The FASEB Journal express article 10.1096/fj.01-0878fje. Published online April 10, 2002.

Microfilament-dependent movement of the !3 integrin subunit within focal contacts of endothelial cells
Daisuke Tsuruta*, Meredith Gonzales*, Susan B. Hopkinson*, Carol Otey, Satya Khuon*, Robert D. Goldman*, Jonathan C.R. Jones* *Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois; Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina Corresponding author: Jonathan C. R. Jones, Department of Cell and Molecular Biology Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. E-mail: j-jones3@nwu.edu ABSTRACT To gain insight into the dynamic properties of focal contacts, we induced expression of green fluorescent protein-tagged !3 integrin (GFP-!3) and actinin-1 (GFP-actinin-1) in endothelial cells. Both tagged proteins localize with "v!3 integrin in focal contacts distributed towards the periphery of transfected cells. Labeled focal contacts migrate at about 0.1 #m/min in stationary live endothelial cells. We compared !3 integrin and actinin-1 dynamics in focal contacts by using fluorescence recovery after photobleaching. Recovery of signal in bleached focal contacts that have incorporated actinin-1 is rapid and occurs within less than 4 min. This recovery is energydependent. In contrast, recovery of bleached focal contacts that contain GFP-!3 integrin takes longer than 30 min. Yet, when a narrow stripe of fluorescence is bleached across a !3 integrinlabeled focal contact, recovery is complete within 16 min. The latter recovery is energydependent and is blocked not only by actin-filament disrupting drugs but also by a myosin light chain kinase inhibitor. Thus, integrins are not immobile when incorporated into focal contacts, as some have suggested. We propose that integrins are mobile within the confines of focal contacts and that this mobility is supported by an actin-associated molecular motor. Key words: matrix adhesion $ matrix receptors $ FRAP $ actin cytoskeleton $ actinin

he interaction of cells with the extracellular matrix plays a critical role in development and during periods of tissue remodeling by regulating tissue architecture and function (14). In many cell types, the molecules involved in cellmatrix adhesion are found concentrated in specific morphological entities called focal adhesions or focal contacts (57). Within each focal contact, cell surface receptors of the integrin family cluster together and interact with extracellular matrix ligands on the outside of the cell and with the actin-microfilament system in the cytoplasm (57). Indeed, focal contacts, or rather their components, not only function to mediate cellmatrix adhesion but also serve as a surface anchor for the cytoskeleton and as transducers of signals from the matrix to the cell and vice versa (5, 6, 810). Numerous workers have followed the fate of fluorescently tagged focal contact molecules in live

cells, and it is now well established that focal contacts show movement in locomotory and stationary cells (1117). However, whereas focal contacts and their associated cytoskeleton are dynamic, numerous reports in the literature suggest that integrins are immobile in the plane of the membrane when they are assembled into focal contacts in stationary cells (11). The latter results are in conflict with the idea that matrix adhesions are mobile structures. To resolve this issue, in the present study we compared the dynamic properties of an integrin subunit with that of an actin-binding protein in the focal contacts of cultured cells. For our studies we chose to assay the !% integrin subunit in endothelial cells. Endothelial cells are ideal for such analyses for a number of reasons. They assume a flattened morphology when plated onto glass coverslips and assemble large focal contacts, thus facilitating detailed observation of focal contact activity. Also, understanding focal contact dynamics in endothelial cells has implications for endothelial cell/blood vessel physiology in vivo because blood vessel development during tissue remodeling and pathological conditions, such as cancer, involve endothelial cell migration and modulation in their matrix adhesion sites (18, 19). Moreover, the !% integrin is a component of the "v!%&integrin heterodimer, which plays a significant, if not crucial, role in blood vessel formation/angiogenesis (1821). MATERIALS AND METHODS Cell culture, transient transfections and drug treatments Immortalized human bone marrow endothelial cells (TrHBMECs) were maintained in Dulbeccos modified Eagle's medium (DMEM) containing a final concentration of 2 mM Lglutamine, 10% fetal bovine serum (FBS), and 1' RPMI 1640 vitamins solution (22, 23). These were obtained from Babette Weksler (Weill Medical College of Cornell University, Ithaca, NY) and Denise Paulin (Universite Paris VII and Institut Pasteur, Paris, France). For transfection, TrHBMEC were trypsinized and resuspended in N-(2-hydroxyethyl) piperazine-2'-(2-ethanesulphonic acid) (HEPES)-buffered DMEM at about 8 ' 106 cells/ml. The cells were electroporated with DNA at 950 #FD, 200 ohm, and 250 V in a BTX Electro Cell Manipulator 600 (BTX, San Diego, CA). Electroporated cells were transferred to 35-mm dishes containing #1 glass coverslips or locator coverslips (Bellco Glass Inc., Vineland, NJ). To deplete energy stores, cells were incubated in glucose-free DMEM (Gibco BRL, Rockville, MD) supplemented with 10% FBS, 0.05% sodium azide, and 50 mM 2-deoxy-D-glucose (24). Cell populations were treated with these reagents for 1530 min before their analysis. To induce disassembly of the microfilament network, cytochalasin D (Sigma Chemical Co., St. Louis, MO; 0.110 #M) or latrunculin B (Calbiochem, San Diego, CA; 0.11 #M) was added to medium from stock solutions, and cells were incubated for 30 min and 15 min, respectively, before analysis (25, 26). Colcemid (Sigma; 2.5 #g/ml) was added from a stock solution to culture medium, and then the medium was incubated with cells for 16 h to induce microtubule deploymerization. At least five focal contacts in at least three cells were analyzed for each treatment. The myosin light chain kinase inhibitor ML-7 (CN Biosciences Inc., La Jolla, CA) was added to culture medium to a final concentration of 100 #M. Cloning of a !3 integrin cDNA and generation of constructs A cDNA encoding full-length human !3 integrin was generated by reverse transcription-

polymerase chain reaction (RT-PCR) from mRNA isolated from subconfluent populations of TrHBMEC by using a forward primer 5-CGGGAAGCTTACGAGATGCGAGCGC and reverse primer 5-TTATAGATCTGTGCCCCGGTACGTGATATTGGTG. Following gel purification, the PCR product was ligated into the Acceptor Vector (Novagen, Madison, WI) and then excised by restriction digestion with Hind III and Bgl II. The fragment was gel-purified and ligated into the HindIII/Bam H I sites of pEGFP-N1 (Clontech Laboratories Inc., Palo Alto, CA). The construct was sequenced to ensure that the green fluorescent protein (GFP)-!3 integrin cDNA was in frame and without error with the use of Big Dye (Applied Biosystems, Foster City, CA) automated sequencing reagents on an ABI Prism Automated sequencer (Applied Biosystems, Foster City, CA). The GFP-actinin-1 expression vector was described elsewhere (27, 28). Antibodies and fluorescent probes Mouse monoclonal antibodies against GFP (JL-8), "v!3 integrin, and "v!( integrin (LM609; P1F6) were purchased from Clontech Laboratories Inc. and Chemicon International, Inc. (Temecula, CA), respectively. An antiserum against myosin II was provided by Yoshio Fukui (Northwestern University Medical School, Chicago, IL). Rhodamine-conjugated phalloidin was obtained from Molecular Probes (Eugene, OR). Secondary antibodies conjugated to fluorescein, rhodamine, or horseradish peroxidase were purchased from Jackson ImmunoResearch Labs Inc. (West Grove, PA). Protein G agarose was purchased from GIBCO Invitrogen Corp. (Carlsbad, CA). Immunoblotting and immunoprecipitation Confluent cell cultures were solubilized in sample buffer consisting of 8 M urea, 1% sodium dodecyl sulfate (SDS), in 10 mM Tris-HCL, pH 6.8, 15% !-mercaptoethanol. Proteins were separated by polyacrylamide gel electrophoresis on 7.5% acrylamide gels, transferred to nitrocellulose, and processed for immunoblotting as previously described (29, 30). TrHBMECs were extracted in immunoprecipitation buffer (IP buffer) (25 mM HEPES, pH 7.5, 1% Brij 97, 150 mM NaCl, 5 mM MgCl2, 0.2% SDS, and protease inhibitors). Primary antibody was added to the extract and incubated at 4C overnight. Subsequently, 50 #l of protein G agarose (Gibco/BRL) was added to the mix for an additional 2 h. The protein G agarose was collected by centrifugation, washed four times in IP buffer, and then solubilized in SDS-PAGE (polyacrylamide gel electrophoresis) sample buffer. The resulting protein solution was processed for SDS-PAGE/Western immunoblotting under reducing conditions as above. Immunofluorescence microscopy Cells on glass coverslips were fixed in 3.7% formaldehyde in phosphate buffered saline (PBS) for 5 min, washed thoroughly in PBS, and then extracted for 2 min in 20C acetone. Antibody or rhodamine-conjugated phalloidin was overlaid onto the cells, and the preparations were incubated at 37C for 60 min. The cells on coverslips were washed in three changes of PBS. Preparations incubated in phalloidin were mounted immediately onto glass slides, while cells incubated in antibody were overlaid with rhodamine-conjugated secondary antibody, placed at 37C for an additional 60 min, washed extensively, and then mounted on slides. All preparations were viewed on a Zeiss laser scan confocal microscope (LSM 510) (Zeiss Inc., Thornwood, NY). Microscope images were exported as TIF files, and figures were generated by using Adobe

Photoshop 4.0 (Adobe Systems Inc., San Jose, CA). Live cell observation and fluorescence recovery after photobleaching (FRAP) techniques For live cell studies, a coverslip, on which cells had been plated, was placed on a slide with a chip of glass supporting each of the corners of the coverslip. HEPES-buffered medium, containing 0.03 units/ml Oxyrase (Oxyrase, Inc., Mansfield, OH), was added to the space between the coverslip and slide and, to prevent leakage and evaporation, the coverslips were sealed along their edges with a mixture of petroleum jelly, beeswax, and lanolin (1:1:1). The slide preparation was maintained at 37C with an air stream incubator (Model ASI 400; NEVTEK, Burnsville, VA) on the stage of the LSM 510. Time-lapse observations were made by using a 100', 1.4 numerical aperture, oil-immersion objective. GFP images were acquired by excitation at 488 nm and emission at 515545 nm. Phase-contrast images of cells were taken both before and after time-lapse observations. Images from cells that had undergone gross morphological changes during the period of observation were discarded. This was a rare occurrence. Motility of focal contacts was assayed by using the Metamorph Imaging System (Universal Imaging Corp., Downingtown, PA) motion analysis and particle tracking software. FRAP analyses were carried out on the LSM 510 microscope as described by Yoon et al. (24). For TrHBMEC expressing GFP-!3 and GFP-actinin-1, regions were bleached at 488 nm with the lowest iteration possible (1218 for GFP-!3 FRAP and 50-200 for GFP-actinin-1 FRAP) and recovery was monitored at 25 min intervals for GFP-!3 and 15-60 seconds intervals for GFPactinin-1. For quantitative analysis, the fluorescence intensity of (1) the photobleached region and (2) three background intensity values were determined by pixel count by using LSM 510 image analysis software and Metamorph Imaging System software at each time point. The data were analyzed by using Microsoft Excel (Microsoft Corporation, Redmond, WA). The mobile fraction (m) of the measured population of tagged molecules was obtained by estimating the degree to which the plateau fluorescence level (F)) approaches the prebleach value (Fpre) using the equation:

where F0 is the fluorescence intensity immediately after bleach (31). Data were adjusted for sample fading according to (32). The t1/2 value for each experiment was the time taken to reach 50% of the final recovery in fluorescence signal (31). Statistical significance was estimated by ANOVA. RESULTS Characterization of GFP-tagged !3 integrin and actinin-1 in TrHBMEC TrHBMECs were transfected with mammalian expression vectors encoding either GFP-!3 integrin subunit or GFP-actinin-1. Western blot analyses of protein extracts of the transiently

transfected TrHBMEC populations confirm expression of proteins of the appropriate molecular mass; 132 kD for GFP-!3 integrin and 127 kD for GFP-actinin-1 (Fig. 1A, lanes 2 and 3). In addition, we have evaluated whether GFP-!3 integrin incorporates into integrin heterodimers in TrHBMECs by using an immunoprecipitation assay. Antibody LM609 against the "v!% integrin heterodimer and antibody P1F6 against the "v!( integrin complex were added to extracts of TrHBMECs induced to express GFP-!3 integrin and extracts of nontransfected control cells. Precipitated proteins were processed subsequently for immunoblotting by using a monoclonal antibody against GFP (Fig. 1B). LM609 antibodies specifically precipitate a 132 kD protein from transfected cell extracts that is recognized by the GFP antibody, whereas antibody P1F6 does not (Fig. 1B). In TrHBMECs both tagged actinin-1 and !3 integrin proteins incorporate into focal contact-like structures primarily at the cell periphery (Fig. 2A, D). These same focal contacts are stained by LM609 antibodies that recognize the "v!3 integrin heterodimer and appear similar to focal contacts assembled by endothelial cells in vitro (Fig. 2B, E) (for example, see 33). However, GFP-actinin-1 localizes not only to focal contacts but also to the microfilament network of the transfected cells (Fig. 2D). All subsequent experiments were performed on cells such as those shown in Figure 2; that is, cells showing normal morphology in which GFP-tagged proteins have incorporated into focal contact structures. To provide evidence that focal contacts in TrHBMEC containing GFP-tagged proteins show dynamic properties comparable with !1 integrin containing adhesion structures as detailed previously (15, 17), we analyzed the assembly and fate of labeled focal contacts in live subconfluent TrHBMECs (Fig. 3). New focal contacts appear to assemble close to the cell periphery, consistent with the work of others (15, 34). Under the conditions of our studies, these cells show limited migration, and all of our analyses were undertaken on stationary cells. GFP!3 integrin containing focal contacts show considerable movement in TrHBMECs (Fig. 3). We analyzed more than 141 focal contacts in three cells. Of these focal contacts, 54 show migration towards the nucleus, 9 move towards the cell periphery, 8 move along the cell edge, and 11 move in apparently random fashion, whereas 59 show no obvious dynamics. The rate of movement of labeled focal contacts is approximately 0.1 #m/min regardless of the type of motion that they show. Focal contacts containing GFP-actinin-1 also undertake a similar range of dynamic translocation (not shown). These observations are consistent with the work of others who have established that focal contacts show dynamic movements in cells (15, 17) and confirm that the behavior of both the tagged !3 integrin and actinin-1 likely recapitulates that of their endogenous equivalents in TrHBMECs. FRAP analyses of GFP-!3 integrin and GFP-actinin-1 in focal contacts We have analyzed !3 integrin and actinin-1 dynamics in focal contacts by using FRAP. All of our studies were performed on focal contacts clustered towards the cell periphery of stationary TrHBMECs, because these all appear to be classical focal contacts possessing both actinin-1 and the !3 integrin subunit and are distinct from fibrillar matrix adhesions in the perinuclear zone (unpublished observations) (7). When an entire GFP-!3 integrin-containing focal contact is bleached, recovery of a detectable signal takes at least 30 min (Fig. 4AD). The mean value for the mobile fraction is 32%, with a t1/2 of about 22 min (Table 1). Because the recovery is quite slow and incomplete, we were concerned that we had disrupted "v!% integrin complexes within focal contacts by our bleaching technique. To assess whether this is the case, live cells in which

we had bleached GFP-!% integrin-labeled focal contacts were processed for immunofluorescence microscopy by using antibody LM609 against the "v!% integrin heterodimer (Fig. 5). LM609 antibodies show intense staining of both the bleached and untreated focal contacts (Fig. 5). Compared with the slow recovery of GFP-!3 signal in the above studies, FRAP of GFP-actinin-1 labeled focal contacts occurs rapidly in live TrHBMECs (Fig. 4EH). The mobile fraction is more than 63% with a t1/2 of less than 1 min (Table 1). Furthermore, these values for GFPactinin-1 recovery in focal contacts are approximately the same, even when about 1/4 of the area of cell body is bleached (data not shown). We next evaluated fluorescence recovery of stripes bleached across moving, labeled matrix adhesion sites. In the case of GFP-!% integrin-containing focal contacts, recovery of the bleached stripe is complete within about 16 min (Fig. 6AC and inset). The mobile fraction 72%, and t1/2 is approximately 5 min (Table 1). Furthermore, in cases where a stripe was bleached within multiple focal contacts in the same cell, all of the bleached focal contacts recover at the same rate (Fig. 6AC). During the recovery of the bleached stripe, there is a decrease in net fluorescence signal of approximately 15% in the nonbleached regions of the same focal contact (Fig. 6). Remarkably, FRAP occurs within 1 min following bleaching of a narrow band of fluorescence across a GFP-actinin-1 labeled focal contact (Fig. 6JL; Table 1). This is significantly more rapid than when the entire GFP-actinin-1 in an individual focal contact is bleached (Table 1). Effects of energy depletion and microfilament disruption on !3 integrin and actinin-1 dynamics in focal contacts We next analyzed the energy requirements necessary to support mobility of the !3 integrin compared with actinin-1 within focal contacts. To deplete cellular stores of ATP, we incubated populations of transfected TrHBMECs in medium containing a combination of sodium azide and 2-deoxy-D-glucose for 1530 min before assay and in the same medium during microscopical analyses. Fluorescence recovery of a bleached stripe across a !3 integrin labeled focal contact is inhibited under these conditions compared with FRAP in non-drug-treated cells (Fig. 6DF; Table 1). The mobile fraction is 42% with a t1/2 of 4.5 min. Similarly, there is significant negative effect on the recovery of signal following bleaching of actinin-1 labeled matrix adhesions under the same conditions (Fig. 7AD; Table 1). The mobile fraction drops to 43% with a t1/2 of 1 min (Table 1). To study the potential involvement of ligand and/or cytoskeleton in FRAP of a stripe across a focal contact that has incorporated GFP-!3 integrin, we used either antibodies that inhibit "v!% integrin/ligand interaction (LM609) or drugs (colcemid, cytochalasin D, and latrunculin B) that perturb the organization of the cytoskeleton. Antibody LM609, which inhibits "v!3 interaction with a variety of extracellular ligands, has no obvious impact on FRAP of GFP-!3 integrin (Table 1) (2022). For our cytoskeleton studies, we first determined the treatment regimen that was sufficient to induce microtubule/vimentin and microfilament network collapse by staining drug-treated cells with anti-tubulin antibody, anti-vimentin antibody, or rhodamine conjugated phalloidin. Colcemid added to culture medium to a final concentration of 2.5 #g/ml and then incubated with cells for 16 h is sufficient to induce both disassembly of the microtubule network and collapse of the vimentin network in TrHBMECs (not shown). The microfilament network in cells incubated for 30 min in medium supplemented with as little as 0.1 #M cytochalasin D or incubated for 15 min in medium containing 0.1 #M latrunculin B appears collapsed and fails to

associate with focal contacts (not shown). FRAP analyses were then performed on TrHBMECs under these drug treatment regimens. Both t1/2 and the mobile fraction of GFP-!3 integrin in TrHBMECs treated with colcemid are comparable with that observed in non-drug-treated cells (Table 1). In contrast, both latrunculin B and cytochalasin D treatment inhibit the extent of recovery of a signal within a bleached stripe across a GFP-!3 integrin labeled focal contact (Fig. 6GI; Table 1). The mobile fraction in cells treated with cytochalasin D is 42% with a t1/2 of 5 min, whereas in cells treated with latrunculin B the mobile fraction is 50% with a t1/2 of 5.9 min (Table 1). The mobile fraction under both conditions is reduced significantly in comparison with results from non-drug-treated cells (Table 1). As a control we also evaluated FRAP in TrHBMECs treated with cytochalasin D and then allowed to recover for 6 h after drug treatment (Table 1). FRAP in such cells is identical to that observed in non-drug-treated cells. We were unable to perform comparable FRAP analyses of GFP-actinin-1 in focal contacts in TrHBMECs incubated in medium supplemented with cytochalasin D or latrunclin B because actinin-1 fails to localize to matrix adhesion sites stained with antibodies against the "v!% integrin complex in drug treated cells (Fig. 8). The above data indicate that !% integrin mobility within the confines of a focal contact is ligand and microtubule-independent while microfilament and energy-dependent. This finding raises the possibility that a molecular motor associated with the actin cytoskeleton may be involved in integrin dynamics in focal contacts. One obvious candidate for such a motor is myosin. To test this hypothesis, we first prepared TrHBMECs for double labeling by using an antiserum against myosin II and monoclonal antibody LM609 against "v!% integrin (Fig. 9A). Myosin II is found associated with the microfilament network, as well as at the sites of focal contacts (yellow color in Fig. 9A) (35). We next used myosin light chain kinase inhibitor ML-7 to assess whether this could inhibit FRAP. ML-7 was used at concentrations that induce some changes in overall cell morphology but fails to produce either microfilament collapse or focal contact disassembly as evaluated by LM609 antibody staining (Fig. 9B). In FRAP studies, ML-7 inhibits recovery of a stripe ablated across a GFP-!3 labeled focal contact in TrHBMECs (Table 1). DISCUSSION Focal contacts are considered to play an active role in adhesion and motility of normal and cancer cells during development, periods of tissue remodeling, and metastasis. They provide a means by which cells adhere to the extracellular matrix over or through which the cells migrate and are involved in producing tractive forces necessary for motility (36). Despite increasing evidence that focal contacts are dynamic, even in stationary cells, numerous reports have concluded that integrin receptors, actin, and actin-associated proteins show low mobility within an intact focal contact (11, 1517). It was our goal to test this long-standing idea by assessing the fate of both an integrin subunit and a cytoskeletal component of focal contacts in stationary live cells. To initiate our analyses, we first expressed GFP-!% integrin and GFP-actinin-1 in endothelial cells and then assessed whether the tagged proteins behave like their untagged counterparts. Both GFP-labeled proteins incorporate into focal contacts. Moreover, GFP-!3 integrin coprecipitates with complexes of "v!3 integrin heterodimers. In addition, others have shown recently that integrin complexes containing an identical GFP-!3 integrin to the one we have produced are fully functional and bind ligand (37).

Consistent with published observations of tagged non-full-length integrins or tagged non-integrin focal contact proteins as markers, focal contacts containing GFP-!3 and GFP-actinin-1 primarily move toward the cell center at speeds of about 0.10.2 #m/min (15, 17). Taken together these data support the notion that, when expressed in endothelial cells, not only GFP-actinin-1 but also GFP-!% integrin exhibit the properties of the endogenous, untagged !3 integrin subunit. Indeed, these observations allowed us to focus our studies on the dynamic properties of the !% integrin subunit and actinin-1 in focal contacts in live stationary cells by using FRAP techniques. Recovery of GFP-actinin-1 into fully bleached focal contacts in endothelial cells occurs within less than 4 min. The mean value for the mobile fraction of labeled actinin-1 molecules, a numerical indicator of recovery, is 63% with a t1/2 of recovery of about 0.9 min. The mobile fraction is significantly larger than that reported by others (38) as is the rate of recovery (27). The rate and extent of recovery is even more rapid when GFP-actinin-1 exchanges into a single bleached stripe across an individual focal contact. Indeed, this value approaches that reported by others who studied actinin exchange into non-focal contact aggregates, termed podosomes, which occur along the substratum attached surface of transformed cells (16). These authors have suggested that this high rate of exchange is by diffusion, limited merely by the cytoplasm (16). However, this is unlikely in our studies because we observe a significant decrease in the mobile fraction of actinin-1 in energy-depleted TrHBMEC. Moreover, because recovery of actinin-1 into focal contacts occurs even when neighboring focal contacts and their associated cytoskeleton are bleached, it is unlikely that there is an exchange of actinin-1 between focal contacts or between focal contacts and cytoskeleton bound actinin-1. Indeed, our data support the idea that there is a rapid, energy-dependent exchange of actinin-1 between a soluble pool of actinin-1 and focal contacts (13). In contrast to actinin-1, recovery of GFP-!% integrin subunits in bleached focal contacts is a slow process, taking more than 60 min or more. This indicates that exchange from cytoplasmic stores of integrin and focal contacts is probably not a simple diffusion event. This is not too surprising because incorporation of new integrin into focal contacts presumably involves movement of internal membrane-bound, newly synthesized, or stored protein to the cell surface and assembly into a specific membrane domain. However, our data provide evidence that, within an individual focal contact, the !% integrin subunit is capable of moving in an energy-dependent manner. Moreover, there is a net loss of fluorescence in the non-bleached regions of a GFP-!% integrincontaining focal contact during recovery of bleached stripe zones. This finding indicates that recovery of signal is a result of movement of protein from non-bleached regions of focal contacts into a bleached zone. This finding was a surprise because it has been reported that integrins are immobile when assembled into a focal contact (11). Our data show otherwise. One possible explanation for the disparity in results is that many of the earlier studies on focal contact dynamics in live cells were undertaken by visualizing fluorescently tagged antibodies bound to integrins; we suggest that these antibodies may impact the dynamics of the proteins to which they bind. Of course, we cannot rule out the possibility that the divergence in results may reflect variability in the dynamics of different integrin receptors in different cell types. Although the recovery of signal into the bleached stripe of a GFP-!% integrin-labeled focal contact is relatively rapid, it is too slow to be accounted for by diffusion alone and is slower than recovery of GFP-actinin-1 under similar circumstances; that is, integrin mobility within a focal contact is restricted. One possibility is that restriction in movement results from !% integrin being tethered to its extracellular ligand and/or to the cytoskeleton. If this were the case,

perturbation of either the cytoskeleton or ligand binding of !3 integrin-containing heterodimers should relieve such constraints, and therefore result in rapid fluorescence recovery (39). However, treatment of TrHBMECs with antibody LM609, which perturbs "v!3 integrin binding to ligand, has a minor inhibitory impact on integrin dynamics. In addition, there is no decrease in the rate of FRAP of the bleached stripe zone of !% labeled focal contacts in TrHBMECs treated with colcemid, which not only perturbs the microtubule network but also induces collapse of the vimentin cytoskeleton (40). In contrast, the mobile fraction of labeled integrin in focal contacts is reduced by 60% in cells treated with microfilament-perturbing drugs. This implies that the microfilament system does not merely position integrins but plays an active role as a positive regulator of mobility of !% integrin within a focal contact. Because FRAP is slowed in energydepleted cells and is microfilament-dependent, this raises the possibility that an actin-associated molecular motor is involved in the dynamic aspects of !3 integrin-containing heterodimers that we have detailed. Our data concerning the localization of myosin II to focal contacts and the ability of the myosin light chain kinase inhibitor ML-7 to inhibit fluorescence recovery suggests that myosin is a good candidate for such a molecular motor. However, because ML-7 perturbs the actin cytoskeleton, we cannot yet rule out the possibility that the effects of this inhibitor on FRAP may be via a local disruption of the actin cytoskeleton. In summary our data reveal that certain cytoskeleton associated proteins, but also integrins, are mobile within the confines of a focal contact. We hypothesize that such movement is crucial to focal contact function. Indeed, mobility of integrins in focal contacts would allow a cycling in receptor/ligand interactions, as focal contacts move along the substratum attached surface of cells even in stationary cells (36, 41). We envision that precise regulation in integrin/ligand binding and modulation in integrin mobility by the cytoskeleton within a focal contact is necessary to permit focal contacts to be dynamic. ACKNOWLEDGMENTS This work was supported by grants to J.C.R.J. from the National Institutes of Health (DE12328) and the American Heart Association (0151136Z). D. T. was supported, in part, by United States Army Medical Research and Materiel Command grant # DAMD17-00-1-0386. We thank Weiming Yu, Yoshio Fukui, Shinichiro Kojima, and Miri Yoon for helping to perform the FRAP studies and analyzing data. We are very grateful for the gift of cells and antibodies from Babette Weksler, Denise Paulin, and Yoshio Fukui and for the technical assistance of Kristin deHahn. REFERENCES 1. 2. 3. 4. Clark, E. A., and Brugge, J. S. (1995) Integrins and signal transduction: the road taken. Science 268, 233-239 Gumbiner, B. M. (1996) Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84, 345-357 Howe, A., Aplin, A. E., Alahari, S. K., and Juliano, R. L. (1998) Integrin signaling and cell growth. Curr. Opin. Cell Biol. 10, 220-231 Schoenwaelder, S. M., and Burridge, K. (1999) Bidirectional signaling between the cytoskeleton and integrins. Curr. Opin. Cell Biol. 11, 274-286

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Received November 20, 2001; revised February 14, 2002.

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38.

39. 40. 41.

Table 1

FRAP Analysis.
FRAP Analysis Drug Treatment n t1/2 (value SE) GFP-3 Full focal contact bleach None 5 22.00 2.79 < 0.01 32 2.7 < 0.0001 p Mobile Fraction (%) (value SE) p

Bleached stripe

None 2-deoxy-D-glucose/azide (energy depletion) Anti-v3 antibody Colcemid (microtubule) Cytochalasin D (microfilament) Cytochalasin D+recovery* Latrunculin B (microfilament) ML-7 (myosin light chain kinase)

11 16 13 19

5.22 0.35 4.54 0.33 6.89 0.47 6.20 0.46

ns < 0.01 ns

72 3.9 42 4.3 68 5.8 71 5.0

- < 0.0001 ns ns

12

5.05 0.77

ns

42 4.9

< 0.0001

10 11

6.37 0.84 5.85 0.67

ns ns

73 4.4 50 6.3

ns < 0.01

14

4.18 0.40

ns

52 4.3

< 0.01

GFP-actinin-1 Full focal contact bleach None 2-deoxy-D-glucose/azide (energy depletion) Bleached stripe None 14 10 8 0.88 0.07 1.08 0.06 0.57 0.05 ns < 0.01 63 4.3 43 5.1 71 6.4 0.013 ns

Cells were treated with cytochalasin D. After a 30 min treatment, media were replaced and the cells were allowed to recover for 6 h before FRAP studies.

Table 1. Quantification of FRAP analyses. Data are presented with standard error of the mean. n, number of focal contacts evaluated. t1/2, minutes taken to
recover to one-half the final intensity value. The target of each drug is listed in parenthesis under the drug name. Statistical analyses are listed in the p columns. Comparisons were made between focal contacts in control cells and drug-treated cells.

Fig. 1

Figure 1. Characterization of GFP-actinin-1 and GFP-3 integrin in TrHBMECs. A) Extracts of nontransfected

TrHBMECs (lane 1) or populations of TrHBMEC transfected either with a construct encoding GFP-3 integrin (lane 2) or GFP-actinin-1 (lane 3) were processed for Western immunoblot analysis by using a GFP monoclonal antibody. Approximately 15 g of each protein sample was loaded per lane of the gel. Molecular weights are indicated to the left. B) Approximately 15 g of protein extracts of control and GFP-3 integrin expressing TrHBMECs were loaded into each lane of a gel. Separated proteins were transferred to nitrocellulose, which was then processed for immunoblotting using GFP antibody (lanes 1 and 2, respectively). Molecular weight markers are indicated to the left. A reactive species is seen in lane 2 only. The same extracts were subjected to immunoprecipitation (IP) by using either antibody LM609 against the v3 integrin complex (lanes 3 and 4) and P1F6 against the v5 integrin complex (lanes 5 and 6). Approximately 15 g of the precipitated proteins were loaded onto each lane of the gel, which was then prepared for immunoblotting (IB) by using the GFP antibody (upper panel, lanes 36). Note that GFP-3 integrin is precipitated by the v3 integrin antibody in lane 4 (upper panel), but not by antibodies against v5 integrin (lane 6; upper panel). Molecular weights are indicated to the left of lane 1. The lower panel (lanes 36) shows reactivity of precipitated immunoglobulin in the same immunoblot by using conjugated secondary anti-mouse antibody and confirms that equal amounts of precipitated antibody were loaded into each lane. A molecular weight marker is indicated to the left of lane 3 in the lower panel.

Fig. 2

Figure 2. GFP-3 integrin and GFP-actinin-1 incorporate into focal contacts in TrHBMECs. TrHBMECs

transfected either with a construct encoding GFP-3 integrin (AC) or GFP-actinin-1 (DF) were fixed and then processed for immunofluorescence microscopy by using antibody LM609 against the v3 integrin heterodimer. The GFP signal in cells is shown in (A) and (D), respectively, whereas LM609 staining is shown in (B) and (E). (C) and (F) are the merged images of the two sets of images. Both GFP-3 (A) and GFP-actinin-1 (D) colocalize precisely with the endogenous v3 integrin complexes in focal contacts towards the cell periphery (B, E). The latter appear yellow in the merged images in (C) and (F). GFP-actinin-1 in D is found in a punctate pattern in association with stress fibers. The nuclear stain in (B) and (E) is nonspecific binding of the antibody. Bar, 10 m.

Fig. 3

Figure 3. The dynamics of v3 integrin-containing focal contacts in live cells. TrHBMECs expressing GFP-3
integrin were followed for 35 min in the live state. Images at 0, 15, and 35 min were coded in blue, green, and red, respectively, and then overlaid following the procedure of (15). Long arrows indicate focal contacts that are moving toward the cell center. This structure appears as a rainbow of blue, green, and red. An arrowhead marks a focal contact moving towards the cell surface, while a small arrow marks a focal contact migrating along the cell edge. Bar, 10 m.

Fig. 4

either GFP-3 integrin (AD) or GFP-actinin-1 (EH) are shown before bleaching (A, E), immediately after bleaching (B, F) and at the indicated time following treatment (C, D, G, H). In (AD), one focal contact was bleached (arrow), while in (EH), three distinct focal contacts were bleached (region marked by arrow). Note that even at 60 min there is minimal recovery in signal in the bleached focal contact in (D). Signal is restored in as little as 4 min into focal contacts shown in H. Bar, 2 m.

Figure 4. GFP-3 integrin and GFP-actinin-1 dynamics in focal contacts using FRAP. Images of cells expressing

Fig. 5

contacts in an individual TrHBMEC expressing GFP-3 integrin were bleached (area outlined by the box in BD) and then the same cell was fixed and processed for indirect immunofluorescence using the anti-v3 integrin antibody LM609. Images of GFP-tagged proteins in the live cell are shown before (A) and after bleaching (B). Localization of LM609 antibodies in the fixed cell is shown in (C). In (D) the images in (B) and (C) have been merged, revealing that the bleached focal contacts are recognized by LM609 antibodies (boxed area in C and D). In (D) these bleached focal contacts appear red; their unbleached counterparts are yellow. Bar, 10 m.

Figure 5. Photobleaching does not affect localization of v3 integrin heterodimers in focal contacts. Several focal

Fig. 6

Figure 6. FRAP of partially bleached focal contacts in TrHBMECs. Images of cells expressing either GFP-3
integrin (AI) or GFP-actinin-1 (JL) are shown before bleaching (A, D, G, J), immediately after bleaching (B, E, H, K) and at the indicated times following treatment (C, F, I, L). A narrow stripe across one or more focal contacts was bleached in the cells. The cell in (DF) was incubated in medium containing 0.05% sodium azide and 50 mM 2-deoxy-Dglucose, whereas the cell in (GI) was incubated in medium supplemented with 0.1 M cytochalasin D. Recovery of signal is inhibited in (F) and (I) compared with (C). There is rapid recovery of the bleached area in (L). The inset in (C) shows an overlay of colorized images of the focal contacts shown in (AC) taken immediately following (blue) and at 8 (green) and 16 (red) min after photobleaching. Note that the focal contacts show movement as indicated by the rainbow color effect. Bar in (C), 2 m. Bar in inset in C, 1 m.

Fig. 7

Figure 7. FRAP of GFP-actinin-1 labeled focal contacts in TrHBMECs is energy-dependent. Images of a cell
expressing GFP-actinin-1 are shown before bleaching (A), immediately after bleaching (B), and at the indicated times following treatment (C, D). In this instance the cell was incubated in medium containing 0.05% sodium azide and 50 mM 2-deoxy-D-glucose. Note that recovery is inhibited (compare with Fig. 4). In these energy-depleted cells, GFP-actinin-1 does not show obvious association with stress fibers. Bar, 2 m.

Fig. 8

Figure 8. GFP-actinin-1 does not localize to focal contacts in TrHBMECs treated with microfilament destabilizing
drugs. At 72 h after transfection of a population of TrHBMECs with a construct encoding GFP-actinin-1, the cells were treated with 0.1 M cytochalasin D and, 30 min later, were prepared for indirect immunofluorescence by using the antiv3 integrin antibody LM609. GFP-actinin-1 (A) shows no obvious association with focal contacts recognized by antibody LM609 (B). (A) and (B) are merged in (C). Bar, 10 m.

Fig. 9

Figure 9. Myosin II localization in TrHBMECs. TrHBMECs were processed for double labeling using an antiserum

against myosin II (green) and LM609 antibodies against the v3 integrin heterodimer (red). Overlaid images of the stains of cells are shown in (A, B). In control cells in (A), myosin II localizes along bundles of microfilaments. In addition, a yellow color indicates myosin II colocalization in association with v3 integrin at the site of focal contacts. In (B), TrHBMECs were treated with 100 M ML-7 for 30 min before processing with antibody. Note that the treated cells possess microfilament bundles and focal contacts stained by LM609 antibodies despite changes in their overall morphology. Bar, 10 m.