Correspondence to Erik H.J. Danen: e.danen{at}nki.nl
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Introduction |
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The dynamic regulation of the F-actin network is crucial to cell migration and is mediated by multiple actin-polymerizing, -capping, -severing, and -cross-linking proteins (Pollard and Borisy, 2003; Ridley et al., 2003). The Arp2/3 complex and cofilin are involved in the generation of propulsive force at the leading edge: the severing activity of cofilin and the branching activity of Arp2/3 act in synergy to stimulate protrusion (DesMarais et al., 2004; Ghosh et al., 2004). Cofilin does not appear to be required for lamellipodia formation, per se, but it is required for directional migration with a single, broad lamellipod at the leading edge (Dawe et al., 2003; Ghosh et al., 2004; Mouneimne et al., 2004). Stabilization of lamellipodia occurs through integrin-mediated adhesion to the ECM. Integrins cluster in cellmatrix adhesions where they connect the ECM to the F-actin network via scaffolding proteins such as talin, vinculin, and paxillin. At the front of a migrating cell, the adhesions are static and act as traction sites, whereas they become more dynamic toward the rear (Ballestrem et al., 2001). Cellmatrix adhesions also recruit signaling intermediates such as FAK, Src, and ERK, which may regulate local dynamics during cell migration (Geiger et al., 2001).
The organization of the F-actin (as well as the microtubule) network and the formation of cellmatrix adhesions in response to extracellular cues are controlled by small GTPases of the Rho family (Etienne-Manneville and Hall, 2002). In their GTP-bound state Rho GTPases can activate multiple downstream effector pathways. Both Rac1 and RhoA have been reported to activate a pathway that results in the inhibition of cofilin through phosphorylation at Ser3 (Edwards et al., 1999; Maekawa et al., 1999), but Rac1 supports lamellipodia extension and formation of nascent adhesions, whereas RhoA stimulates stress fiber formation and maturation of cellmatrix adhesions (Rottner et al., 1999). In a migrating cell the activities of the different Rho GTPases and/or their effector pathways must be controlled in a temporal and spatial manner, but it is incompletely understood how this is brought about (Ridley et al., 2003).
Integrin expression profiles might affect the migratory response to FN through integrin-specific effects on the activities of Rho family GTPases. Integrin-mediated adhesion to FN stimulates Rac1 activation and membrane extension (Price et al., 1998; del Pozo et al., 2000), whereas it triggers an abrupt reduction in the levels of GTP-bound RhoA (Ren et al., 1999; Arthur et al., 2002). At later stages of cell spreading on FN, RhoA GTP levels increase through an unknown mechanism that supports further actin cytoskeletal rearrangement. In leukocytes and CHO cells, overexpression of vß3 has been linked to increased RhoA GTP levels (Miao et al., 2002; Butler et al., 2003), but using epithelial and fibroblastic cells derived from ß1 knockout mice we have shown that ß1 integrins are required to support RhoA activation (Danen et al., 2002).
We and others have also shown that cell migration is strongly inhibited when ß1 integrins are absent (Gimond et al., 1999; Sakai et al., 1999; Raghavan et al., 2003). Here, we show that an increased expression of vß3 effectively stimulates migration of ß1 null cells, arguing against a specific requirement for ß1 integrins, by themselves, for migration. However, ß1 and ß3 integrins promote dramatically different modes of migration on FN. Lamellipodia formation, cellmatrix adhesion dynamics, and cofilin-mediated actin cytoskeletal polarization all are differently affected by ß1 and ß3 integrins. This involves the differential activation of Rho GTPases and ultimately affects the persistence of migration. Our findings demonstrate that the alterations in the expression of FN-binding integrins, as observed in vivo, can profoundly affect multiple parameters of cell migration through changes in the activity of Rho GTPases.
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Results |
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Notably, the motile behavior in wounding assays differed dramatically for cells adhering by 5ß1 or
vß3: GEß1 cells moved randomly as single cells extending protrusions in multiple directions, whereas GEß3 cells moved as a sheet of cells that maintained directionality (Video 1). To rule out effects due to differences in cellcell adhesion, the migration of sparsely seeded cells was analyzed. The velocity under these conditions was
25 µm/h for both cell types. However, similar to the results obtained in wounding assays, GEß1 cells moved randomly whereas GEß3 cells moved in a much more persistent fashion (Fig. 1 AC; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). To examine if the lack of directionality of GEß1 cells in wounding assays was due to a defect in polarization, we analyzed if these cells responded to wounding by polarizing their microtubule-organizing center (MTOC) (Gotlieb et al., 1981). GEß1 and GEß3 cells at the wound edge polarized their MTOC with similar efficiency within 3 h after wounding (Fig. 1 D; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1), indicating that GEß1 cells could "sense" the wound and orient their MTOC accordingly. However, although GEß3 cells maintained their polarized phenotype as they migrated into the wound area, MTOC polarity in the direction of the wound was lost in GEß1 cells during this process (Fig. 1 D, 9 h).
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Together, these findings demonstrate that in the same cellular context, ß1 and ß3 integrins promote distinct migratory strategies. Integrin vß3 supports a persistent lamellipodial mode of migration, whereas cells adhering by
5ß1 do not maintain polarity and migrate randomly.
Integrin-specific regulation of cytoskeletal polarization and cofilin activity
We next tested if differences in actin cytoskeletal polarization could explain the maintenance of directionality in GEß3 versus its loss in GEß1 cells. Indeed, GEß3 cells formed broad lamellipodia at the leading edge that were devoid of stress fibers and contained numerous small cellmatrix adhesions, whereas no such remodeling of the actin cytoskeleton was observed in GEß1 cells: these cells extended long, thin membrane protrusions with actin stress fibers ending at elongated cellmatrix adhesions (Fig. 2 A). To analyze whether GEß1 cells intrinsically lacked the ability to undergo extensive actin cytoskeletal remodeling, we treated the GEß1 and GEß3 cells with EGF, HGF, and the phorbol ester PMA, which is known to cause a reorganization of the actin cytoskeleton (Schliwa et al., 1984). Although EGF did not noticeably affect the morphology of either cell type, HGF induced ruffling in GEß3 cells but not in GEß1 cells (unpublished data). Moreover, although GEß3 cells responded to PMA treatment with a dramatic reorganization of the actin cytoskeleton and formation of a large lamellipod that was devoid of actin stress fibers, PMA hardly affected the actin cytoskeleton in GEß1 cells (Fig. 2 B). It has been reported that the distribution of microtubules is changed to conform to the altered cellular shape upon PMA stimulation, but that the microtubule cytoskeleton is not functionally implicated in the morphological response to PMA (Schliwa et al., 1984). Indeed, GEß3 (but not GEß1) cells also underwent a dramatic reorganization of their microtubule cytoskeleton in response to PMA (Fig. 2 B).
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Integrin-specific regulation of cellmatrix adhesion dynamics
In addition to actin cytoskeletal polarization, persistent migration also requires cellmatrix adhesions at the leading edge to be sufficiently static to stabilize the lamellipod and generate traction forces. Therefore, we analyzed if differences in the dynamics of cellmatrix adhesions in GEß1 and GEß3 could explain the distinct migratory behavior of these cells. Time lapse confocal imaging of GFP-paxillin in migrating GEß1 cells showed cellmatrix adhesions sliding away from the main cell body in membrane protrusions that extended in multiple directions (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). These extensions were often short-lived, and their collapse was accompanied by a rapid retraction of the adhesions. By contrast, cellmatrix adhesions at the leading edge of GEß3 cells were relatively inert, whereas large adhesions were observed to slide inwards at the rear. The same phenomenon could be observed during cell spreading on FN: adhesions in GEß1 cells were seen sliding outwards, whereas adhesions in GEß3 did not obviously move. Rather, new adhesions accumulated in GEß3 cells while existing adhesions remained intact (Fig. 4 A; Video 4). The apparent sliding of adhesions in GEß1 cells was correlated with a relatively fast turnover rate (within 34 min) as compared with that of adhesions in GEß3 cells (stable for at least 10 min) (Fig. 4 A and unpublished data).
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Together, these findings show that cellmatrix adhesions formed on FN in stationary as well as in spreading or migrating cells adhering by 5ß1 are highly dynamic, whereas those formed by cells adhering by
vß3 are relatively static.
Inhibition of Rho signaling induces a switch from ß1- to ß3-associated behavior
We previously showed that RhoA activity is supported by ß1 but not by ß3 integrins, whereas Rac-GTP levels are similar (Fig. 5 A; Danen et al., 2002), and we examined whether this difference might explain the distinct modes of cell migration supported by these integrins. Expression of a dominant-inhibitory RhoA mutant (RhoT19N) or expression of the Rho inhibitor C3 toxin in GEß1 cells induced a conversion to more circularly spread cells, reminiscent of a GEß3-like phenotype (Fig. 5 B; Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). Vice versa, expression of an activated mutant of RhoA (RhoAQ63L) or the catalytic DH/PH domain of p190RhoGEF in GEß3 cells caused a switch toward a GEß1-like phenotype, though cells expressing these constructs at too high levels were unable to spread on FN at all (Fig. 5 B; Fig. S4). Inhibition of signaling downstream from Rho using the Y27632 Rho kinase inhibitor caused extension of extremely long protrusions in line with an important function for Rho kinase in tail retraction. However, Y27632-treated GEß1 cells were also observed to switch to a GEß3-like morphology and a ß3-like migration pattern when seeded sparsely, a phenomenon that was never observed in nontreated GEß1 cells (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1). In such cells the mobility of GFP-paxillin in cellmatrix adhesions was also significantly decreased, with a -value that became almost as high as that observed in GEß3 cells (Fig. 5 C). Inhibition of RhoA or of its downstream effector Rho kinase also restored the ability of GEß1 cells to reorganize their actin cytoskeleton in response to PMA treatment or wounding: expression of C3 toxin or treatment with Y27632 supported the generation of broad lamellipodia in GEß1 cells, similar to those seen in GEß3 cells (Fig. 5, D and E).
Because cofilin activity was regulated in an integrin-specific fashion and was required for the formation of broad lamellipodia in GEß3 cells (Fig. 3), we investigated if the high level of phosphorylation of cofilin on Ser3 in GEß1 cells was also due to the relatively high activity of the Rho/Rho kinase pathway in GEß1. Indeed, inhibition of Rho kinase caused a strong and significant decrease in cofilin pSer3 levels to a level normally seen in GEß3 (Fig. 5, F and G).
Together, these findings show that the differences in lamellipodia formation, cellmatrix adhesion dynamics, and cofilin-mediated actin cytoskeletal polarization observed between cells adhering to FN by ß1 or ß3 integrins are due to the difference in Rho signaling.
Rac1 activation causes a partial conversion from ß1- to ß3-associated behavior
Extensive cross talk takes place between effector pathways downstream of Rho and Rac. In GEß1 and GEß3 cells, the balance between Rho and Rac activities differs due to the higher amounts of Rho-GTP in GEß1 cells (Fig. 5 A). To test if this balance, rather than RhoA activity by itself, was responsible for the different modes of migration of GEß1 and GEß3 cells, we transiently expressed an activated mutant of Rac1 in GEß1 cells. Expression of RacQ61L caused a conversion from a ß1- to a ß3-associated morphology (Fig. 6 A). Moreover, the conversion to a GEß3-like morphology observed in GEß1 cells transiently expressing RacQ61L was accompanied by a decrease in the dynamics of GFP-paxillin in their cellmatrix adhesions to a level that became indistinguishable from that observed in GEß3 (Fig. 6 B).
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In agreement with the notion that cofilin activity is implicated in actin cytoskeletal polarization (Fig. 3), phosphorylation of cofilin on Ser3 was not reduced in GEß1 cells expressing RacQ61L (Fig. 6, E and F). This suggested that these cells lacked sufficient cofilin activity to polarize their actin cytoskeleton. Therefore, we tested if expression of the activated cofilinS3A mutant could induce polarization in GEß1RacQ61L cells. Indeed, GEß1 cells expressing both RacQ61L and cofilinS3A underwent a dramatic reorganization of their actin cytoskeleton in response to PMA. In some cells this resulted in a distorted shape, but many others showed a phenotype that was indistinguishable from GEß3 (Fig. 6 G). Moreover, sparsely seeded GEß1RacQ61L cells transfected with cofilinS3A were considerably more dynamic and initiated membrane extensions at the leading edge that were highly reminiscent of GEß3 cells. However, this did not result in efficient lamellipodial migration but caused cellular disruption, most likely due to unusually high levels of Rac and cofilin activity throughout the entire cell (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).
These findings demonstrate that increased Rac activity, although causing enhanced membrane ruffling and cellmatrix adhesion stability, is unable to support polarization and directional migration in cells adhering to FN by 5ß1. This appears to be at least partly due to the fact that it does not stimulate an increase in active cofilin levels.
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Discussion |
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We show that vß3-mediated adhesion to FN supports high cofilin activity, which plays a permissive role in the F-actin cytoskeletal reorganization induced by wounding or by other stimuli such as PMA treatment. This allows for the cytoskeletal polarization observed in persistently migrating cells, with a single broad lamellipod at the leading edge under which cellmatrix adhesions are relatively static. By contrast, cofilin activity is low, polarization does not occur, and cellmatrix adhesions are highly dynamic in cells adhering by
5ß1, leading to a random mode of migration. We show that integrin-specific regulation of Rho GTPases underlies the distinct modes of migration: inhibition of Rho signaling promotes membrane ruffling, cofilin-mediated actin cytoskeletal polarization, and cellmatrix adhesion stability and hence, can cause a conversion from ß1- to ß3-associated migratory behavior. Increased Rac activity promotes membrane ruffling but does not increase cofilin activity, and therefore causes only a partial conversion from ß1- to ß3-like migration (Fig. 7).
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Rather than ß1 integrins being required for migration in general, our findings suggest that ß1 integrins contribute to specific aspects of cell migration, which may be important under certain conditions in vivo. In three-dimensional substrata, cells can adopt a persistent mode of migration with a pseudopod (the three-dimensional variant of a lamellipod) at the leading edge attaching to the ECM, but, alternatively, they can move randomly, undergoing amoeboid shape changes (Sahai and Marshall, 2003; Wolf et al., 2003). We find that high levels of RhoA activity are associated with random migration in two dimensions, and the work by Sahai and Marshall (2003) demonstrates that RhoA activity is associated with the random amoeboid migration in three dimensions. Thus, in both situations RhoA activity stimulates a pattern of migration in which cells often change direction. Our present findings connect ß1 integrins to such a RhoA-mediated random mode of migration. In agreement with this, silencing of the gene encoding the Fos family member Fra-1 in colon carcinoma cells leads to increased ß1 integrinmediated RhoA activation and suppression of Rac-mediated polarized lamellipodia extension (Vial et al., 2003). Clearly, for optimal migration, the balance between Rho and Rac activities must be tightly regulated. For instance, we show that expression of constitutively activated Rac does not necessarily support persistence of migration, due to the excessive formation of lamellipodia in multiple directions. Based on our work and the above-mentioned studies, we propose that ß1 integrins support RhoA activity, which is associated with a random mode of migration. Binding to FN by vß3 instead is associated with low levels of RhoA activity, shifting the balance to Rac-mediated, highly polarized, persistent migration. In vivo, modulation of the expression profiles of these integrins may thus allow cells to optimize their mode of migration.
Regulation of cofilin activity
The severing activity of cofilin and the branching activity of Arp2/3 act in synergy to drive the extension of lamellipodia (Bamburg, 1999; Pollard and Borisy, 2003; Ghosh et al., 2004). Recently, cofilin activity has been reported to be particularly important for the maintenance of a polarized cytoskeleton and thus for directional cell migration (Dawe et al., 2003; Ghosh et al., 2004; Mouneimne et al., 2004). In accord with this, we show that the persistent mode of migration of cells bound to FN by vß3 is associated with relatively high levels of cofilin activity. Adhesion by
5ß1 instead stimulates an increase in cofilin Ser3 phosphorylation and supports random migration.
Rho, Rac, and Cdc42, via their effectors (Rho kinase or PAK), can each stimulate the activity of LIM kinase that inactivates cofilin by phosphorylation at Ser3 (Agnew et al., 1995; Arber et al., 1998; Yang et al., 1998; Maekawa et al., 1999). In the cells used for our study, the Rho/Rho kinase pathway is responsible for cofilin phosphorylation. Adhesion to FN by 5ß1 causes high levels of Rho/Rho kinasemediated cofilin phosphorylation, whereas adhesion by
vß3 supports low levels of RhoA activity, leaving a relatively large proportion of cofilin in the nonphosphorylated, active form. The level of Rac activity in GEß3 or in Y27632-treated GEß1 cells is apparently too low to cause high levels of cofilin phosphorylation. One explanation for our findings would be increased Rho kinasemediated activation of LIM kinase in GEß1 cells. However, we were unable to detect an increase in LIM kinase phosphorylation in GEß1 cells versus GEß3 cells (unpublished data). An alternative explanation would be that the levels or activity of Slingshot, the cofilin phosphatase (Niwa et al., 2002), are differentially affected, but this remains to be examined.
Regulation of cellmatrix adhesion dynamics
We find that the dynamic behavior of cellmatrix adhesions is strongly affected by their integrin composition. Adhesion to FN by either 5ß1 or
vß3 occurs with similar efficiency and kinetics. Nevertheless, adhesions containing
5ß1 appear to be much more dynamic than those containing
vß3, as indicated by the differences in the mobile fraction of paxillin and vinculin, both in motile and fully spread, nonmotile cells. Others have shown that adhesions at the leading edge of migrating cells (most likely containing a mix of various integrins) are relatively static, whereas those at the trailing edge have a high turnover rate and slide inwards (Ballestrem et al., 2001). It has also been reported that the active form of
vß3 localizes preferentially at the edges of lamellipodia through a Rac-dependent mechanism (Kiosses et al., 2001). Moreover, local concentrations of Rac and coupling of GTP-Rac to downstream effectors were found to be increased in pseudopodia and lamellipodia (Cho and Klemke, 2002). Based on these reports and our current finding that expression of Rac1Q61L or treatment with Y27632 causes a decrease in the dynamics of cellmatrix adhesions in GEß1 cells to levels similar to those in GEß3 cells, we propose that the activated
vß3 integrins found in lamellipodia may locally promote Rac activity and increased stability of cellmatrix adhesions, which would contribute to persistence of migration.
Little is known about the molecular mechanism involved in the assembly and disassembly of cellmatrix adhesions. The tyrosine kinases Src and FAK and the adaptor protein p130Cas are present in cellmatrix adhesions and have been implicated in their turnover (Ilic et al., 1995; Fincham and Frame, 1998; Webb et al., 2004). We have not observed any striking differences in the localization or phosphorylation of these proteins except for a delayed phosphorylation of FAK at the auto-phosphorylation site, Y397, in GEß3 cells, which may contribute to the decreased adhesion turnover rates observed in these cells (Danen et al., 2002 and unpublished data). Alternative mechanisms may involve different localization and/or activation of PTP-PEST or SHP-2 because mouse embryonic fibroblasts deficient in these phosphatases develop abnormally high numbers of cellmatrix adhesions and their migration is impaired (Yu et al., 1998; Angers-Loustau et al., 1999). Finally, local ERK-mediated activation of myosin light-chain kinase as well as cleavage of the cellmatrix adhesion constituent, talin, by the calcium-dependent protease calpain have been proposed to contribute to cellmatrix adhesion dynamics (Franco et al., 2004; Webb et al., 2004). Future studies should clarify how all these events are affected by integrin-regulated Rho GTPase activities.
In conclusion, our study provides evidence that the differential modulation of Rho GTPases by 5ß1 and
vß3 affects three important parameters of cell migration: lamellipodia formation, cellmatrix adhesion dynamics, and cofilin-mediated actin cytoskeletal polarization. As a result, these integrins promote distinct modes of migration, with ß1 integrins supporting random and ß3 integrins promoting persistent migration. Based on these findings, we propose that the alterations in the expression profiles of FN-binding integrins as observed during wound healing, angiogenesis, and cancer metastasis allow cells to adopt a mode of migration that fits the local conditions.
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Materials and methods |
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The cDNAs encoding GFP paxillin and GFP vinculin (provided by Dr. Kenneth Yamada; National Institutes of Health, Bethesda, MD) were cloned into the LZRS-neo bicistronic retroviral vector, and transfected to ecotrophic packaging cells to generate virus-containing culture supernatants. Subsequently, GEß1 and GEß3 cells were transduced and selected for stable expression of the GFP fusion proteins in cellmatrix adhesions.
Expression plasmids encoding C3 toxin, RhoAT19N, Rac1Q61L, and RhoAQ63L were provided by Dr. Sylvio Gutkind (National Institutes of Health, Bethesda, MD), GFP-tagged cofilin expression constructs were provided by Dr. James Bamburg (Colorado State University, Fort Collins, CO; Agnew et al., 1995), HA-tagged cofilin expression constructs were from Dr. Kenji Moriyama (Moriyama et al., 1996; Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) and the p190RhoGEFDH/PH expression plasmid was a gift from Dr. Wouter Moolenaar (Netherlands Cancer Institute, Amsterdam, Netherlands). For transient expression of these cDNAs, transfections were performed using the Effectene kit from QIAGEN according to the manufacturer's protocol.
Random migration analysis
GEß1 or GEß3 cells were plated sparsely (3 x 104 cells) on 24-mm glass coverslips coated with FN. Human plasma FN was purified as described previously (Danen et al., 2000) and used at 10 µg/ml in PBS, a concentration at which GEß1 and GEß3 cells adhere with identical efficiency (Danen et al., 2002). 3 h later, coverslips were incubated for 2 h with 10 mg/ml mitomycin-C (Sigma-Aldrich) to inhibit cell division, washed, and incubated overnight in culture medium covered with mineral oil at 37°C and 5% CO2. A 10x dry lens objective was used and phase-contrast images were taken every 15 min on a Widefield CCD system (Carl Zeiss MicroImaging, Inc.); tracks of individual cells were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). The migration speed was calculated as [total path length (µm)/time (hour)] and the persistence of migration was calculated as [net displacement (µm)/total path length (µm)].
Analysis of migration and polarization in wounding assays
GE11, GEß1, or GEß3 cells were plated overnight at high density (2 x 105 cells) on 24-mm glass coverslips coated with FN. Confluent cultures were wounded using a blue pipette tip, washed, and incubated with fresh culture medium. A 10x dry lens was used and phase contrast images were taken every 15 min on a Widefield CCD system; videos were generated using ImageJ software.
For the analysis of MTOC polarization, wounded cultures were generated as above, maintained at 37°C and 5% CO2 for different times, and fixed in ice-cold methanol. Coverslips were incubated with an anti-pericentrin antibody (Covance Research Products) for MTOC staining and counterstained with DAPI to visualize nuclei. In three independent experiments, 100 cells at the wound edge were analyzed and the percentage of cells with the MTOC positioned in the quadrant facing the wound relative to the position of the nucleus was calculated (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).
Time-lapse confocal microscopy
GEß1 or GEß3 cells stably expressing GFP-paxillin were plated on FN-coated glass coverslips and imaged using a DM-IRE2 inverted microscope fitted with TCS-SP2 scan head (Leica). After identification of a cell that had started to adhere, confocal images of GFP at the cellsubstrate adhesion level were taken with a 63x oil objective every 15 s for 45 min. Videos were generated using ImageJ software.
FRAP and FLIP experiments
GEß1 or GEß3 cells stably expressing GFP-paxillin or GFP-vinculin were plated on FN-coated glass coverslips in complete medium for 24 h and subsequently imaged through a 63x oil objective using a DM-IRE2 inverted microscope fitted with a TCS-SP2 scan head in bicarbonate-buffered saline (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 1 mM CaCl2, 23 mM NaHCO3, and 10 mM Hepes, pH 7.2) at 37°C and 5% CO2.
For FRAP analysis, a spot of 1.3 µm (full width half-maximum) in a GFP-containing cellmatrix adhesion was bleached using an external 488-nm argon laser line for 0.5 s. Subsequently, fluorescence intensity in that spot was measured every 250 ms for 2 min at low laser power (<5% background bleaching). Recovery of fluorescence was analyzed while correcting for background bleaching and stage drifting by analysis of control (nonbleached) cellmatrix adhesions.
For FLIP analysis, a spot in the cytoplasm was bleached using an internal 488-nm laser at high power for 4 s. Subsequently, fluorescence in 10 selected cellmatrix adhesions and control regions was measured at low laser power. This cycle was repeated every 25 s. Loss of fluorescence from cellmatrix adhesions was analyzed while correcting for background bleaching and stage drifting by analysis of background fluorescence and fluorescence in control (nonbleached) cells (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1).
Immunofluorescence and Western blotting
For immunofluorescence, cells were fixed in 4% PFA, permeabilized in 0.2% Triton X-100, blocked with 2% BSA, and incubated with AlexaFluor 568conjugated Phalloidin (Molecular Probes, Inc.), mAbs against paxillin (Transduction Laboratories), or -tubulin (Sigma-Aldrich) followed by FITC- or Texas redlabeled secondary antibodies. Preparations were mounted in MOWIOL 488 solution supplemented with DABCO (Calbiochem) and analyzed using a DM-IRE2 inverted microscope fitted with a TCS-SP2 confocal scan head. Images were obtained using a 63x oil objective and were imported in Adobe Photoshop.
For Western blotting, total cell lysates were prepared in SDS sample buffer, resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and analyzed by Western blotting using mAbs against GFP (Covance Research Products) or -tubulin, or with polyclonal antisera against cofilin (provided by Dr. James Bamburg) or pSer3 cofilin (Cell Signaling) followed by HRPO-labeled secondary antibodies (Amersham Biosciences) and ECL using the SuperSignal system (Pierce Chemical Co.).
Biochemical assays for activity of Rho GTPases
The activities of Rac1 and RhoA were determined in GTP pull-down assays as described previously (Danen et al., 2002).
Online supplemental material
Examples of MTOC polarization and FLIP experiments, as well as quantification of the results obtained in transient transfection experiments are provided as supplementary figures. In addition, supplementary videos of migration experiments are provided. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200412081/DC1.
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Acknowledgments |
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This work was funded by the Dutch Cancer Society (grant NKI 2003-2858).
Submitted: 13 December 2004
Accepted: 29 March 2005
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References |
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