The eukaryotic expression vectors pEGFP-N3, pECFP-N1, and pEYFP-N1 were obtained from CLONTECH Laboratories, Inc. For α5 and paxillin cDNA (provided by L. Reichardt and C.E. Turner, SUNY Upstate Medical University), we inserted a KpnI restriction site before the stop codon using a mutagenic PCR primer with a noncomplementary KpnI site at its 5′ end. A minimal amount of this 3′ cDNA fragment was religated to the original cDNA using either pCR2.1 (Invitrogen) or pCRScript (Stratagene) as a cloning intermediate. We used the KpnI site and one upstream within the original cDNA to ligate each cDNA into the polylinker 5′ to the start codon of the respective GFP variant. The entire PCR fragment and the junction region between the protein and GFP was sequenced. We obtained a similarly prepared α-actinin–GFP construct in the pEGFP-N1 vector from C. Otey and M. Edlund (University of North Carolina, Chapel Hill, NC). This fusion scheme created a 10–amino acid linker between α5 or paxillin (LQAGPGSIAT) and EGFP, a 13–amino acid linker between α5 or paxillin and enhanced yellow fluorescent protein (EYFP) or enhanced cyan fluorescent protein (ECFP) (AAVPRARDPPVAR), and a 20–amino acid linker between α-actinin and EGFP, ECFP, or EYFP (KLRILQSTVPRARDPPVAT). Paxillin and α-actinin ECFP and EYFP were prepared by cloning the cDNAs into the mammalian expression vector pCDNA3.1/Zeo (Invitrogen). The head and rod domains of α-actinin fused to GFP were provided by C. Otey and M. Edlund.

CHO K1 and CHO B2 cells were cultured in DME (GIBCO BRL) supplemented with 10% FBS, 4 mM l-glutamine, 1 mM sodium pyruvate, and nonessential amino acids (Sigma-Aldrich). WI38 cells were maintained in high glucose DME supplemented with 10% FBS and 1 mM sodium pyruvate. Transfections were performed with lipofectamine (GIBCO BRL), and the cells were selected with 1 mg/ml G418 (Sigma-Aldrich) or 0.5 mg/ml zeocin, where appropriate. Stably transfected cells were maintained in 0.5 mg/ml G418 and/or 0.25 mg/ml zeocin and sorted for expression levels by flow cytometry.

Cells were plated for >4 h in HyQ-CCM1 serum-free medium (Hyclone) on coverslips coated with 20 μg/ml fibronectin (Fn), fixed, and stained as described previously (Sastry et al. 1999). Focal adhesion proteins were detected with the VIN-11-5 antivinculin antibody (Sigma-Aldrich), antipaxillin 165 (Transduction Laboratories), or anti–α-actinin A5044 (Sigma-Aldrich). Human α5 was stained with monoclonal antibody 6F4 and endogenous and expressed α5 were detected with polyclonal anti-α5 Ab1949 (Chemicon). CHO endogenous β1 was detected using monoclonal 7E2 (University of Iowa Developmental Studies Hybridoma Bank, Iowa City, IA) and polyclonal 36E3 (a gift from D. DeSimone, University of Virginia, Charlottesville, VA). The transferrin receptor was stained with monoclonal antibody B65.3 (provided by S. Green, University of Virginia, and I. Trowbridge, Salk Institute, La Jolla, CA). Actin fibers were visualized using rhodamine-phalloidin.

CHO B2 cells transfected with untagged α5 or α5-GFP were washed twice with EBSS, 25 mM Hepes, pH 7.4 (EBS-H), and biotinylated for 60 min at 4°C with 0.5 mg/ml NHS-biotin (Pierce Chemical Co.). After washing the cultures, the cells were extracted with 100 mM N-octylglucoside in 25 mM Hepes, 150 mM NaCl, pH 7.4 (HBS), with protease inhibitors (aprotinin, leupeptin, and E-64) for 30 min at 4°C and centrifuged for 10 min at 14,000 g. α5 integrin was then immunoprecipitated by incubation with 6F4 monoclonal antibody (5 μg) for 12 h at 4°C and protein G agarose (Santa Cruz Biotechnology, Inc.) for 4 h at 4°C. The samples were washed five times with 25 mM N-octylglucoside in HBS and centrifuged for 10 min at 14,000 g. The immunoprecipitates were subjected to SDS-PAGE on 5% slabs, transferred to nitrocellulose, and detected by Western blot analysis.

Tissue culture dishes were modified to facilitate microscopic observation of living cells as described previously (Palecek et al. 1996). The dishes were coated with Fn for 16 h at 4°C and then blocked with 2% BSA. 10⁵ cells were plated in either CCM1 (Hyclone) or DME/F-12 for 1 h at 37°C. During microscopy, cells were maintained at 37°C, pH 7.4, and illuminated with a halogen lamp. To visualize EGFP, an endow GFP filter cube (ex HQ470/40, em HQ525/50, Q495LP dichroic mirror) was used (Chroma). FM 4-64 was visualized using a rhodamine/TRITC cube (ex BP520-550, barrier filter BA580IF, dichroic mirror DM565). Exposure times ranged from 0.05 to 0.20 s. For two-color fluorescence, ECFP (ex D436/10, em D470/30) and EYFP (ex HQ500/20, em HQ535/30) were positioned in “dual” filter wheels (Ludl Electronic Products), and a JP4 bandpass filter cube was used to visualize CFP/YFP, respectively. Using the 40× (NA 0.75; Nikon) and the 60× (NA 1.40; Nikon) objectives, images were acquired from a cooled CCD camera (Photometrics CH250 or Hammatsu OrcaII) attached to an Olympus IX-70 or Nikon TE-300 inverted microscope. Image acquisition was controlled using the Inovision ISee software program interfaced to a Ludl modular automation controller (Inovision).

For migration experiments, cell paths were tracked using the nanotrack tool in ISee, which records the x and y pixel coordinates of the approximate centurion of the somitic cell cortex. The average speed for each cell was determined by computing the average net centroid translation divided by the time interval at each 5- or 10-min time point. This protocol was adapted for calculating rates of movement of paxillin- or α-actinin–containing adhesion complexes.

Time-lapse images are included as online videos, which further depict Fig. 2 (Videos 1 and 2), 3 (Videos 3 and 4), 5 (Video 5), 6 (Video 6), and 10 (Videos 8–10). Video 7 shows α-actinin adhesions moving inward toward the cell center. Additional time-lapse images for Fig. 3, showing vesicles emanating from membrane protrusions (Video 2) and vesicles moving to and from the base of the lamellipodia (Video 11) are included. The time intervals for each video are as follows: Videos 1, 2, 6, and 10, 5 s; Videos 3, 5, and 8, 10 s; Video 9, 15 s; Video 4, 30 s; Videos 7 and 11, 60s. Supplemental Fig. 1, Fig. 2, and Fig. 3 are also provided. All videos and supplemental figures are available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1.

To study the formation and breakdown of adhesive structures, we prepared expression constructs that encode α5 integrin, paxillin, and α-actinin as GFP fusion proteins. Previous studies have examined the properties of cells expressing GFP fusions with paxillin and α-actinin; however, α5-GFP has not been characterized (Dabiri et al. 1997; Kiosses et al. 1999; Salgia et al. 1999; Katz et al. 2000; Knight et al. 2000). To demonstrate that GFP was not cleaved in our fusion constructs, cell surface proteins on untagged α5- and α5-GFP–transfected CHO B2 cells were biotinylated, extracted, and immunoprecipitated with the α5 antibody 6F4. Two major bands were biotinylated in each sample, and the larger of these surface-expressed proteins cross-reacted with a polyclonal α5 antibody (Fig. 1 a). The α5-GFP band was ∼30-kD larger than the band precipitated from CHO B2 cells transfected with untagged α5, indicating that GFP was not cleaved (Fig. 1 a). The smaller biotinylated band was ∼98 kD, which is the size expected for coprecipitated β1 integrin (Fig. 1 a). Furthermore, cleaved GFP was not observed in total protein extracts from α5-GFP–expressing CHO B2 and CHO K1 cells (data not shown). Similarly, cleaved GFP was not detected in cells expressing moderate (<2× over expression) levels of α-actinin and paxillin; these cells were subsequently used in our experiments.

We compared cell surface levels of α5 integrin in our transfected CHO B2 and CHO K1 cells by FACS^® analysis. As shown in Fig. 1 b, using saturating concentrations of the antibodies, the α5 integrin expression level in the transfected CHO B2 cells was comparable to the level of endogenous α5 integrin in the CHO K1 cells. Thus, these results indicate that α5 integrin is not significantly overexpressed in the CHO B2 cells when compared with the expression of endogenous α5 integrin in CHO K1 cells.

To determine whether the α5-GFP fusion retained integrin function, we performed migration assays and tested its ability to rescue cell spreading of CHO B2 cells, a cell line isolated for its low expression levels of α5 integrin. These cells do not spread on Fn-coated substrates unless α5 is expressed ectopically (Schreiner et al. 1989; Zhang et al. 1993), making this system useful for studies of α5 function (Cao et al. 1998). The GFP fusion had no detectable effect on migration, since cells expressing GFP-tagged α5 and GFP-tagged paxillin and α-actinin migrate indistinguishably from the cells expressing similar levels of the untagged proteins (Table ). Expression of α5-GFP restored the ability of CHO B2 cells to spread similarly to cells expressing untagged α5 integrin (Table ). To confirm that the ectopic expression of α5 integrin rescues CHO B2 spreading on Fn, we used antibodies that perturb the integrin–Fn interaction. This rescue was inhibited by 16G3 Mab (a gift from K. Yamada, National Institutes of Health, Bethesda, MD), which recognizes the RGD sequence in Fn and disrupts the α5–Fn interaction (Table ). The α5 integrin function-blocking antibodies, VD1 and VD10 (provided by R. Isberg, Tufts University, Boston, MA), inhibited spreading of α5-GFP–expressing CHO B2 cells on Fn by 99 ± 2% and 97 ± 3%, respectively. The nonfunction perturbing α5 antibody, 6F4, had no effect on cell spreading (Table ). Thus, the spreading of α5-GFP–transfected CHO B2 cells is mediated by a direct interaction between α5 and Fn.

Each fusion protein reproduced the localization patterns of its untagged counterpart when expressed in CHO B2, CHO K1, and NIH-3T3 cells under conditions that promote formation of prominent focal adhesions, such as plating on Fn concentrations >5 μg/ml or longer than 6 h. When CHO K1 cells expressing α5-GFP, α-actinin–GFP, or paxillin-GFP were allowed to adhere under focal adhesion–promoting conditions, all three fusion proteins colocalized with vinculin in focal adhesions with α-actinin also localizing along fibrous structures (Fig. 1, c–e). This confirms previous studies using paxillin- and α-actinin–GFP (Dabiri et al. 1997; Kiosses et al. 1999; Zamir et al. 1999, Zamir et al. 2000; Katz et al. 2000; Knight et al. 2000; Edlund et al. 2001).

We then compared the kinetics of adhesive assembly of untagged α5- and α5-GFP–expressing cells by allowing cells to adhere for 0.5, 1.5, and 18 h in CCM1 on substrates coated with 5 μg/ml Fn. Cells were stained for endogenous and exogenous α5 and endogenous β1, F-actin, vinculin, paxillin, and α-actinin. Untransfected CHO B2 cells did not adhere to the coverslips even after 18 h of plating, demonstrating that α5 expression is necessary for these cells to adhere to Fn. In CHO B2 cells transfected with either α5-GFP or untagged α5, the integrins, actin, and focal adhesion proteins were visible at the periphery of cells within 30 min of plating (data not shown; Fig. S1, a–c and g–i, available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). When cells were plated for 1.5 h, an increase in organized adhesions was observed (Fig. S1, d–f, available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). However, at both time points the intensity of staining suggested that the integrins were not as highly organized as vinculin. Importantly, the time course of organization was similar regardless of ectopic expression of the different adhesion molecules in all of the cell types studied. Both untagged α5- and α5-GFP–expressing CHO B2 cells organized more slowly than the parental CHO K1 cells, probably reflecting a clonal difference. Integrin α5-GFP–expressing CHO B2 and CHO K1 cells viewed live after plating on Fn concentrations >5 μg/ml or for longer than 6 h exhibited clear focal adhesion–like structures. At lower Fn concentrations or at shorter time points, clearly discernible α5 organization was rarely seen.

Since integrin organization in CHO cells was observed only at longer time points after plating and on higher concentrations of Fn, we complemented our CHO cell studies by examining the adhesive organization in WI38 cells. Organized adhesions as indicated by vinculin and α5 integrin staining were apparent 60 min after plating on 2 μg/ml Fn (Fig. S2, a and b, available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). As with the CHO cells, the intensity of vinculin staining was much greater than that observed for α5 integrin. The GFP fusion had no effect on α5 integrin localization in the WI38 cells since the localization pattern of α5-GFP and endogenous α5 integrin was similar (Fig. S2, a–c, available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1).

To obtain insight into the mechanisms by which adhesions form and stabilize at the cell front, we investigated the dynamics of the GFP probes in protrusive regions under migration-promoting conditions: Fn concentrations <5 μg/ml plated for <2 h. CHO K1 and CHO B2 cells were plated in CCM1 on 1–5 μg/ml Fn for 1–2 h and then observed in fluorescence every 5 or 30 s for 2–10 min. Under these conditions, both cell types are motile and show prominent protrusive activity. α-Actinin–GFP and α5-GFP localized prominently along the cell border in membrane protrusions (Fig. 2, a and b; Videos 1 and 2 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). This localization was observed in ∼70% of the cells viewed and in >90% of the cells that showed protrusive activity. In both CHO and WI38 cells, α-actinin–CFP and α5-YFP colocalized to membrane protrusions along the cell edge (Fig. 2, c–f). α-Actinin–GFP also localized along fibrous-like structures (Fig. 2 a).

Previous reports using fixed cells suggest that integrin-containing vesicles are present at the cell rear and gather in a perinuclear region in migrating cells (Regen and Horwitz 1992; Lawson and Maxfield 1995; Palecek et al. 1996; Bretscher and Aguado-Velasco 1998; Pierini et al. 2000). In contrast, our observations in CHO cells revealed prominent large and small α5-GFP vesicle-like structures that emanated from the leading edge in protrusions (Fig. 3 a; Video 3 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). These structures colocalized with the fluorescent lipophilic dye, FM 4-64, indicating that they were membrane-enclosed endocytic vesicles. The α5-containing vesicles moved centripetally toward the cell center and concentrated in a perinuclear region that colocalized with FM 4-64 and the transferrin receptor. The preponderance of GFP-labeled vesicles in CHO cells originated from lamellipodial regions and moved centripetally; this was also observed in WI38 cells in regions where membrane ruffling was apparent. However, in migrating WI38 cells in which minimal membrane ruffling was observed α5-containing vesicles moved from the perinuclear region to the base of the lamellipodium but not into it (Fig. 3 b; Video 4 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). α5 vesicles were also observed in the rear of the cells. These vesicles moved quickly, often along the lateral edge, toward the perinuclear region where we could no longer track them due to the volume of the cell in that region and the large number of vesicular structures that accumulate there. We did not observe the α5-containing vesicles entering the lamellipodium. Interestingly, we observed substantially more vesicles in cells either expressing higher α5 integrin levels or plated on lower substrate concentrations; this coincides with increased membrane protrusive activity. Membrane-bound palmitoylated GFP also emanated from protrusions and moved inward toward the cell body, suggesting membrane internalization.

The observation that integrin-containing vesicles can emanate from the leading lamella prompted us to ask whether the inclusion of α5-GFP in vesicles was caused by fusion of α5 with GFP. Untagged α5-expressing CHO B2 cells were stained for α5 or endogenous β1 and costained for endogenous transferrin receptor. Using deconvolution (not shown) and confocal microscopy, α5 (Fig. 4) and β1 (not shown) were observed to colocalize with the transferrin receptor. After a pulse of FM 4-64, a similar number of FM 4-64–containing vesicles was observed in cells expressing either untagged α5 or α5-GFP. Thus, the vesicular and endosomal localization of α5-GFP is a property of endogenous integrins.

The leading edge of a protrusion is a site where adhesions form and stabilize. Using either GFP- or CFP- and YFP-tagged α5, α-actinin, and paxillin-transfected CHO B2 or CHO K1 cells, we observed the differing dynamics of these molecules. Of the three fusion proteins examined, paxillin appeared first. Paxillin was observed initially in a wave of diffuse fluorescence and then localized in small clusters (∼1 μm) near the leading edge of the lamellipodium (Fig. 5 a). These small clusters rapidly reached maximum intensity in ∼300 s. Once formed, individual foci did not join together to make larger adhesive complexes.

Paxillin-containing adhesions at the base of new protrusions were highly dynamic and tended to turn over. We viewed numerous rounds of adhesion, protrusion, and reformation in paxillin-GFP–transfected cells. In nearly every observation, the intensity of the paxillin clusters at the base of the new protrusion diminished and often disappeared as adhesions formed in a newly protruded region of the lamellipodium (Fig. 5 a; Video 5 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). The rates at which the intensity decreased paralleled an increase of intensity in new adhesions (Fig. 5 b). Interestingly, these paxillin clusters were visible by total internal reflection microscopy, demonstrating their proximity to the substrate (Fig. 5 d).

Organized α-actinin was either not apparent or very weak in the highly dynamic paxillin adhesions. This may be due to differential turnover of α-actinin and paxillin in the adhesions or the lack of visibly organized α-actinin in the newly forming paxillin adhesions. To distinguish between these, CHO K1 cells were transfected with α-actinin–CFP and paxillin-YFP. Clearly organized α-actinin was not detected or was very weak in the paxillin adhesions that turned over (Fig. 6, a–c; Fig. S3 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). Furthermore, the paxillin adhesions that formed at the lamellipodial tip as old adhesions broke down also lacked organized α-actinin (Fig. 6, a–c).

However, when protrusive activity ceased, α-actinin–GFP began to localize in small ∼0.5-μm foci at the edge of the former lamellipodium. These structures developed and persisted as the protrusive activity, as seen in edge-enriched localization, dissipated (Fig. 6 d; Video 6 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). The individual foci did not join together to make larger adhesive complexes but often grew gradually in size and intensity and extended filamentous structures toward the cell body. When visibly organized α-actinin entered the adhesions, the individual clusters tended to move centripetally from their original location, and further intensification was observed. Unlike the highly dynamic paxillin adhesions, α-actinin–containing adhesions did not turn over but instead tended to move inward toward the cell body. These results suggest paxillin dynamics differed from that of α-actinin and that some adhesive components at the lamellipodial base are turned over as adhesions form at the leading edge.

In CHO cells, we were unable to detect visibly organized α5 integrin colocalizing with α-actinin in newly forming adhesions (Fig. 7 a). It is unlikely that another integrin is involved, since clearly organized αv and β1 integrins were also not observed in the new adhesions. In addition, αvβ3 was not detected in the CHO cells, which is consistent with previous reports that these cells express little β3 or other integrins (Ylanne et al. 1993; Takagi et al. 1997). A more likely possibility is that the α5 integrin, although present in new adhesions, is not highly concentrated and thus is not readily observed. Comparing the intensity of integrin staining with that of other adhesion molecules supports this hypothesis, since even in cells with highly organized adhesions the integrin staining is considerably less intense than that of paxillin or α-actinin.

Since endogenous α5 was more visibly organized in the WI38 cells than in the CHO cells, we examined the dynamics of the fusion proteins as these cell migrated. Paxillin-GFP was observed near the leading edge and turned over in regions where new protrusions formed, which is similar to our observations in the CHO cells. As membrane ruffling dissipated, organized α-actinin and subsequently α5 integrin were observed in the adhesions. Unlike the highly dynamic paxillin-containing adhesions, the α5-containing adhesions were stable and did not turn over. In WI38 cells expressing α5-YFP and α-actinin–CFP, some α-actinin adhesions slide inward from the cell perimeter (Fig. 7, b–e; Video 7 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1); however, those that also contained α5 integrin remained stationary (Fig. 7, f–i). Thus, in these cells highly organized α5 was not visible in all adhesions, and when it was the adhesions did not translocate.

Since clearly organized integrin was not observed in the forming adhesions, we examined the role of α5 ligation in the formation of paxillin clusters. When paxillin-GFP–expressing CHO K1 cells were plated on poly-l-lysine, paxillin-GFP–containing adhesions were not detected; however, paxillin clusters were observed when the cells were plated on 2 μg/ml Fn (Fig. 8, a and b). Paxillin clusters were no longer apparent after addition of the function-blocking antibody, VD1, which disrupts integrin binding to Fn (Fig. 8e and Fig. f). The nonfunction-blocking antibody, 6F4, had no effect on paxillin adhesions (Fig. 8c and Fig. d). These results suggest that the integrin–Fn interaction is necessary for paxillin clustering.

As adhesions break down, a fraction of the integrins can be left behind the cell in tracks (Regen and Horwitz 1992; Palecek et al. 1996, Palecek et al. 1998); however, the fate of the cytoskeletal proteins is not known. Thus, we examined the fate of adhesive components at the cell rear as adhesions broke down. When α5-GFP integrin-expressing CHO B2 and CHO K1 cells were plated on 2.5–5 μg/ml Fn, we observed fluorescent fibers extending behind retracting cell regions (Fig. 9 a). A network of “retraction” fibers greater than a cell length was also observed when α5-GFP–expressing WI38 cells were plated on 0.5–2 μg/ml Fn. In some cases, the fibers were left behind as the cells migrated. The length of these fibers was variable. These fibrous structures were membranous, since similar structures were seen behind cells transfected with a palmitoylated membrane-bound GFP (Moriyoshi et al. 1996), but FM 4-64 was not observed in fibers. When CHO K1 cells expressing similar levels of soluble GFP were plated under the same conditions, “retraction” fibers were not observed, indicating that the cytoplasmic volume of these structures is small. Paxillin-CFP and α-actinin–CFP do not localize with α5-YFP in these fibrous networks (Fig. 9, b–e). This suggests that the adhesion is breaking down closer to the integrin than to the actin.

Previous studies have also reported the movement of aggregates of integrins along the cell edge (Regen and Horwitz 1992; Palecek et al. 1996). One interpretation is that the weakening or release of integrin–ligand interactions can be an early event in the process of adhesion breakdown. Thus, one expects to see organized cytoplasmic adhesion complexes remaining. We observed clusters of paxillin-GFP and α-actinin–GFP moving along the lateral edge of the cell (Fig. 10, a and b; Videos 8 and 9 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). These clusters averaged 1–2 μm in length and moved at ∼30 μm/h from the cell rear toward the cell front, although there was variation in speed between individual clusters. The clusters remained intact for over 30 min, indicating their stability (Fig. 10 c; Video 10 available at http://www.jcb.org/cgi/content/full/153/7/1427/DC1). Small clusters would often appear to coalesce and move up the tail as it retracted behind them. The clusters would gradually decrease in intensity and eventually disperse. Paxillin-YFP and α-actinin–CFP colocalized in sliding adhesive structures on the ends of α-actinin–containing fibers. The connection between stress fibers and paxillin and α-actinin complexes suggests that they remained connected to actin, which likely mediated their movement. Since visibly organized α5 adhesions were seldom seen at these early times in CHO cells, we also examined the breakdown of adhesive structures in WI38 cells. When WI38 cells were transfected with α-actinin–CFP and α5-YFP, α-actinin–containing adhesive structures slide inward toward the cell center as the rear of the cell retracted; however, the α5 integrin remained associated with the substrate.