent cell migration, and the ability of 4β1-integrin to promote directionally persistent migration is abolished by disrupting both 4-integrin phosphorylation and paxillin binding but not by the single mutations. Consistent with the enhancing effect of Y991A on fanning activities, under shear flow, the mean PT of CHO-4Y991A cells was significantly higher than that of CHO-4WT cells (P < 0.05). Surprisingly, no statistical differences were observed between mean PT values of CHO-4WT and CHO-4S988A cells under shear flow (P > 0.3), although the double mutation had a strong inhibitory effect (P < 0.005). These results show that 1) paxillin binding negatively regulates the shear flow-induced directional persistence (as shown by CHO-4Y991A cells), 2) 4-integrin phosphorylation is dispensable for 4β1-integrin to promote shear flow-induced directional persistence when paxillin can bind to the 4-tail (as shown by CHO-4S988A cells), and 3) 4-integrin phosphorylation is required for 4β1-integrin to promote shear flow-induced directional persistence when paxillin binding is inhibited (as shown by CHO-4DM cells compared with cells with single mutations). These results suggest that both 4-integrin phosphorylation and paxillin binding/dissociation contribute to the ability of 4β1-integrin to promote directionally persistent cell migration in response to shear force, but via a complex mechanism where the two events act in a compensatory manner.
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4Y991A correlated with enhanced fanning activity. To directly assess the correlation between the cell shape and directional persistence, we calculated the mean PT for cells that formed the fan or teardrop shape or for those with neither shape (Fig. 4B). For all cell types except CHO-4DM cells, cells with polarized shapes (fan or teardrop shape) had similar mean PTs (P > 0.3) and much higher mean PTs than cells without polarized shapes (P < 0.001). Thus, there is a tight correlation between stably polarized shapes and directional persistence. We conclude that 4β1-integrin promotes directionally persistence migration by stabilizing front-back cell polarity.
4β1-Integrin enhances cell migration speed but does not influence cell adhesion strength on fibronectin under shear flow.
It is possible that 4β1-integrin-promoted cell shape changes also influence the speed of cell migration. Using the coordinates of the centroid positions of the cells, we calculated, for each cell, RMS speed, defined as the square root of the averaged square of cell speeds at each time step value (Fig. 4C). This calculation reflects the migration speed of a cell along the migration path. As observed for the directional persistence, for all cell types except CHO-4DM cells, cells with polarized shapes (fan or teardrop shape) had significantly higher migration speeds than cells without these shapes (P < 0.001). Furthermore, fan-shaped CHO-4WT and teardrop-shaped CHO-4S988A cells, which had similar directional persistence, had no significant differences in migration speeds (P > 0.5). Differing from directional PTs for specific cell shapes (Fig. 4B), fan-shaped CHO-4Y991A cells migrated at a faster pace than fan-shaped CHO-4WT cells (P < 0.001), indicating that cell shape and migration speed do not have a tight causal relationship.
Adhesive strength is known to play critical roles in regulating cell migration. The mutations at the 4-tail may confer changes in adhesion strength that could potentially affect the motile activities of the cells. To test this possibility, the adhesion strength of 4β1-integrin-expressing cells on CS-1 under shear flow was determined by assessing the percentage of cells detaching from CS-1-coated surfaces under increasing shear stresses up to 96 dyn/cm2, a force much stronger than the 4 dyn/cm2 force used in our shear flow assays (Fig. 4D). Among the wild-type and mutant 4-integrin-expressing cell types, no significant differences were observed in the adhesion strength on CS-1, even at 96 dyn/cm2 (P > 0.5). We conclude that in our CHO cell system, paxillin binding and 4-integrin phosphorylation do not play a major role in modulating the adhesive strength of the cells under shear flow.
Distribution of paxillin, 4-integrin, and phospho-4-integrin under shear flow.
It has been reported that, in randomly migrating cells, paxillin and 4β1-integrin colocalized at the sides and trailing edge but not at the leading edge, whereas phospho-4-integrin is localized preferentially at the leading edge (12). This observation is consistent with a model in which 4-integrin phosphorylation inhibits 4-integrin/paxillin binding at the leading edge, thus promoting the formation of leading edge lamellipodia (26). Our shear flow experiments suggested that 4-integrin phosphorylation may have additional functions. To understand how 4-integrin phosphorylation and paxillin binding regulate shear flow-induced motile activities, we examined the distribution of 4-integrin, phospho-4-integrin, and paxillin in CHO-4WT cells when cells were plated on fibronectin and subjected to shear flow. Cells began to polarize in shape after 15 min of shear flow stimulation, and, after 4 h, many cells formed the fan shape. At both time points, 4-integrin localized throughout the cell surface with higher intensity of staining at the leading and trailing edges and phospho-4-integrin colocalized with 4-integrin (Fig. 5, d–i). The staining of phospho-4-integrin was specific since there was little staining of phospho-4-integrin on the plasma membrane of CHO-4S988A cells (Fig. 5b). The distribution of phospho-4-integrin in CHO-4WT cells is consistent with 4-integrin phosphorylation acting at both the leading and trailing edges of the cells under shear flow.
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4WT cells responded to the shear flow and began to protrude lamellipodia at the leading edge. As reported for CHO-4WT cells stimulated with scratch wounding (12, 28), when the cells were plated on fibronectin and stimulated with shear flow, 4-integrin was localized in punctate structures at the leading edge of protruding lamellipodia, where most of the 4-integrin positive puncta had little overlap with paxillin-positive focal complexes and focal adhesions (Fig. 6, c and f, marked by arrows). In the areas at the leading edge flanking the protruding membrane, however, prominent colocalization of the two proteins was observed (Fig. 6, c and f, marked by asterisks). At the 4-h time point, 4-integrin and paxillin had little apparent colocalization at the leading edge (Fig. 6l). These results are consistent with 4-integrin/paxillin binding playing a negative regulatory role in the formation of lamellipodia. At both the 15-min and 4-h time points, little apparent colocalization of 4-integrin and paxillin was observed at the trailing edge, suggesting that these two proteins had little interactions in this area.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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4-tail in regulating directionally persistent cell migration under shear flow.
In this study, we showed that 4β1-integrin is necessary and sufficient to promote directionally persistent cell migration in response shear flow when ectopically expressed in CHO cells. In addition to the reported role of 4β1-integrin in facilitating the formation of lamellipodia at the leading edge (12, 29), we found that 4β1-integrin also regulated trailing edge detachment/retraction. Notably, we found that disruption of serine phosphorylation alone or together with disruption of paxillin binding at the 4-tail led to the formation of long trailing tails, which is in contrast to the flattened and retracted trailing edges formed by cells expressing wild-type 4-integrin. This trailing tail phenotype is reminiscent of that caused by inhibiting RhoA and its downstream effectors (37) and by knockdown of nonmuscle myosin IIA (34). We propose that 4-integrin phosphorylation at the trailing edge transduces signals into cells via the Rho-mediated contractility pathway, which regulates trailing edge retraction and/or focal adhesion dynamics (4), and this function is likely to be independent of the binding between 4-integrin and paxillin.
In a recent model proposed by Ginsberg and colleagues (11), restriction of 4-integrin phosphorylation to the leading edge of a migrating cell limits 4-integrin-paxillin binding to the sides and trailing edge of the cell; the 4-integrin-paxillin complex inhibits Rac1 activities, thus confining lamellipodia formation to the leading edge (26). This model is based on studies of randomly migrating cells. In this study, we examined cells stimulated with shear flow and provided evidence for a more complex mechanism. Consistent with Ginsberg et al.'s model, we showed that inhibiting paxillin binding to the 4-tail enhances lamellipodia protrusion and that there is little colocalization of 4-integrin and paxillin at the edges of protruding membrane at the leading edge, whereas the two proteins colocalize in areas flanking the protruding membrane. These results support a role for paxillin binding/dissociation in regulating the protrusive activities at the leading edge. However, we also found that 4-integrin phosphorylation functions at the trailing edges of shear flow-stimulated cells. Furthermore, the double mutation, which disrupts both 4-integrin phosphorylation and paxillin binding, impaired the ability of 4β1-integrin to promote directionally persistent cell migration, whereas the single mutations had no inhibitory effects. Therefore, both 4-integrin phosphorylation and paxillin binding/dissociation contribute to stabilizing front-back polarity and directional persistence, but they are likely to act via independent pathways. Our results, nevertheless, do not rule out a possible role of 4-integrin phosphorylation in regulating paxillin binding to establish the polarity of migrating cells. Although we showed that 4-integrin phosphorylation can occur at both the leading and trailing edges, the possibility that there are subtle temporal and spatial differences in 4-integrin phosphorylation levels between the front and back of shear flow-stimulated cells still remains.
Role of 4β1-integrin in transducing signals in response to shear stress.
The physiological role of the 4β1-integrin-dependent shear flow response has been implicated in blood vessel remodeling (13). This integrin also contributes to the functions of endothelial cells during tumor angiogenesis (9) and may mediate the shear flow-induced migration of these cells. In the present study, we demonstrated that the role of 4β1-integrin in shear flow-induced cell migration is recapitulated when 4β1-integrin is ectopically expressed in CHO cells. Analysis of this model system allowed us to elucidate the mechanism by which 4β1-integrin facilitates the shear flow response. One possible mechanism is that shear flow may regulate cell migration by modulating the adhesive activities of 4β1-integrin. Shear flow has been shown to activate 4β1-integrin in leukocytes to facilitate adhesion of these cells to endothelial cells (40), which is regulated by 4-integrin- paxillin binding (1, 25). However, under shear flow, CHO cell types expressing wild-type and mutant 4-integrin did not have any significant differences in their cell adhesion strengthening to the CS-1 fragment of fibronectin, the 4β1-integrin-specific ligand, yet the cells display different shear flow-induced motile activities. Therefore, the shear flow-induced motile activities we observed are not likely due to changes of adhesion strengthening. A similar observation has also been made in primary pericytes isolated from mouse embryos. Knockout of the 4-integrin gene impaired the ability of pericytes to migrate in a directionally persistent manner under shear flow but had no significant effect on the adhesion strength of the cells (13). Therefore, in CHO cells and pericytes, the mechanism by which 4β1-integrin responds to shear force is different from that in leukocytes. 4β1-Integrin may transduce forces when a directional shear flow exerts pulling and pushing forces on the trailing and leading edges of the cell, respectively. Alternatively, 4β1-integrin may act downstream of other mechanosensors or mechanotransducers to regulate directionally persistent cell migration.
In summary, we showed that 4β1-integrin is sufficient and required for promoting directionally persistent migration of CHO cells in response to shear flow by facilitating leading edge protrusions and trailing edge detachment/retraction. Furthermore, we found a novel role of 4-integrin phosphorylation in trailing edge retraction/detachment that is likely to be independent of paxillin binding and a requirement for 4-integrin phosphorylation and paxillin binding in stabilizing the front-back polarity of shear flow-induced cells. These results provide new insight into the role of integrins in directed cell migration.
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ACKNOWLEDGMENTS |
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4-integrin antibody. We are also grateful to Douglas Robinson and Lew Romer for suggestions and critical comments on this article. |
FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* D. A. Dikeman, L. A. Rivera Rosado, and T. A. Horn contributed equally to this work. 
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REFERENCES |
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2. Arroyo AG, Yang JT, Rayburn H, Hynes RO. Differential requirements for alpha4 integrins during fetal and adult hematopoiesis. Cell 85: 997–1008, 1996.[CrossRef][Web of Science][Medline]
3. Bloom L, Ingham KC, Hynes RO. Fibronectin regulates assembly of actin filaments and focal contacts in cultured cells via the heparin-binding site in repeat III13. Mol Biol Cell 10: 1521–1536, 1999.
4. Chrzanowska-Wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol 133: 1403–1415, 1996.
5. Del Pozo MA, Kiosses WB, Alderson NB, Meller N, Hahn KM, Schwartz MA. Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat Cell Biol 4: 232–239, 2002.[CrossRef][Web of Science][Medline]
6. Diamond SL, Eskin SG, McIntire LV. Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science 243: 1483–1485, 1989.
7. Dickinson R, Tranquillo R. Optimal estimation of cell movement indices from the statistical analysis of cell tracking data. AIChE J 39: 1995–2010, 1993.[CrossRef]
8. Euteneuer U, Schliwa M. Persistent, directional motility of cells and cytoplasmic fragments in the absence of microtubules. Nature 310: 58–61, 1984.[CrossRef][Medline]
9. Garmy-Susini B, Jin H, Zhu Y, Sung RJ, Hwang R, Varner J. Integrin alpha4beta1-VCAM-1-mediated adhesion between endothelial and mural cells is required for blood vessel maturation. J Clin Invest 115: 1542–1551, 2005.[CrossRef][Web of Science][Medline]
10. Giancotti FG, Tarone G. Positional control of cell fate through joint integrin/receptor protein kinase signaling. Annu Rev Cell Dev Biol 19: 173–206, 2003.[CrossRef][Web of Science][Medline]
11. Ginsberg MH, Partridge A, Shattil SJ. Integrin regulation. Curr Opin Cell Biol 17: 509–516, 2005.[CrossRef][Web of Science][Medline]
12. Goldfinger LE, Han J, Kiosses WB, Howe AK, Ginsberg MH. Spatial restriction of 4 integrin phosphorylation regulates lamellipodial stability and 4β1-dependent cell migration. J Cell Biol 162: 731–741, 2003.
13. Grazioli A, Alves CS, Konstantopoulos K, Yang JT. Defective blood vessel development and pericyte/pvSMC distribution in alpha 4 integrin-deficient mouse embryos. Dev Biol 293: 165–177, 2006.[CrossRef][Web of Science][Medline]
14. Han J, Liu S, Rose DM, Schlaepfer DD, McDonald H, Ginsberg MH. Phosphorylation of the integrin alpha 4 cytoplasmic domain regulates paxillin binding. J Biol Chem 276: 40903–40909, 2001.
15. Healy ZR, Lee NH, Gao X, Goldring MB, Talalay P, Kensler TW, Konstantopoulos K. Divergent responses of chondrocytes and endothelial cells to shear stress: cross-talk among COX-2, the phase 2 response, and apoptosis. Proc Natl Acad Sci USA 102: 14010–14015, 2005.
16. Hu YL, Li S, Miao H, Tsou TC, del Pozo MA, Chien S. Roles of microtubule dynamics and small GTPase Rac in endothelial cell migration and lamellipodium formation under flow. J Vasc Res 39: 465–476, 2002.[CrossRef][Web of Science][Medline]
17. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687, 2002.[CrossRef][Web of Science][Medline]
18. Kil SH, Krull CE, Cann G, Clegg D, Bronner-Fraser M. The alpha4 subunit of integrin is important for neural crest cell migration. Dev Biol 202: 29–42, 1998.[CrossRef][Web of Science][Medline]
19. Kiosses WB, Shattil SJ, Pampori N, Schwartz MA. Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration. Nat Cell Biol 3: 316–320, 2001.[CrossRef][Web of Science][Medline]
20. Kole TP, Tseng Y, Wirtz D. Intracellular microrheology as a tool for the measurement of the local mechanical properties of live cells. Methods Cell Biol 78: 45–64, 2004.[Web of Science][Medline]
21. Konstantopoulos K, Kukreti S, McIntire LV. Biomechanics of cell interactions in shear fields. Adv Drug Delivery Res 33: 141–164, 1998.[CrossRef][Web of Science][Medline]
22. Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell 84: 359–369, 1996.[CrossRef][Web of Science][Medline]
23. Li S, Butler P, Wang Y, Hu Y, Han DC, Usami S, Guan JL, Chien S. The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells. Proc Natl Acad Sci USA 99: 3546–3551, 2002.
24. Liu S, Thomas SM, Woodside DG, Rose DM, Kiosses WB, Pfaff M, Ginsberg MH. Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses. Nature 402: 676–681, 1999.[CrossRef][Medline]
25. Manevich E, Grabovsky V, Feigelson SW, Alon R. Talin 1 and paxillin facilitate distinct steps in rapid VLA-4-mediated adhesion strengthening to vascular cell adhesion molecule 1. J Biol Chem 282: 25338–25348, 2007.
26. Nishiya N, Kiosses WB, Han J, Ginsberg MH. An alpha4 integrin-paxillin-Arf-GAP complex restricts Rac activation to the leading edge of migrating cells. Nat Cell Biol 7: 343–352, 2005.[CrossRef][Web of Science][Medline]
27. Nishizaka T, Shi Q, Sheetz MP. Position-dependent linkages of fibronectin- integrin-cytoskeleton. Proc Natl Acad Sci USA 97: 692–697, 2000.
28. Pinco KA, He W, Yang JT. alpha4beta1 integrin regulates lamellipodia protrusion via a focal complex/focal adhesion-independent mechanism. Mol Biol Cell 13: 3203–3217, 2002.
29. Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112: 453–465, 2003.[CrossRef][Web of Science][Medline]
30. Sengbusch JK, He W, Pinco KA, Yang JT. Dual functions of 4β1 integrin in epicardial development: initial migration and long-term attachment. J Cell Biol 157: 873–882, 2002.
31. Shyy JY, Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res 91: 769–775, 2002.
32. Tseng Y, Kole TP, Wirtz D. Micromechanical mapping of live cells by multiple-particle-tracking microrheology. Biophys J 83: 3162–3176, 2002.[Web of Science][Medline]
33. Tzima E, Del Pozo MA, Kiosses WB, Mohamed SA, Li S, Chien S, Schwartz MA. Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression. EMBO J 21: 6791–6800, 2002.[CrossRef][Web of Science][Medline]
34. Vicente-Manzanares M, Zareno J, Whitmore L, Choi CK, Horwitz AF. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J Cell Biol 176: 573–580, 2007.
35. Webb DJ, Parsons JT, Horwitz AF. Adhesion assembly, disassembly and turnover in migrating cells–over and over and over again. Nat Cell Biol 4: E97–E100, 2002.[CrossRef][Web of Science][Medline]
36. Wojciak-Stothard B, Ridley AJ. Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases. J Cell Biol 161: 429–439, 2003.
37. Worthylake RA, Lemoine S, Watson JM, Burridge K. RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol 154: 147–160, 2001.
38. Yang JT, Rayburn H, Hynes RO. Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development 121: 549–560, 1995.[Abstract]
39. Zaidel-Bar R, Kam Z, Geiger B. Polarized downregulation of the paxillin-p130CAS-Rac1 pathway induced by shear flow. J Cell Sci 118: 3997–4007, 2005.
40. Zwartz GJ, Chigaev A, Dwyer DC, Foutz TD, Edwards BS, Sklar LA. Real-time analysis of very late antigen-4 affinity modulation by shear. J Biol Chem 279: 38277–38286, 2004.
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