microfilament dependet movement of the b3 integrin subunit within focal contacts of endothelial...

8/21/2019 Microfilament Dependet Movement of the B3 Integrin Subunit Within Focal Contacts of Endothelial Cells

1/22

The FASEB Journalexpress 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 BiologyNorthwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611.

E-mail: [email protected]

ABSTRACT

To gain insight into the dynamic properties of focal contacts, we induced expression of greenfluorescent protein-tagged !3 integrin (GFP-!3) and actinin-1 (GFP-actinin-1) in endothelialcells. Both tagged proteins localize with "v!3 integrin in focal contacts distributed towards theperiphery of transfected cells. Labeled focal contacts migrate at about 0.1 #m/min in stationarylive endothelial cells. We compared !3 integrin and actinin-1 dynamics in focal contacts by usingfluorescence recovery after photobleaching. Recovery of signal in bleached focal contacts thathave incorporated actinin-1 is rapid and occurs within less than 4 min. This recovery is energy-dependent. 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 integrin-labeled focal contact, recovery is complete within 16 min. The latter recovery is energy-dependent and is blocked not only by actin-filament disrupting drugs but also by a myosin lightchain kinase inhibitor. Thus, integrins are not immobile when incorporated into focal contacts, assome have suggested. We propose that integrins are mobile within the confines of focal contactsand that this mobility is supported by an actin-associated molecular motor.

Key words: matrix adhesion $matrix receptors $FRAP $actin cytoskeleton $actinin

T

he interaction of cells with the extracellular matrix plays a critical role in development andduring periods of tissue remodeling by regulating tissue architecture and function (14). Inmany cell types, the molecules involved in cellmatrix adhesion are found concentrated in

specific morphological entities called focal adhesions or focal contacts (57). Within each focalcontact, cell surface receptors of the integrin family cluster together and interact withextracellular matrix ligands on the outside of the cell and with the actin-microfilament system inthe cytoplasm (57). Indeed, focal contacts, or rather their components, not only function tomediate cellmatrix adhesion but also serve as a surface anchor for the cytoskeleton and astransducers 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


2/22

cells, and it is now well established that focal contacts show movement in locomotory andstationary cells (1117). However, whereas focal contacts and their associated cytoskeleton aredynamic, numerous reports in the literature suggest that integrins are immobile in the plane ofthe membrane when they are assembled into focal contacts in stationary cells (11). The latterresults are in conflict with the idea that matrix adhesions are mobile structures. To resolve thisissue, in the present study we compared the dynamic properties of an integrin subunit with thatof an actin-binding protein in the focal contacts of cultured cells. For our studies we chose toassay the !%integrin subunit in endothelial cells. Endothelial cells are ideal for such analyses fora number of reasons. They assume a flattened morphology when plated onto glass coverslips andassemble large focal contacts, thus facilitating detailed observation of focal contact activity.Also, understanding focal contact dynamics in endothelial cells has implications for endothelialcell/blood vessel physiology in vivo because blood vessel development during tissue remodelingand pathological conditions, such as cancer, involve endothelial cell migration and modulation intheir 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 vesselformation/angiogenesis (1821).

MATERIALS AND METHODS

Cell culture, transient transfections and drug treatments

Immortalized human bone marrow endothelial cells (TrHBMECs) were maintained inDulbeccos modified Eagle's medium (DMEM) containing a final concentration of 2 mM L-glutamine, 10% fetal bovine serum (FBS), and 1'RPMI 1640 vitamins solution (22, 23). Thesewere 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. Thecells were electroporated with DNA at 950 #FD, 200 ohm, and 250 V in a BTX Electro CellManipulator 600 (BTX, San Diego, CA). Electroporated cells were transferred to 35-mm dishescontaining #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 inducedisassembly 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 mediumfrom stock solutions, and cells were incubated for 30 min and 15 min, respectively, beforeanalysis (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 microtubuledeploymerization. At least five focal contacts in at least three cells were analyzed for eachtreatment. 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-


3/22

polymerase chain reaction (RT-PCR) from mRNA isolated from subconfluent populations ofTrHBMEC by using a forward primer 5-CGGGAAGCTTACGAGATGCGAGCGC and reverseprimer 5-TTATAGATCTGTGCCCCGGTACGTGATATTGGTG. Following gel purification, thePCR product was ligated into the Acceptor Vector (Novagen, Madison, WI) and then excised byrestriction digestion with Hind III and Bgl II. The fragment was gel-purified and ligated into theHindIII/Bam H I sites of pEGFP-N1 (Clontech Laboratories Inc., Palo Alto, CA). The constructwas sequenced to ensure that the green fluorescent protein (GFP)-!3 integrin cDNA was inframe 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 wasobtained 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% sodiumdodecyl sulfate (SDS), in 10 mM Tris-HCL, pH 6.8, 15% !-mercaptoethanol. Proteins wereseparated by polyacrylamide gel electrophoresis on 7.5% acrylamide gels, transferred tonitrocellulose, and processed for immunoblotting as previously described (29, 30).

TrHBMECs were extracted in immunoprecipitation buffer (IP buffer) (25 mM HEPES, pH7.5, 1% Brij 97, 150 mM NaCl, 5 mM MgCl2, 0.2% SDS, and protease inhibitors). Primaryantibody was added to the extract and incubated at 4C overnight. Subsequently, 50 #l of proteinG agarose (Gibco/BRL) was added to the mix for an additional 2 h. The protein G agarose wascollected by centrifugation, washed four times in IP buffer, and then solubilized in SDS-PAGE(polyacrylamide gel electrophoresis) sample buffer. The resulting protein solution was processedfor 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. Antibodyor rhodamine-conjugated phalloidin was overlaid onto the cells, and the preparations wereincubated at 37

C 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 cellsincubated in antibody were overlaid with rhodamine-conjugated secondary antibody, placed at37

C for an additional 60 min, washed extensively, and then mounted on slides. All preparationswere 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


4/22

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 achip 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 spacebetween the coverslip and slide and, to prevent leakage and evaporation, the coverslips weresealed along their edges with a mixture of petroleum jelly, beeswax, and lanolin (1:1:1). Theslide 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 byusing a 100', 1.4 numerical aperture, oil-immersion objective. GFP images were acquired byexcitation at 488 nm and emission at 515545 nm. Phase-contrast images of cells were takenboth before and after time-lapse observations. Images from cells that had undergone grossmorphological changes during the period of observation were discarded. This was a rareoccurrence. 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 thelowest iteration possible (1218 for GFP-!3 FRAP and 50-200 for GFP-actinin-1 FRAP) andrecovery was monitored at 25 min intervals for GFP-!3 and 15-60 seconds intervals for GFP-actinin-1. For quantitative analysis, the fluorescence intensity of (1) the photobleached regionand (2) three background intensity values were determined by pixel count by using LSM 510image analysis software and Metamorph Imaging System software at each time point. The datawere 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)) approachesthe prebleach value (Fpre) using the equation:

where F0 is the fluorescence intensity immediately after bleach (31). Data were adjusted forsample fading according to (32). The t1/2 value for each experiment was the time taken to reach50% 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-!3integrin subunit or GFP-actinin-1. Western blot analyses of protein extracts of the transiently


5/22

transfected TrHBMEC populations confirm expression of proteins of the appropriate molecularmass; 132 kD for GFP-!3 integrin and 127 kD for GFP-actinin-1 (Fig. 1A, lanes 2 and 3). Inaddition, we have evaluated whether GFP-!3 integrin incorporates into integrinheterodimers inTrHBMECs by using an immunoprecipitation assay. Antibody LM609 against the "v!%integrinheterodimer and antibody P1F6 against the "v!( integrin complex were added to extracts ofTrHBMECs induced to express GFP-!3 integrin and extracts of nontransfected control cells.Precipitated proteins were processed subsequently for immunoblotting by using a monoclonalantibody against GFP (Fig. 1B). LM609 antibodies specifically precipitate a 132 kD protein fromtransfected 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-likestructures primarily at the cell periphery(Fig. 2A, D). These same focal contacts are stained byLM609 antibodies that recognize the "v!3 integrin heterodimer and appear similar to focalcontacts assembled by endothelial cells in vitro (Fig. 2B, E) (for example, see 33). However,GFP-actinin-1 localizes notonly to focal contacts but also to the microfilament network of the

transfected cells (Fig. 2D).All subsequent experiments were performed on cells such as thoseshown in Figure 2; that is, cells showing normal morphology in which GFP-tagged proteins haveincorporated into focal contact structures.

To provide evidence that focal contacts in TrHBMEC containing GFP-tagged proteins showdynamic properties comparable with !1 integrin containing adhesion structures as detailedpreviously (15, 17), we analyzed the assembly and fate of labeled focal contacts in livesubconfluent TrHBMECs (Fig. 3). New focal contacts appear to assemble close to the cellperiphery, consistent with the work of others (15, 34). Under the conditions of our studies, thesecells 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). Weanalyzed 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 movein apparently random fashion, whereas 59 show no obvious dynamics. The rate of movement oflabeled focal contacts is approximately 0.1 #m/min regardless of the type of motion that theyshow. Focal contacts containing GFP-actinin-1 also undertake a similar range of dynamictranslocation (not shown). These observations are consistent with the work of others who haveestablished that focal contacts show dynamic movements in cells (15, 17) and confirm that thebehavior of both the tagged !3 integrin and actinin-1 likely recapitulates that of their endogenousequivalents in TrHBMECs.

FRAP analyses of GFP-3 integrin and GFP-actinin-1 in focal contactsWe 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 stationaryTrHBMECs, because these all appear to be classical focal contacts possessing both actinin-1 andthe !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 isbleached, recovery of a detectable signal takes at least 30 min (Fig. 4AD). The mean value forthe mobile fraction is 32%, with a t1/2 of about 22 min(Table 1). Because the recovery is quiteslow and incomplete, we were concerned that we had disrupted "v!%integrin complexes withinfocal contacts by our bleaching technique. To assess whether this is the case, live cells in which


6/22

we had bleached GFP-!%integrin-labeled focal contacts were processed for immunofluorescencemicroscopy by using antibody LM609 against the "v!% integrin heterodimer (Fig. 5). LM609antibodies 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-1labeled focal contacts occurs rapidly in live TrHBMECs (Fig. 4EH). The mobile fraction ismore than 63% with a t1/2 of less than 1 min (Table 1). Furthermore, these values for GFP-actinin-1 recovery in focal contacts are approximately the same, even when about 1/4 of the areaof cell body is bleached (data not shown).

We next evaluated fluorescence recovery of stripes bleached across moving, labeled matrixadhesion sites. In the case of GFP-!%integrin-containing focal contacts, recovery of the bleachedstripe is complete within about 16 min(Fig. 6ACandinset). The mobile fraction 72%, and t1/2is approximately 5 min (Table 1). Furthermore, in cases where a stripe was bleached withinmultiple 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 fluorescenceacross a GFP-actinin-1 labeled focal contact (Fig. 6JL;Table 1). This is significantly more rapidthan 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-1dynamics in focal contacts

We next analyzed the energy requirements necessary to support mobility of the !3 integrincompared with actinin-1 within focal contacts. To deplete cellular stores of ATP, we incubatedpopulations of transfected TrHBMECs in medium containing a combination of sodium azide and2-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 isinhibited 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 negativeeffect on the recovery of signal followingbleaching of actinin-1 labeled matrix adhesions underthe 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 afocal 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) thatperturb the organization of the cytoskeleton. Antibody LM609, which inhibits "v!3 interactionwith 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 thatwas sufficient to induce microtubule/vimentin and microfilament network collapse by stainingdrug-treated cells with anti-tubulin antibody, anti-vimentin antibody, or rhodamine conjugatedphalloidin. Colcemid added to culture medium to a final concentration of 2.5 #g/ml and thenincubated with cells for 16 h is sufficient to induce both disassembly of the microtubule networkand collapse of the vimentin network in TrHBMECs (not shown). The microfilament network incells incubated for 30 min in medium supplemented with as little as 0.1 #M cytochalasin D orincubated for 15 min in medium containing 0.1 #M latrunculin B appears collapsed and fails to


7/22

associate with focal contacts (not shown). FRAP analyses were then performed on TrHBMECsunder these drug treatment regimens. Both t1/2 and the mobile fraction of GFP-!3 integrin inTrHBMECstreated 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 ofrecovery 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 5min, whereas in cells treated with latrunculin B themobile fraction is 50% with a t1/2 of 5.9 min(Table 1). The mobile fraction under both conditions is reduced significantly in comparison withresults from non-drug-treated cells (Table 1). As a control we also evaluated FRAP inTrHBMECs 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 wereunable to perform comparable FRAP analyses of GFP-actinin-1 in focal contacts in TrHBMECsincubated in medium supplemented with cytochalasin D or latrunclin B because actinin-1 fails tolocalize to matrix adhesion sites stained with antibodies against the "v!% integrin complex indrug treated cells (Fig. 8).

The above data indicate that !%integrin mobility within the confines of a focal contact is ligandand microtubule-independent while microfilament and energy-dependent. This finding raises thepossibility that a molecular motor associated with the actin cytoskeleton may be involved inintegrin dynamics in focal contacts.One obvious candidate for such a motor is myosin. To testthis hypothesis, we first prepared TrHBMECs for double labeling by using anantiserum againstmyosin II and monoclonal antibody LM609 against "v!%integrin (Fig. 9A). Myosin II is foundassociated with the microfilament network, as well as at the sites of focal contacts (yellow colorin Fig. 9A) (35). We next used myosin light chain kinase inhibitor ML-7 to assess whether thiscould inhibit FRAP. ML-7 was used at concentrations that induce some changes in overall cellmorphology but fails to produce either microfilament collapse or focal contact disassembly asevaluated by LM609 antibody staining (Fig. 9B). In FRAP studies, ML-7 inhibits recovery of astripe ablated across a GFP-!3 labeled focal contactin TrHBMECs (Table 1).

DISCUSSION

Focal contacts are considered to play an active role in adhesion and motility of normal andcancer cells during development, periods of tissue remodeling, and metastasis. They provide ameans by which cells adhere to the extracellular matrix over or through which the cells migrateand are involved in producing tractive forces necessary for motility (36). Despite increasingevidence that focal contacts are dynamic, even in stationary cells, numerous reports haveconcluded that integrin receptors, actin, and actin-associated proteins show low mobility withinan intact focal contact (11, 1517). It was our goal to test this long-standing idea by assessing thefate of both an integrin subunit and a cytoskeletal component of focal contacts in stationary livecells.

To initiate our analyses, we first expressed GFP-!% integrin and GFP-actinin-1 in endothelialcells and then assessed whether the tagged proteins behave like their untagged counterparts. BothGFP-labeled proteins incorporate into focal contacts. Moreover, GFP-!3 integrin coprecipitateswith complexes of "v!3 integrin heterodimers. In addition, others have shown recently thatintegrin complexes containing an identical GFP-!3 integrin to the one we have produced arefully functional and bind ligand (37).


8/22

Consistent with published observations of tagged non-full-length integrins or tagged non-integrinfocal contact proteins as markers, focal contacts containing GFP-!3 and GFP-actinin-1 primarilymove toward the cell center at speeds of about 0.10.2 #m/min (15, 17). Taken together thesedata support the notion that, when expressed in endothelial cells, not only GFP-actinin-1 but alsoGFP-!%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 !%integrinsubunit 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 withinless than 4 min. The mean value for the mobile fraction of labeled actinin-1 molecules, anumerical indicator of recovery, is 63% with a t1/2 of recovery of about 0.9 min. The mobilefraction 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 singlebleached stripe across an individual focal contact. Indeed, this value approaches that reported byothers 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 mobilefraction of actinin-1 in energy-depleted TrHBMEC. Moreover, because recovery of actinin-1 intofocal contacts occurs even when neighboring focal contacts and their associated cytoskeleton arebleached, it is unlikely that there is an exchange of actinin-1 between focal contacts or betweenfocal contacts and cytoskeleton bound actinin-1. Indeed, our data support the idea that there is arapid, energy-dependent exchange of actinin-1 between a soluble pool of actinin-1 and focalcontacts (13).

In contrast to actinin-1, recovery of GFP-!%integrin subunits in bleached focal contacts is a slowprocess, taking more than 60 min or more. This indicates that exchange from cytoplasmic storesof 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 ofinternal membrane-bound, newly synthesized, or stored protein to the cell surface and assemblyinto a specific membrane domain. However, our data provide evidence that, within an individualfocal 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-!%integrin-containing focal contact during recovery of bleached stripe zones. This finding indicates thatrecovery of signal is a result of movement of protein from non-bleached regions of focal contactsinto a bleached zone. This finding was a surprise because it has been reported that integrins areimmobile when assembled into a focal contact (11). Our data show otherwise. One possibleexplanation for the disparity in results is that many of the earlier studies on focal contactdynamics in live cells were undertaken by visualizing fluorescently tagged antibodies bound tointegrins; 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 reflectvariability 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 focalcontact is relatively rapid, it is too slow to be accounted for by diffusion alone and is slower thanrecovery of GFP-actinin-1 under similar circumstances; that is, integrin mobility within a focalcontact is restricted. One possibility is that restriction in movement results from !% integrinbeing tethered to its extracellular ligand and/or to the cytoskeleton. If this were the case,


9/22

perturbation of either the cytoskeleton or ligand binding of !3 integrin-containing heterodimersshould relieve such constraints, and therefore result in rapid fluorescence recovery (39).However, treatment of TrHBMECs with antibody LM609, which perturbs "v!3 integrin bindingto ligand, has a minor inhibitory impact on integrin dynamics. In addition, there is no decrease inthe rate of FRAP of the bleached stripe zone of !%labeled focal contacts in TrHBMECs treatedwith colcemid, which not only perturbs the microtubule network but also induces collapse of thevimentin cytoskeleton (40). In contrast, the mobile fraction of labeled integrin in focal contacts isreduced by 60% in cells treated with microfilament-perturbing drugs. This implies that themicrofilament system does not merely position integrins but plays an active role as a positiveregulator of mobility of !%integrin within a focal contact. Because FRAP is slowed in energy-depleted cells and is microfilament-dependent, this raises the possibility that an actin-associatedmolecular motor is involved in the dynamic aspects of !3 integrin-containing heterodimers thatwe have detailed. Our data concerning the localization of myosin II to focal contacts and theability of the myosin light chain kinase inhibitor ML-7 to inhibit fluorescence recovery suggeststhat myosin is a good candidate for such a molecular motor. However, because ML-7 perturbsthe 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, aremobile within the confines of a focal contact. We hypothesize that such movement is crucial tofocal contact function. Indeed, mobility of integrins in focal contacts would allow a cycling inreceptor/ligand interactions, as focal contacts move along the substratum attached surface ofcells even in stationary cells (36, 41). We envision that precise regulation in integrin/ligandbinding and modulation in integrin mobility by the cytoskeleton within a focal contact isnecessary 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 StatesArmy Medical Research and Materiel Command grant # DAMD17-00-1-0386. We thankWeiming Yu, Yoshio Fukui, Shinichiro Kojima, and Miri Yoon for helping to perform the FRAPstudies and analyzing data. We are very grateful for the gift of cells and antibodies from BabetteWeksler, Denise Paulin, and Yoshio Fukui and for the technical assistance of Kristin deHahn.

REFERENCES

1. Clark, E. A., and Brugge, J. S. (1995) Integrins and signal transduction: the road taken.Science268, 233-239

2. Gumbiner, B. M. (1996) Cell adhesion: the molecular basis of tissue architecture andmorphogenesis. Cell84, 345-357

3. Howe, A., Aplin, A. E., Alahari, S. K., and Juliano, R. L. (1998) Integrin signaling andcell growth. Curr. Opin. Cell Biol.10, 220-231

4. Schoenwaelder, S. M., and Burridge, K. (1999) Bidirectional signaling between thecytoskeleton and integrins. Curr. Opin. Cell Biol.11, 274-286


10/22

5. Dogic, D., Eckes, B., and Aumailley, M. (1999) Extracellular matrix, integrins and focaladhesions. Curr. Top. Path. 93, 7585

6. Simon, K. O., and Burridge, K. 1994. Interactions between integrins and thecytoskeleton: structure and regulation, p. 49-78. In D. A. Cheresh, and R. P. Mecham(ed.), Integrins: Molecular and Biological Responses to the Extracellular Matrix.Academic Press, San Diego, CA.

7. Zamir, E., Katz, B.-Z., Aota, S., Yamada, K. M., Geiger, B., and Kam, Z. (1999)Molecular diversity of cell-matrix adhesions.J. Cell Sci.112, 1655-1669

8. Giancotti, F. G., and Ruoslahti, E. (1999) Integrin signaling. Science285, 1028-1032

9. Hynes, R. O. (1992) Integrins: Versatility, modulation, and signaling in cell adhesion.Cell69, 11-25

10. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Integrins: emergingparadigms of signal transduction.Ann. Rev. Cell Dev. Biol.11, 549-599

11. Duband, J. L., Nuckolls, G. H., Ishihara, A., Hasegawa, T., Yamada, K. M., Thiery, J. P.,and Jacobson, K. (1988) Fibronectin receptor exhibits high lateral mobility in embryoniclocomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells.J. Cell Biol.107, 1385-1396

12. Geiger, B., Avnur, Z., and Schlessinger, J. (1982) Restricted mobility of membraneconstituents in cell-substrate focal contacts of chicken fibroblasts. J. Cell Biol.93, 495-500

13. Kreis, T. E., Geiger, B., and J., S. (1982) Mobility of microinjected rhodamine actinwithin living chicken gizzard cells determined by fluorescence photobleaching recovery.Cell29, 835-845

14. Schlessinger, J., and Geiger, B. (1983) The dynamic interrelationships of actin andvinculin in cultured cells. Cell Motil.3, 399-403

15. Smilenov, L. B., Mikhailov, A., Pelham, R. J., Marcantonio, E. E., and Gunderson, G. G.(1999) Focal adhesion motility revealed in stationary fibroblasts. Science286, 1172-1174

16. Stickel, S. K., and Wang, Y.-L. (1987) Alpha-actinin-containing aggregates in

transformed cells are highly dynamic structures.J. Cell Biol.104, 1521-1526

17. Zamir, E., Katz, M., Posen, Y., Erez, N., Yamada, K. M., Batz, B.-Z., Lin, S., Lin, D. C.,Bershadsky, A., Kam, Z., and Geiger, B. (2000) Dynamics and segregation of cell-matrixadhesions in cultured fibroblasts.Nature Cell Biol.2, 191-197


11/22

18. Brooks, P. C., Montgomery, A. M. P., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G.,

and Cheresh, D. A. (1994) Integrin "v!3 antagonists promote tumor regression byinducing apoptosis of angiogenic blood vessels. Cell79, 1157-1164

19. Ruoslahti, E., and Engvall, E. (1997) Integrins and vascular extracellular matrixassembly.J. Clin. Invest.99, 1149-1152

20. Brooks, P. C., Clark, R. A. F., and Cheresh, D. A. (1994) Requirement of vascularintegrin "v!3 for angiogenesis. Science264, 569-571

21. Brooks, P. C., Strmblad, S., Klemke, R., Visscher, D., Sarkar, F. H., and Cheresh, D. A.(1995) Antiintegrin "v!3 blocks human breast cancer growth and angiogenesis in humanskin.J. Clin. Invest.96, 1815-1822

22. Gonzales, M., Weksler, B., Tsuruta, D., Goldman, R. D., Yoon, K. J., Hopkinson, S. B.,Flitney, F. W., and Jones, J. C. R. (2001) Structure and function of a vimentin-associated

matrix adhesion in endothelial cells.Mol. Biol. Cell12, 85-100

23. Schweitzer, K. M., Vicart, P., Delouis, C., Paulin, D., Drger, A. M., Langenhuijsen, M.M. A. C., and Weksler, B. B. (1997) Characterization of a newly established human bonemarrow endothelial cell line: Distinct adhesive properties for hematopoietic progenitorscompared with human umbilical vein endothelial cells.Lab. Invest.76, 25-36

24. Yoon, M., Moir, R. D., Prahlad, V., and Goldman, R. D. (1998) Motile properties ofvimentin intermediate filament networks in living cells.J. Cell. Biol.143, 147-157

25. Cooper, J. A. (1987) Effects of cytochalasin and phalloidin on actin. J. Cell Biol.105,1473-1478

26. Spector, I., Shochet, N. R., Blasberger, D., and Kashman, Y. (1989) Latrunculins - novelmarine macrolides that disrupt microfilament organization and affect cell growth: I.Comparison with cytochalasin D. Cell Motil. Cytoskel.13, 127-144

27. Edlund, M., Lotano, M. A., and Otey, C. A. (2001) Dynamics of "-actinin in focaladhesions and stress fibers visualized with "*actinin green fluorescent protein. CellMotil. Cytoskel.48, 190-200

28. Ren, X.-D., Kiosses, W. B., Sieg, D. J., Otey, C. A., Schlaepfer, D. D., and Schwartz, M.A. (2000) Focal adhesion kinase suppresses Rho activity to promote focal adhesionturnover.J. Cell Sci.113, 3673-3678

29. Harlow, E., and Lane, D. 1988. Antibodies: A Laboratory Manual, p. 92-121. ColdSpring Harbor Laboratory, Cold Spring Harbor, New York.

30. Klatte, D. H., Kurpakus, M. A., Grelling, K. A., and Jones, J.C.R. (1989)Immunochemical characterization of three components of the hemidesmosome and theirexpression in cultured epithelial cells.J. Cell Biol.109, 3377-3390


12/22

31. Schootemeijer, A., van Willigen, G., van der Vuurst, H., Tertoolen, L.G.J., De Laat, S.

W., and Akkerman, J.-W.N. (1997) Lateral mobility of integrin "++b!% (glycoproteinIIb/IIIa) in the plasma membrane of a human megakaryocyte. Thrmob. Haem.77, 143-149

32. Vikstrom, K. L., Lim, S. S., Goldman, R. D., and Borisy, G. G. (1992) Steady statedynamics of intermediate filament networks.J. Cell Biol.118, 121-129

33. Tzima, E., del Pozo, M. A., Shattil, S. J., Chien, S., and Schwartz, M. A. (2001)Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependentcytoskeletal alignment.EMBO J.20, 4639-4647

34. Rottner, K., Hall, A., and Small, J.V. (1999) Interplay between Rac and Rho in thecontrol of substrate contact dynamics. Curr. Biol.17, 640-648

35. Fujiwara, K., and Pollard, T. D. (1976) Fluorescent antibody localization of myosin in the

cytoplasm, cleavage furrow, and mitotic spindle of human cells.J. Cell Biol.71, 848-875

36. Sheetz, M. P., Felsenfeld, D. P., and Galbraith, C. G. (1998) Cell migration: regulation offorce on extracellular matrix-integrin complexes. Trends Cell Biol.8, 51-54

37. Plancon, S., Morel-Kopp, M. C., Schaffner-Reckinger, E., Chen, P., and Kieffer, N.(2001) Green fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb orbeta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect ofbeta3GFPon alphaIIb(beta3) ligand binding.Biochem. J.15, 529-536

38. Geiger, B., Avnur, Z., Rinnerthaler, G., Hinssen, H., and Small, V. J. (1984)Microfilament-organizing centers in areas of cell contact: cytoskeletal interactions during

cell attachment and locomotion.J. Cell Biol.99, 83s-91s

39. Broday, D. M. (2000) Diffusion of clusters of transmembrane proteins as a model offocal adhesion remodeling.Bull. Math. Biol.62, 891-924

40. Goldman, R. D., and Knipe, D. M. (1973) Functions of cytoplasmic fibers in non-musclecell motility. Cold Spring Harbor Symp. Quant. Biol.37, 523-534

41. Felsenfeld, D. P., Choquet, D., and Sheetz, M. P. (1996) Ligand binding regulates thedirected movement of !,integrins on fibroblasts.Nature383, 438-440

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


13/22

Table 1

FRAP Analysis.

FRAP Analysis Drug Treatment n t1/2

(value SE)

p Mobi

(va

GFP-3

Full focal contact bleach None 5 22.00 2.79 < 0.01 32 2

Bleached stripe None 11 5.22 0.35 - 72 3

2-deoxy-D-glucose/azide (energy depletion) 16 4.54 0.33 ns 42 4

Anti-v3 antibody

Colcemid

(microtubule)

Cytochalasin D

(microfilament)

Cytochalasin D+recovery*

Latrunculin B

(microfilament)

ML-7

(myosin light chain kinase)

13

19

12

10

11

14

6.89 0.47

6.20 0.46

5.05 0.77

6.37 0.84

5.85 0.67

4.18 0.40

< 0.01

ns

ns

ns

ns

ns

68 5

71 5

42 4

73 4

50 6

52 4

GFP-actinin-1

Full focal contact bleach None 14 0.88 0.07 - 63 4

2-deoxy-D-glucose/azide (energy depletion) 10 1.08 0.06 ns 43 5

Bleached stripe None 8 0.57 0.05 < 0.01 71 6

Cells were treated with cytochalasin D. After a 30 min treatment, media were replaced and the cells were allowed 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 contarecover to one-half the final intensity value. The target of each drug is listed in parenthesis under the drug name. Statistica

Comparisons were made between focal contacts in control cells and drug-treated cells.


14/22

Fig. 1

Figure 1.Characterization of GFP-actinin-1 and GFP-3 integrin in TrHBMECs. A)Extracts of nontransfectedTrHBMECs (lane 1) or populations of TrHBMEC transfected either with a construct encoding GFP-3 integrin (lane 2) orGFP-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 eachlane 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 seenin lane 2 only. The same extracts were subjected to immunoprecipitation (IP) by using either antibody LM609 against the

v3integrin complex (lanes 3 and 4) and P1F6 against the v5integrin complex (lanes 5 and 6). Approximately 15 gof 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 v3integrin antibody

in lane 4 (upper panel), but not by antibodies against v5integrin (lane 6; upper panel). Molecular weights are indicatedto 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.


15/22

Fig. 2

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


16/22

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.


17/22

Fig. 4

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


18/22

Fig. 5

Figure 5.Photobleaching does not affect localization ofv3 integrin heterodimers in focal contacts. Several focalcontacts 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 antibodyLM609. 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 Cand D). In (D)these bleached focal contacts

appear red; their unbleached counterparts are yellow. Bar, 10 m.


19/22

Fig. 6

Figure 6. FRAP of partially bleached focal contacts in TrHBMECs. Images of cells expressing either GFP-3integrin (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-D-

glucose, whereas the cell in (GI)was incubated in medium supplemented with 0.1 M cytochalasin D. Recovery ofsignal 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.


20/22

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 timesfollowing 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.


21/22

Fig. 8

Figure 8. GFP-actinin-1 does not localize to focal contacts in TrHBMECs treated with microfilament destabilizingdrugs. 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 anti-

v3 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.


22/22

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 v3integrin heterodimer (red). Overlaid images of thestains 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 v3integrin 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 cellspossess microfilament bundles and focal contacts stained by LM609 antibodies despite changes in their overall

morphology. Bar, 10 m.

microfilament dependet movement of the b3 integrin subunit within focal contacts of endothelial...

Documents