IntroductionFocal adhesions (FAs) are elongated adhesion structures at theventral plasma membrane that enable cells to adhere to theextracellular matrix (Burridge et al., 1988). FAs are linked tothe termini of contractile actomyosin filaments termed stressfibers and the tension and traction generated through stressfiber contraction allows cells to polarize and to migrate.Furthermore, focal adhesions form important signaling centerswhere integrins transduce signals from the cellularenvironment to regulate different cellular functions, such ascell growth, survival and gene expression (Adams, 2002;Cukierman et al., 2002).

Within FAs integrin �� heterodimers are the main mediatorsof adhesion to the surrounding substratum. Integrins use theirextracellular domain to interact with ECM components,whereas their cytoplasmic tail associates either directly orindirectly with a number of structural, cytoskeletal proteins,such as talin (Burridge and Connell, 1983), paxillin (Turner etal., 1990), vinculin (Geiger, 1979) and �-actinin (Lazaridesand Burridge, 1975). These cytoskeletal proteins in turnconnect integrins to actin-containing microfilaments, therebyestablishing a link between the ECM and the actincytoskeleton.

FAs have been studied intensively usingimmunofluorescence and electron-labeling techniques, whichshowed the localization of more than 50 different proteins tothese structures (Zamir and Geiger, 2001). Fusing FA proteins

with fluorescent proteins has made it possible to study thedynamics of FA assembly and disassembly (Wehrle-Haller andImhof, 2002). In addition, biochemical studies have elucidateda large number of protein-protein interactions important for FAfunction (Zamir and Geiger, 2001). In contrast to these detailedcell biological and biochemical findings, less is known aboutthe FA ultrastructure.

Early studies using interference reflexion microscopy (IRM)and transmission electron microscopy established that FAsoccur at the points of closest cell-to-substrate contact wherethey are associated with the distal ends of stress fibers(Abercrombie et al., 1971; Curtis, 1964; Heath and Dunn,1978; Izzard and Lochner, 1976). At their proximal end, themicrofilaments in FAs appear to be continuous with the lineararrays of microfilaments in stress fibers (Izzard and Lochner,1976), whereas the distal ends of the microfilament bundlesoften spread out (Heath and Dunn, 1978). Investigations ofplatinum replicas of detergent-extracted fibroblasts confirmedthat at the distal ends of stress fibers, microfilaments aretransformed into flattened plaques closely associated with thesubstrate (Svitkina et al., 1984). In addition, thin transversefilaments bridging the gap between microfilaments weredescribed and later identified as plectin sidearms connected tovimentin cores (Svitkina et al., 1998).

Immunological electron microscopic (immuno-EM) studieshave provided further insight into the vertical and laterallocalization of several FA components within the adhesion

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Atomic force microscopy (AFM) can produce high-resolution topographic images of biological samples inphysiologically relevant environments and is therefore wellsuited for the imaging of cellular surfaces. In this work wehave investigated focal adhesion complexes by combinedfluorescence microscopy and AFM. To generate high-resolution AFM topographs of focal adhesions, REF52 (ratembryo fibroblast) cells expressing YFP-paxillin as amarker for focal adhesions were de-roofed and paxillin-positive focal adhesions subsequently imaged by AFM. Theimproved resolution of the AFM topographs complementedthe optical images and offered ultrastructural insight intothe architecture of focal adhesions. Focal adhesions had acorrugated dorsal surface formed by microfilamentbundles spaced 127±50 nm (mean±s.d.) apart andprotruding 118±26 nm over the substratum. Within focal

adhesions microfilaments were sometimes branched andarranged in horizontal layers separated by 10 to 20 nm.From the AFM topographs focal adhesion volumes couldbe estimated and were found to range from 0.05 to0.50 ��m3. Furthermore, the AFM topographs show thatfocal adhesion height increases towards the stress-fiber-associated end at an angle of about 3°. Finally, bycorrelating AFM height information with fluorescenceintensities of YFP-paxillin and F-actin staining, we showthat the localization of paxillin is restricted to the ventralhalf of focal adhesions, whereas F-actin-containingmicrofilaments reside predominantly in the membrane-distal half.

Key words: AFM, Focal adhesions, Paxillin

Summary

Analyzing focal adhesion structure by atomic forcemicroscopyClemens M. Franz and Daniel J. Müller*Center of Biotechnology, University of Technology Dresden, Tatzberg 49, 01307 Dresden, Germany*Author for correspondence (e-mail: mueller@biotec.tu-dresden.de)

Accepted 18 August 2005Journal of Cell Science 118, 5315-5323 Published by The Company of Biologists 2005doi:10.1242/jcs.02653

Research Article

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plaque. Double immunogold labeling performed on ultrathinfrozen sections from chick heart fibroblasts showed thatvinculin is situated closer to the membrane than �-actinin inFAs (Chen and Singer, 1982) whereas in wet-cleaved chickenembryo fibroblasts, both talin and vinculin are present at highconcentrations in a dense network close to the plasmamembrane (Feltkamp et al., 1991). Rotary replication incombination with immunogold labeling of sheared Xenopusfibroblasts demonstrated that FA microfilaments are highlybundled and these bundles appear to be linked to the plasmamembrane by laterally attached discrete protein aggregatescontaining vinculin, talin and �1-integrin (Samuelsson et al.,1993). In agreement, �1-integrin was found to localize inlinearly arranged discrete clusters by immuno-EM experimentsperformed on wet-cleaved fibroblasts (Meijne et al., 1994).However, these techniques have so far either only allowed theanalysis of a subsection of the entire FA structure or failed toprovide a quantitative three-dimensional (3D) model of FAarchitecture. In addition, the EM techniques require samplepreparation protocols that may distort the native array of actinfilaments in FAs, such as detergent extraction, sample dryingand coating, staining and cutting (Heuser and Kirschner, 1980;Small et al., 1999; Svitkina et al., 1995).

Atomic force microscopy (AFM) produces sampletopographies by scanning the surface with a sharp, nanometerscale probe (tip) attached to a flexible cantilever (Binnig et al.,1986). AFM permits imaging in a fluid environment, thereforeavoiding artifacts caused by drying and/or coating of samples,and has the advantage of maintaining biological systems andtheir functionality in physiological conditions (Horber andMiles, 2003; Lesniewska et al., 1998; Muller et al., 2002).Furthermore, cells and biological materials can be imageddirectly by AFM with very little sample preparation and aresolution of less than 1 nm. The combination of AFM imagingwith fluorescence microscopy is becoming a valuable tool inthe investigation of complex cellular structures, in whichfluorescence labeling serves to identify protein complexes ofinterest which can then be imaged at superior resolution byAFM (Kassies et al., 2005; Sharma et al., 2005). We used acombination of fluorescence microscopy and AFM to imageFAs in de-roofed cells under physiologically relevantconditions. The resolution of the AFM topographs surpassesthat of the light microscope images and provides structuralinformation about the 3D organization of microfilaments inFAs. Furthermore, we show that by correlating the heightinformation of AFM topographs with the fluorescence stainingintensities of FA components, differences in their verticallocalization can be detected.

Materials and MethodsREF52 cells (kindly provided by Alexander Bershadsky, WeizmannInstitute of Science, Rehovot, Israel) were maintained in DMEMcontaining 10% FCS, 100 IU/ml penicillin and 100 �g/mlstreptomycin. For de-roofing, 5�104 cells were seeded on ethanol-cleaned glass coverslips (22 mm diameter) inserted into 35 mm tissueculture dishes and grown for 72 hours. Cells were washed three timeswith warm PBS, followed by incubation for 15 seconds with lowmolecular weight (15,000-30,000) poly-L-lysine. Subsequently, cellswere washed three times for 30 seconds with 1/3 strength intracellularbuffer (ICB; 20 mM HEPES pH 7.6, 1.5 mM MgCl2, 5 mM EGTA,5 mM NaCl, 2 mM CaCl2, 140 mM potassium glutamate) and then

transferred to a 92 mm tissue culture dish containing 10 ml ICB. Usinga Hielscher microsonicator, cells were de-roofed with several short(<1 second) ultrasonic bursts at minimum amplitude. After threewashes with ICB buffer, cells were fixed for 45 seconds in 2%glutaraldehyde/ICB, followed by 10 minutes in 4%paraformaldehyde/ICB. Using a custom-made metal holder,coverslips were mounted on an inverted Zeiss Axiovert 200microscope (Carl Zeiss, Göttingen, Germany), on which an AFM(NanowizardTM AFM, JPK Instruments, Berlin, Germany) wasmounted. Coverslips were overlaid with PBS containing 9 mM propylgallate to minimize photo bleaching and fluorescence images werecollected using a Carl Zeiss 63� oil-immersion objective. Using afluid cell, AFM contact mode images were recorded in liquid using200 �m long V-shaped cantilevers, with nominal spring constants of0.01 N/m (MSCT-AUHW, Veeco Instruments, Santa Barbara, CA).The force applied to the cantilever was adjusted manually to ~50 pN(Muller et al., 1999) and the feedback gains were manually adjustedto obtain the best resolution both on height and deflection channels.Images were collected at a line-scan rate ranging from 0.3 to 0.6 Hz.Light microscopy images were processed using MetaMorph software(Universal Imaging Corporation, Downingtown, PA). AFM imageswere analyzed using the JPK Image Processing software and FAvolumes were estimated using the SPIP software (Image Metrology,Lyngby, Denmark).

ResultsPreparing FAs for AFM imagingREF52 cells develop an extensive contractile apparatus with amultitude of stress fibers terminating in large FAs when grownon glass surfaces (Turner et al., 1989). REF52 cells stablyexpressing YFP-paxillin as a marker for focal adhesions weregrown on glass coverslips for 3 days to enable them to developmature adhesive contacts (Fig. 1A-C). To make FAs accessiblefor AFM imaging, it was necessary to remove the apical plasmamembrane, the nucleus and the cytoplasm of these cells. Cellde-roofing was achieved by using short ultrasonic bursts basedon a method developed by Heuser (Heuser, 2000). Thesonication step was performed in a buffer establishingintracellular ionic conditions to preserve the integrity ofintracellular structures once cells were disrupted. Cells wereusually de-roofed, rinsed and fixed within 1 minute. Sonicationcaused varying degrees of de-roofing, with some cellsremaining intact, some showing partial lysis and some cellsbeing completely de-roofed as judged by phase-contrastmicroscopy. Alternative de-roofing methods, such as wetcleaving (Brands and Feltkamp, 1988) or low-ionicstrength/detergent extraction (Katoh et al., 1998) producedsimilar results to the sonication protocol but frequently led toless complete removal of cytoplasmic organelles. In thesesamples subsequent AFM scanning was difficult because theloosely bound organelles frequently contaminated the AFMtip.

In de-roofed REF52 cells, paxillin-YFP continued tolocalize to oblong structures with dimensions typical for FAsin these cells (usually 0.5-2 �m by 3-8 �m), indicating that FAintegrity was preserved during the de-roofing step (Fig. 1B).Paxillin-containing complexes also stained positive forvinculin (data not shown) and F-actin (Fig. 1D), furthersuggesting that FA structure was resistant to the de-roofingprocedure. Stress fibers were rarely retained during the cell de-roofing and usually sheared off precisely at the interface withthe FA (Fig. 1D). Addition of protease inhibitors or F-actin

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stabilizing agents, such as phalloidin orjasplakinolide to the sonication buffer had nodetectable influence on the FA structure (datanot shown).

Overview AFM images of completely de-roofed cells (Fig. 2A,B) showed retention ofdifferent cellular structures verticallyprotruding between 20 and 500 nm from thesubstratum. A brief incubation of the cells with poly-L-lysine prior to sonication caused partial preservationof the basal membrane around the cell perimeter (Fig.2B), whereas the basal membrane was usuallycompletely removed when this step was omitted. Inorder to identify FAs among the preserved basalprotein complexes, the localization of YFP-paxillinwas determined by fluorescence microscopy (Fig. 2C).A cluster of eight structures identified as FAs byoverlaying the AFM topograph with the paxillinfluorescence image (Fig. 2D) were subsequentlyimaged at increased resolution (Fig. 2E,F).Filamentous structures were observed that extendedthroughout the entire lengths of some FAs. Filamentswere not always oriented parallel to each other butfanned out towards the cell perimeter at angles rangingfrom 5 to 8 degrees. Actin-containing microfilamentsconstitute the predominant filamentous structures inFA and are typically arranged in a fan-shaped pattern(Heath and Dunn, 1978; Svitkina et al., 1984). Weconsequently assumed that the filamentous structuresobserved in the AFM topographs were composed ofactin-containing microfilaments.

Revealing microfilament organization in FAsTo investigate the organization of microfilaments inFAs in more detail, FAs in de-roofed cells wereidentified as before by paxillin fluorescence (Fig. 3A)and staining for F-actin using TRITC-phalloidin (Fig.3B) and then imaged by AFM (Fig. 3D,E). An overlayof the merged fluorescence image (Fig. 3C) with theAFM deflection image (Fig. 3F) demonstrates thegood correlation between the two image-generatingtechniques. A central region within an FA wassubsequently imaged with increasing resolution (Fig.3G,H). Scanning at the highest magnification revealedthe predominantly parallel array of filaments in thecentral part of the FA (Fig. 3H). Occasionally filamentsappeared to branch and individual filaments crossed the

parallel arrays at different angles (Fig. 3G,H). These crossingfilaments could be either actin-containing microfilaments or

Fig. 1. Near-confluent cells were de-roofed by asonication procedure (D-F) or left untreated (A-C),fixed and stained for F-actin using TRITC-phalloidin (A,D). Untreated cells showed anabundance of stress fibers (A) terminating inpaxillin-containing plaques (B). The size and shapeof paxillin-containing patches was unchanged afterthe de-roofing step (E), indicating the preservationof FA complexes. FAs also stained positive for F-actin although the link between FAs and stressfibers was generally broken during de-roofing (D).Bar, 10 �m.

Fig. 2. Cells were de-roofed and an AFM image recorded (A, deflection andB, height). Parts of the ventral plasma membrane were preserved during thede-roofing procedure (B, arrows). (C) The localization of paxillin-YFP withinan area corresponding to the dashed box in (B) was determined byfluorescence microscopy. (D) Overlaying the AFM height image with thepaxillin fluorescence image highlights paxillin-containing FAs. (E) Severalpaxillin-positive protein complexes (arrows) were subsequently imaged athigher resolution by AFM (E, height and F, deflection signal; scan areacorresponding to the dashed box in D). Filamentous structures arranged atslight angles to each other can be distinguished in some of the FAs. The fullrange of the height scale corresponds to heights of 600 nm (B,D) and 350 nm(E). Bar, 10 �m (A-D) and 1 �m (E,F).

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thinner intermediate filaments (Svitkina et al., 1984; Svitkinaet al., 1998). The apparent diameter of the parallel-arrangedfilaments varied from 90 nm down to 20 nm, which was thelimit of the lateral resolution achieved. The variation of theapparent filament width suggests that the observed filamentsconsist of microfilament bundles in which individualmicrofilaments could not be resolved because of the limitedresolution achieved (see Discussion). However, occasionallyseveral adjacent filaments displaying the minimal apparentwidth of 20 nm could be resolved (Fig. 3J,K), probably becauseof slightly looser microfilament bundling in these areas. Thesesmallest filaments are likely to represent single microfilamentswhose apparent width in the AFM scans is increased as a resultof non-linear tip convolution effects (Schwarz et al., 1994).

Cross sections taken perpendicularly to the microfilamentarray showed height variations ranging from 10 to 20 nm betweenadjacent, parallel microfilaments (Fig. 3J) or microfilamentscrossing each other (Fig. 3K), suggesting that microfilaments areorganized in layers in the FA plaque. The minimal heightdifference measured between crossing microfilaments of 10 nmis close to the microfilament diameter of 7 nm (Moore et al.,

1970), supporting the notion that individual microfilaments couldbe resolved by our AFM approach.

Along the microfilaments laterally attached globularstructures with diameters of around 50 nm could be detected(Fig. 3H), which could represent integrin-containingaggregates observed by immuno-EM (Samuelsson, 1993),although no regularity in the spacing of these globularstructures was found. The abundance of these globularstructures varied (compare Fig. 3J,K) but was usually highestin the central part of FAs.

The de-roofing method we used maintained the FAstructures in physiological conditions during the entirepreparation protocol but included an ultimate glutaraldehydefixation step. Fixation was necessary because higher structureswere frequently too pliable for reproducible AFM scanning inunfixed FAs. In order to minimize potential structure artifactsdue to glutaraldehyde fixation, such as microfilament fusion(Svitkina et al., 1995), we used a short glutaraldehyde fixationtime, followed by longer paraformaldehyde fixation.Nevertheless, at higher magnification, the AFM image qualitysometimes became compromised even after fixation because of

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Fig. 3. Fluorescence images of YFP-paxillin (A) and actin filaments (B) inde-roofed cells. AFM topographs (D)and deflection image (E) of the samearea. (F) Overlay of the mergedfluorescence images (C) with theAFM deflection image. (G) Theboxed area in D scanned at higherresolution. Arrows indicate filamentsthat cross parallel arrays of actinfilaments at different angles.(H,J) Boxed area in G scanned atfurther increased resolution.Microfilaments occasionally branch atangles between 30° and 45°(H, branching points indicated byasterisks). The trace and retracetopographies show predominantlyparallel filaments exhibiting apparentdiameters ranging from 20 to 90 nm.A merge of the trace (green) andretrace topographs (red) demonstratesgood correlation between bothscanning directions. (J,K) Filamentsare decorated by globular structureswith apparent diameters between 50and 80 nm. A cross section (solidblack line) through a group ofadjacent ~20-nm-wide filaments(arrows) and the corresponding heightprofile indicates a height difference of12 nm between two neighboringfilaments. (K) Height profiles (blacksolid lines) taken perpendicular to twofilaments (black and white dashedlines) before and after their crossing point demonstrate a height difference of between 10 and 16 nm. The full range of the height scale correspondsto heights of 450 nm (D), 160 nm (G), 60 nm (H) and 50 nm (J,K). Bar, 3 �m (A-F); 500 nm (G); 50 nm (H); 100 nm (J,K).

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continued sample pliability and adhesion to the AFM tip. Thiseffect was strongest at the distal end of FAs wheremicrofilaments may not be anchored to the substrate as firmlyas in the central part because of active FA assembly ordisassembly processes. To reduce the lateral movement offilamentous and globular structures through tip movement, thescan speed had to be kept low (0.3 Hz) and the scanning forcehad to be continuously minimized. However, comparison ofheight trace and retrace topographs (Fig. 3H) generated fromthe central region of an FA demonstrates that optimizing thescan parameters enabled us to image the filament array withoutany significant structural distortions. Varying the scan angle atsuch optimized imaging conditions generally had no noticeableeffect on the generated topographies.

Analyzing overall FA architectureTo illustrate overall FA structure, the AFM topography of arepresentative FA was presented in a relief (Fig. 4B). The

finger-like branching of microfilaments at the end of the FAopposite to the stress fibers could be resolved clearly. A sideview of a 3D reconstruction with true (1:1) aspect ratiobetween the Y and Z axis shows a gradual, constant increase inheight from about 50 nm at the front end to about 180 nm atthe stress-fiber-associated end of the FA, resulting in anincrease of approximately 3°. At the very end of the stress-fiber-associated side, FAs regularly showed a steep increase inheight (Fig. 4C). This may not be an intrinsic structural FAfeature but rather represent structural damage inflicted on theFA structure as a result of the forceful removal of the stressfiber interface during the preparation protocol. An overlay offive cross sections taken across the FA at intervals of 500 nmshows that FAs are low and wide at the front, but high andnarrow at the stress-fiber-associated end (Fig. 4D).

Moving from the distal to the proximal end, FA heightincreased 2.2-fold, whereas FA width decreased by about thesame factor (Fig. 4F). In contrast, the cross section areas variedonly by a factor of 1.4 over the length of the FA, reaching a

Fig. 4. FA topographs (A) and 3D-reconstruction (B) showing the wedge-shaped morphology and the finger-like array of microfilaments at thedistal end of the SF. (C) Side view of the same FA with Y and Z dimensions at the same scale. The height of the FA increases towards the SF-associated end at an angle of approximately 3°. From the AFM topograph the area (1.9�106 nm2) and the volume (1.2�108 nm3) of the FA wasdetermined. (D) Location of X-Z cross sections taken through the FA topograph every 500 nm in the Y direction. (E) Overlay of the crosssection demonstrates that the FA structure is low and wide at the front and high and narrow at the SF-associated end. (F) Graphs showing therelative changes in FA height, width and cross-section area moving from the distal to the proximal end of the FA. (G) Schematic representationof the 3D-array of microfilaments in FAs. Height scale in A, 200 nm.

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maximum in the central part. The greater constancy of thecross-section area compared to cross-section height and widthsuggests that the change in FA shape may be the result of agradual reorganization of structural elements extendingthroughout the entire FA structure, i.e. microfilaments, ratherthan being caused by the presence of different structuralelements in different parts of the FA. Fig. 4G shows a modelof the microfilament organization within FAs. Microfilamentsare bundled into a circular array at the stress-fiber-associatedend and then gradually flatten out into a planar array towardsthe opposite end. From the AFM topography, the area of theFA in contact with the underlying substratum (1.9�106 nm2)and the FA volume (1.2�108 nm3) could be estimated. Overall,the FA volumes analyzed from 15 FAs ranged from 5�107 to5�108 nm3.

Determining microfilament height and spacing in FAsIn order to quantify the height and spacing of microfilamentbundles at the dorsal FA surface, bisecting cross sectionsperpendicular to the FA long axis were taken as exemplifiedfor a cluster of FAs in Fig. 5A. The corresponding heightprofiles are shown in Fig. 5B. Because of the high vertical

resolution of the AFM, the average height of microfilamentsover the surrounding substrate (corresponding to the averagevalue of the local maxima in the cross-section graphs) couldbe precisely determined. Their average peak height was118±26 nm (mean±s.d.; 30 FAs analyzed), whereas the averageoverall height of the cross section was 84±16 nm (mean±s.d.).However, because of spatial constrictions, the AFM tipprobably could not fully penetrate between narrowly spacedmicrofilaments or microfilament bundles, leading to a slightoverestimation of the sample height in the interjacent areas.The overall average height is therefore expected to be lowerthan measured. The distance between neighboringmicrofilament bundles was determined by measuring thedistance between neighboring local maxima in the cross-section graphs and varied from 40 to 240 nm, with an averageof 127±50 nm (Fig. 5C).

Determining differential vertical localization of paxillinand F-actin within the FA by correlating fluorescenceintensities with the AFM height informationOccasionally FAs in de-roofed cells contained areas in whichthe F-actin staining intensity was markedly decreased.Generally, such areas with lower F-actin staining correspondedto regions exhibiting decreased heights in the AFM topographs.These circular indentations could be the result of mechanicaldisruption of FA structure during de-roofing. An example of anFA complex displaying a local reduction in F-actin staining andheight is given in Fig. 6A,D. In contrast to the F-actin stainingintensity, the paxillin signal in this area remained unchanged(Fig. 6B). A line scan through the AFM topograph and thecorresponding fluorescence images (Fig. 6E) demonstrates apositive correlation between the height and F-actin signal butnot the paxillin signal (Fig. 6F). As the removal of the upper

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Fig. 5. (A) Topograph and3D-reconstruction of a clusterof four FAs. Cross sectionsalong the indicated lines(bisecting FAsperpendicularly) were taken.(B) Height profilescorresponding to the crosssections taken in (A). Theaverage height of bisectingcross sections corresponds to84±16 nm (average height),whereas the filamentousstructures extended over thesubstratum by 118±27 nm.The dashed lines indicate theaverage values generated fromanalysis of more than 30 FAs.(C) Histogram showing thedistribution of distancesbetween neighboringfilament/filament bundles(>30 FAs analyzed). Themean spacing betweenfilaments/filament bundles atthe FA half-length was127±50 nm.

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(dorsal) part of the FA architecture (~50 nm) does not decreasethe paxillin signal, paxillin localization must be restricted tothe lower, membrane-proximal half of the FA. In the oppositesituation, because F-actin staining intensity was almostreduced to background levels when the upper FA structure wasremoved, F-actin appears to be mainly localized in themembrane-distal half of the FA.

DiscussionWe have combined fluorescence microscopy and AFM togenerate high-resolution images of FA complexes in de-roofedfibroblasts. Fluorescence microscopy was first used to identifypaxillin-containing FAs, which were subsequently imaged byAFM at a resolution surpassing the limits of conventional lightmicroscopy. The AFM topographs yielded structural detail thatwas not obtainable from light microscopic images andprovided insight into the 3D organization of microfilamentswithin FAs.

AFM topographs of FAs showed the presence of a multitudeof filaments spanning the entire length of the FA. Thesefilaments were arranged predominantly in parallel within thecentral part of the FA but frequently fanned out towards thedistal end of the FA, an arrangement consistent with theorganization of actin microfilaments in FAs as described in EMstudies (Heath and Dunn, 1978; Svitkina et al., 1984).However, whereas transmission EM images of FAs show atight lateral packing of microfilaments throughout the lengthof the FA (Heath and Dunn, 1978; Singer, 1979), our AFMtopographs suggest that the dorsal FA surface at least, is notformed by a homogeneously dense microfilament layer.Instead, FAs had a corrugated dorsal surface formed byfilamentous structures spaced by an average of 127 nm and

protruding by 10 to 40 nm over the interjacent areas. Theapparently dense packing of microfilaments observed intransmission-EM images may be the result of the projection ofthe 3D microfilament lattice onto a plane. Such a projectioneliminates the information about the vertical separationbetween microfilaments and consequently reduces the apparentdistances between microfilaments. In addition, thesuperimposition of different horizontal layers ofmicrofilaments may further reduce the apparent spacingbetween microfilaments in transmission EM images.

The apparent width of filaments observed by AFM variedfrom 20 to 90 nm. It is well known that the finite size of theAFM tip can cause a non-linear convolution effect in whichhigh, steeply increasing structures appear severely broadened(Schwarz et al., 1994). Considering such tip-inducedbroadening of the imaged object, the true diameter of filamentsshould be expected to be much smaller. According to modelsdescribing the contribution of AFM tip radius and geometry tothe convolution of cylindrical structures in AFM images(Schwarz et al., 1994), the thinnest filaments we observed(apparent width 20 nm) probably correspond to single actinfilaments (~7 nm). Depending on the true radius of the tip usedin a particular scan (ranging from 10 to 40 nm, according tothe manufacturer’s instructions), the tip broadening effectcould even account for the apparent widening of single actinfilaments into the thicker filaments observed in our AFMimages. However, the presence of different filament diameterswithin the same image (i.e. generated with the same AFM tip,see Fig. 3H-K) suggests that the larger filament diameters werecaused by the grouping of different numbers of microfilaments.In these larger filamentous structures individual microfilamentsmay have been bundled tightly so that they could not beresolved by the AFM tip. Consistent with this idea, EM

Fig. 6. F-actin andpaxillin fluorescenceimages of an FA in a de-roofed cell (A-C).Corresponding topograph(D) andfluorescence/heightoverlay image (E). Thecircular area withdecreased F-actin staining(A, asterisk) correspondsto an area of decreasedheight in the AFMtopograph (D). Thisdefect may be the resultof mechanical disruption

of the FA structure during de-roofing. In contrast to the F-actin signal, thepaxillin signal in this area is not decreased (B). (F) A line scan through theoverlay image (E) demonstrates a correlation between the height and F-actin signal but not the paxillin signal. The shaded area (light green)indicates a higher relative fluorescence intensity of paxillin-YFP comparedto the F-actin staining. As the disruption of FA architecture down to aheight of less than 50 nm does not decrease the paxillin signal, the paxillinlocalization must be restricted to a membrane-proximal region of the FA.F-actin-containing structures, on the other hand, must be localizedpredominantly in the membrane-distal half, as disruption of the FAstructure down to about 50 nm reduces F-actin staining to almostbackground levels. Bar, 500 nm.

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pictures of rotary replicas show tight bundling ofmicrofilaments within the FA plaque (Samuelsson et al., 1993).Thus, in FAs, microfilaments may be grouped into discretefunctional units.

In the membrane-proximal region of the FA, �-actininfunctions as a linker between actin filaments and thecytoplasmic tails of integrin subunits (Lazarides and Burridge,1975; Otey et al., 1990; Rajfur et al., 2002). Nevertheless, �-actinin predominantly localizes to the membrane-distal regionof FAs (Chen and Singer, 1982; Geiger et al., 1981) where thebulk of actin filaments is located (Fig. 6A). This points to anadditional role of �-actinin as an actin filament crosslinker inFAs. In addition to �-actinin, microfilaments could also bebundled by other actin crosslinkers present in FAs, such asfimbin (Bretscher and Weber, 1980) or filamin (Geiger et al.,1984) and the different dimensions of these linkers could thengive rise to filament bundles of varying diameters.

Microfilaments were usually arranged in parallel or at slightangles to each other. Occasionally, however, they werebranched and could cross each other (Fig. 3G). We measuredheight differences ranging from 10 to 20 nm between adjacentmicrofilaments. This suggests a layered microfilamentarchitecture in which the vertical separation between the layersis in the order of the diameter of a single actin filament. Thevertical separation between neighboring microfilaments in FAsmay therefore be considerably smaller than the lateral.

Exhibiting a vertical resolution of 1 nm or better, AFMtopographs also provided accurate information about FA heightand enabled us to determine the FA volume. The average heightof bisecting cross sections perpendicular to the FA structurewas 84±16 nm, whereas the microfilament bundles protruded118±27 nm over the substratum (Fig. 5A,B). Given a plasmamembrane thickness of 5 to 10 nm and a distance of 10 to 15nm between the plasma membrane and the substratum at sitesof FAs (Heath and Dunn, 1978), the heights we measured haveto be reduced by 15 to 25 nm in order to give an accurateaccount of the thickness of the cytoplasmic part of the adhesionplaque. The FA height we determined is in good agreementwith thin section EM studies showing a ~60-nm-thick densityat the site of FAs (Chen and Singer, 1982). FA heights of lessthan 120 nm also re-emphasize that FAs are ideally suited forimaging by total internal reflection internal fluorescencemicroscopy (TIRFM) because their height is well within thezone excited by the evanescent wave, which typicallypenetrates some 150 nm into the cytoplasm of the cell (Adams,2002; Krylyshkina et al., 2003).

The AFM topographs showed that FA height increases towardsthe stress-fiber-associated end, whereas the width oftendecreased. This wedge shape could reflect the functionalrequirement of FAs to constitute a link between a cylindricalstress fiber and a planar substrate. By flattening towards the stressfiber distal end and the finger-like fanning out of microfilaments,the contact area with the substratum is maximized, whereas thecircular cross section at the opposite end assures maximumconnectivity with the stress fiber. The relative change in cross-section shape contrasted with much smaller relative changes incross-section area, indicating that the shape change over the FAlength could be caused by a reorganization of microfilamentsextending over the entire FA (Fig. 4G).

The ultrastructure of the stress fiber/AF interface is unknownand it remains to be investigated whether there is continuity of

actin filaments from stress fibers into FAs. This seems unlikelyhowever, as the stress fiber region immediately next to the FAis a highly dynamic region characterized by intense actinpolymerization. Furthermore, stress fiber contraction coincideswith twisting of the entire fiber (Katoh et al., 1998). Actinfilaments in FAs and stress fibers are functionally distinct. Forinstance, myosin II, responsible for the force generation instress fibers is absent from FAs. During our preparationprotocol, stress fibers were usually removed from the FAs,although occasionally stress fibers remained attached to FAs(see Fig. 3B). In the future it will be interesting to investigatethe structure of the FA/stress fiber interface in more detail.

Disruption of FA structure down to a height of 50 nm abovethe surrounding substratum left the paxillin-YFP fluorescencesignal unaffected (Fig. 6B), indicating that paxillin localizes tothe membrane-proximal region of FAs. Allowing a total of 25nm for the thickness of the plasma membrane plus its distancefrom the substratum, paxillin localization appears to be furtherrestricted to within 25 nm of the plasma membrane.Membrane-proximal localization of paxillin is consistent withthe idea that upon recruitment to FAs paxillin binds to vinculin(Turner et al., 1990), an interaction partner shown to localizeto the membrane-proximal part of the FA (Chen and Singer,1982). In FAs, paxillin may also bind to the cytoplasmic tailsof integrin subunits (Chen et al., 2000; Schaller et al., 1995),which would equally confine paxillin to a region immediatelyadjacent to the plasma membrane. In contrast, F-actin stainingwas almost completely lost when the membrane-distal half ofthe FA was disrupted, pointing to a localization of actinfilaments primarily in the upper (membrane-distal half) of theFA. Correlating fluorescence intensities with the heightinformation from AFM imaging thus provides informationabout the vertical localization of FA components within theadhesive plaque.

It is of great interest to understand how changes in thepacking density of FA components in the adhesion plaque affectFA function. For ‘2D’ markers (proteins confined to the plasmamembrane or a thin adjacent horizontal plane), fluorescenceintensities give a measure of how tightly they are packed in theFA. In contrast, fluorescence intensities yield no informationabout the clustering of ‘volume’ markers (proteins distributedthroughout the entire adhesion plaque) (Wehrle-Haller andImhof, 2002). Because paxillin appears to localize close to theplasma membrane, it can be essentially regarded a 2D markerand consequently, changes in the paxillin fluorescence intensityshould correlate with changes in its lateral packing.

In conclusion, we have obtained AFM topographs of FAsusing a simple preparation technique that did not require harshsample treatment and allowed imaging under physiologicallyrelevant conditions. The high vertical resolution of the AFMprovided accurate FA height information and offered insightinto the organization of microfilaments, such as their layeredarray and apparent bundling at the dorsal FA surface. Bycorrelating the paxillin-YFP fluorescence intensity with theheight information contained in the AFM topographs, we couldassign paxillin localization to the membrane-proximal part ofthe FA. It will now be interesting to investigate the FAultrastructure with alternative imaging techniques able toprovide high-resolution, 3D structural information of nativebiological samples, such as cryo-electron tomography(Baumeister, 2005; Resch et al., 2002).

Journal of Cell Science 118 (22)

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5323Analyzing focal adhesion structure

This work was supported by the Deutsche Volkswagenstiftung, bythe EC and the Free-State of Saxony. We would like to thank KatePoole, Pierre-Henri Puech and Christian Le Grimellec for helpfuldiscussions and JPK Instruments for their fruitful and collaborativesupport.

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