Experimental Cell Research 302 (2005) 83–95 www.elsevier.com/locate/yexcr

Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of filament reorganization

Bodo Borm, Robert P. Requardt, Volker Herzog, Gregor Kirfel*

Institute for Cell Biology, University of Bonn, 53121 Bonn, Germany

Received 14 June 2004, revised version received 25 August 2004 Available online 30 September 2004

Abstract

During epithelial cell migration, membrane ruffles can be visualized by phase contrast microscopy as dark waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body. Despite the common use of the term membrane ruffles, their structure, molecular composition, and the mechanisms leading to their formation remained largely unknown. We show here that membrane ruffles differ from the underlying cell lamella by more densely packed bundles of actin filaments that are enriched in the actin cross-linkers filamin and ezrin, pointing to a specific bundling process based on these cross-linkers. The accumulation of phosphorylated, that is, inactivated, cofilin in membrane ruffles suggests that they are compartments of inhibited actin filament turnover. High Rac1 and low RhoA activities were found under conditions of suboptimal integrin–ligand interaction correlating with low lamellipodia persistence, inefficient migration, and high ruffling rates. Based on these findings, we define membrane ruffles as distinct compartments of specific composition that form as a consequence of inefficient lamellipodia adhesion. D 2004 Elsevier Inc. All rights reserved.

Keywords: Cell migration; Membrane ruffle; Lamellipodia; Rac; Rho; Integrin; Cell adhesion; Filamin; Motility assay

Introduction contraction of the cell body to be finally completed by the release of cell-substrate adhesions at the cell rear [1,2]. Cell migration is central not only to many biological The formation of lamellipodia and at the cell processes, including embryogenesis, the inflammatory front is driven by the assembly of actin filaments from the response, tissue repair, and regeneration, but also to cytoplasmic pool of monomeric actin bound to sequestering pathological processes such as cancer and metastasis. The proteins such as profilin and thymosin h4 [3]. The signals onset of migration is accompanied by the acquisition of a for actin assembly to start are provided by constituents of spatial asymmetry that is manifested in a polarized cell the extracellular matrix (ECM) such as fibronectin (FN) and morphology, that is, a clearly distinguishable front and rear by soluble motogens from the large families of growth of the cell. Cell migration can be viewed as a multistep cycle factors including the epidermal growth factor (EGF) [4,5]. beginning with the extension of membrane protrusions Both kinds of signals are sensed by appropriate trans- (lamellipodia, filopodia) at the cell lamella, which is the flat, membrane receptors that, upon ligand binding, activate almost organelle free front region of motile cells. The cycle different signaling pathways [6,7]. The most prominent is continued by the formation of cell substrate adhesions receptors for ECM components are the proteins of the near the tips of protrusions and the acto-myosin powered integrin gene family [6] that, upon activation by ligand binding, can activate the small GTPases of the Rho family such as RhoA, Rac1, and Cdc42 [5,8]. Rac1 and Cdc42 * Corresponding author. Institute for Cell Biology, University of Bonn, have been shown to act as regulators of actin assembly Ulrich-Haberlandstr. 61a, 53121 Bonn, Germany. essential for lamellipodia and filopodia protrusion [9].Of E-mail address: [email protected] (G. Kirfel). the many effector proteins that interact with activated Rac1

0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.08.034 84 B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 and Cdc42, the Wiskott–Aldrich syndrome protein (WASP) membrane ruffles. To gain insight into the process of ruffle and its ubiquitous family member N-WASP provide a direct formation and the potential reorganization of the actin link to actin assembly by activating the Arp2/3 complex filament system, we have analyzed their protein composi- [10]. This complex acts as a nucleation point for the tion, particularly in relation to that of lamellipodia. Our assembly of the branched actin network that is the observations show that ruffles contain densely packed arrays predominant cytoskeletal element of growing lamellipodia. of actin filaments endowed with a specific set of associated According to their role as promoters of actin assembly, proteins including the cross-linkers filamin and ezrin, and Arp2/3 and WASP are concentrated at the tips of lamelli- the assembly regulator ADF/cofilin. To elucidate the func- podia of migrating cells [11,12]. tional significance of ruffle formation during cell migration, Since the cell is a bounded compartment, rapid assembly we have quantified simultaneously with highest resolution of actin filaments cannot continue for long without being in space and time cell migration velocity, the frequency of balanced by rapid and highly regulated disassembly. The ruffle formation, the persistence of lamellipodia, and their ubiquitous proteins of the ADF/cofilin family have been dependence on the adhesive properties of the substrate shown to bind to actin filaments and, thereby, promote their [20,22]. Our findings show that conditions of suboptimal disassembly [13]. The activity of ADF/cofilin depends on cell-substrate adhesion that lead to a significantly reduced LIM-kinase-controlled phosphorylation that blocks its inter- lamellipodia persistence and an inefficient cell migration actions with actin [14]. Since LIM-kinase is a downstream result in a dramatic increase in ruffle frequency. Our data effector of Rho family GTPases, actin assembly is promoted point to integrin-dependent and RhoGTPases-mediated in two ways: by activating the Arp2/3 nucleation complex signaling pathways controlling lamellipodia protrusion and and by inhibiting disassembly via ADF/cofilin regulation. ruffle dynamics. We conclude that ruffles are formed as a Recent findings additionally point to a positive engagement consequence of inefficient integrin–ligand interaction at the of cofilin in actin assembly by generating free barbed ends leading edge of lamellipodia and that they act as compart- that are a prerequisite for filament elongation by the addition ments of actin reorganization. of actin monomers [15]. For sustained migration to occur, the newly formed membrane protrusions have to be stabilized by the Materials and methods formation of firm adhesions to the substrate [16]. The initial step during the establishment of these adhesions has Cells and cell culture been shown to be the aggregation of ligand-bound integrins in the form of small focal contacts (FC) at the leading edge Normal human epidermal keratinocytes from neonatal of protrusions. This aggregation of integrins then triggers foreskin were purchased from Cambrex Bio Science the activation of signaling molecules such as the small (Verviers, Belgium). Subcultures were grown at 378C and GTPase RhoA that is responsible for the recruitment of 5% CO2 in complete keratinocyte basal medium (Cambrex cytoskeletal- and FC-associated proteins, resulting in the Bio Science). Cells were harvested for subculturing or for formation of contractile acto-myosin bundles and the subsequent experiments using 0.025% Trypsin and 0.01% generation of the larger and more organized focal adhesions EDTA in HBSS (Cambrex Bio Science) only between (FA) [8,9,16,17]. The small FCs at the cell front are thought passages 2 and 4. For immunofluorescence staining and to be essential for cell locomotion and act as anchoring sites scanning EM, cells were subconfluently plated on glass for the transmission of acto-myosin-driven propulsive cover slips or CELLocatek cover slips (Eppendorf, forces, whereas the larger FAs seem to inhibit cell migration Hamburg, Germany). For quantification of cell motility, by forming stable and less dynamic anchoring structures cells were plated into chambered cover slips (Nalge Nunc [18,19]. International, Wiesbaden, Germany). For biochemical Lamellipodia that fail to establish stable adhesions are measurements of Rac1 and Rho activity, the cells were believed to become detached from the substrate and plated on 10-cm culture plates. All cells were plated on retracted toward the cell body [6,16,17]. It has been uncoated or FN-coated surfaces (see below). One hour speculated that during this retraction process, membrane before starting the quantification of cell motility, staining of ruffles are formed that are clearly visible in the light integrin h1, or performing Rac1 and RhoA activity assays, microscope as waves of dark contrast on the frontal surface the cells were stimulated with 50 nM human recombinant of cells. These ruffles emerge at the cell edge, move EGF (Sigma-Aldrich, Deisenhofen, Germany) to induce cell centripetally, and finally disappear at the border between the migration. cell lamella and the main cell body [20,21]. What still remained largely unknown are their structure and their Cell adhesion substrates and anti-integrin antibodies molecular composition as well as the signals and the mechanisms leading to ruffle formation. We have applied Coating with human FN (Cell-Systems, St. Katharinen, high-resolution scanning and transmission electron micro- Germany) at concentrations of 2.5, 5, 7.5, or 10 Ag/cm2 scopy (EM) to reveal the structural organization of was done as described previously [23]. To inhibit the B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 85 interaction between integrins and their ligands, function- membrane ruffles appeared as dark descending lines (Fig. blocking antibodies against integrin a5 (clone P1D6) and 1c, black line), whereas lamellipodia protrusions were h1 (clone JB1A) (Chemicon, Temecula, USA) were used at represented by linear ascending contours (Fig. 1c, white 10 and 40 Ag/ml, respectively, after saturation with 1% line). The slope of these lines corresponds to the velocity of BSA for 30 min. ruffle and lamellipodia movement (v =dx (Am)/dt (min)). Projections of these lines along the x-axis (time) were used Quantification of cell motility to calculate the persistence of lamellipodia, that is, the period a protrusion lasts before its retraction begins (Fig. Phase contrast image series of motile keratinocytes were 1c, dashed white line). The number of lines per time was obtained using an inverted LSM 510 (Zeiss, Oberkochen, counted to determine the frequency of ruffle formation. Cell Germany) equipped with a 63Â 1.4 NA Ph3 plan migration velocity was determined by exploiting the clear apochromat objective and an incubation chamber for halo that represents the border between the main cell body constant temperature, and were controlled by the LSM and the lamella. Marking this halo (Fig. 1c, dashed black 510 standard software. Lamella dynamics and cell migra- line) allowed to calculate the cell body displacement in tion velocity were analyzed by the computer-assisted relation to the substrate, which is a common definition of stroboscopic analysis of cell dynamics (SACED) (Fig. 1) cell translocation [25]. that has been described before [20,22] and renamed recently as kymographic assay [24]. For each substrate, at Immunofluorescence staining least 15 individual cells were monitored over a 10-min period by capturing digital images every 2 s. Subsequently, For immunofluorescence staining of integrin h1 (mouse four areas of interest were marked on each image by lines monoclonal antihuman integrin h1, clone P5D2, Chemi- that crossed the cell lamella (Fig. 1a). The resulting 1-pixel- con), filamin (mouse monoclonal anti-filamin, clone PM6/ wide areas were cut (Fig. 1b) and lined up in time space 317, Chemicon), and ezrin (mouse monoclonal anti-ezrin, plots (Fig. 1c) that allowed the quantification of relevant clone 3C12, Sigma-Aldrich), the cells were fixed in 4% motility data. In these stroboscopic time space plots, freshly made paraformaldehyde (PFA) in buffer [26] (150 mM NaCl, 5 mM MgCl2 5 mM EGTA, 5 mM Glucose, 10 mM MES pH 6.1) for 20 min at 378C, washed in PBS containing 30 mM Glycin (G-PBS), permeabilized in PBS/0.2% Triton X-100 for 10 min, rinsed in PBS, and blocked in 3% BSA/PBS for 30 min. The samples were incubated with primary antibodies diluted in 0.3% BSA in PBS for 45 min at 378C, washed three times with PBS, and incubated with goat anti-mouse Cy3- conjugated secondary antibodies (Jackson Laboratories, West Grove, PA, USA) for 45 min at 378C together with Alexa-488 Phallodin (Molecular Probes, Leiden, Nether- lands) for correlative actin filament staining. Immunofluore- scence staining of cofilin and phosphorylated ADF/cofilin was modified from Ref. [27]. The cells were fixed with 3% PFA together with 0.1% (vol/vol) glutaraldehyde in Fig. 1. The SACED motility assay. Cells are monitored over periods of up cytoskeleton buffer for 1 h, washed extensively in PBS, to 10 min by digitally capturing phase contrast images every 2 s. and permeabilized with 0.5% Triton X-100 in PBS for Subsequently, an area of interest is marked on each image by lines that 15 min. After three times washing in PBS, once in TBS cross the cell lamella and the leading edge (a). The resulting 1-pixel-wide areas were cut out (b) and lined up in time space plots (c). These plots allow (20 mM Tris, 145 mM NaCl, pH 8) and twice with 2 mg/ml the simultaneous quantification of different motility parameters. In these NaBH4 in TBS for 15 min, the samples were rinsed four stroboscopic time space plots, membrane ruffles appeared as dark times in PBS and blocked in 3% BSA/PBS for 30 min. Cells descending lines (c, black line) whereas lamellipodia protrusions were were further incubated for 45 min at 378C with anti-cofilin represented by linear ascending contours (c, white line). The slope of these antibody (rabbit polyclonal anti-cofilin, Cytoskeleton Inc., lines corresponds to the velocity of ruffle and lamellipodia movement (v = dx (Am)/dt (min)). Projections of these lines along the x-axis (time) were Denver, USA) and anti-phospho ADF/cofilin antibody used to calculate the persistence of lamellipodia, that is, the period a (rabbit antiserum raised against a phoshopeptide epitope protrusion lasts before its retraction begins (c, dashed white line). The of ADF/cofilin [28], gift from J.R. Bamburg, Colorado State number of lines per time was counted to determine the frequency of ruffle University, USA), respectively, followed by incubation with formation. Cell migration velocity was determined by exploiting the clear a Cy2-conjugated anti-rabbit antibody and Alexa546 Phal- halo that represents the border between the main cell body and the lamella. Marking this halo (c, dashed black line) allowed to calculate cell body loidin for 1 h at 378C. All samples were mounted in anti- displacement in relation to the substrate, which is a common definition of fade reagent (Biomeda Corporation, Foster City, CA, USA) cell translocation [25]. and analyzed by using a LSM 510 equipped with a 63Â NA 86 B. Borm et al. / Experimental Cell Research 302 (2005) 83–95

1.4 plan apochromat objective. All images were obtained as 1 h and lysed with lysis buffer (50 mM TRIS, 0.5 M NaCl, confocal z stacks spanning the whole cell in its z range with 1% Triton X-100, 10 mM MgCl2, pH 7.5). After addition subsequent projection of the image series into a single of agarose beads to the cell lysates and incubation at 48C overlay using the LSM 510 standard software. For for 60 min, the agarose beads were washed and sub- fluorescence quantification using Image-Pro Plus 5 (Media sequently collected by centrifugation at 5000 g. The beads Cybernetics), staining conditions and microscope settings were then resuspended in Laemmli-buffer, separated by were identical for all samples. All measurements were made SDS-PAGE, and detected by Western blot using rabbit on unprocessed images. In brief, the total fluorescence of an polyclonal anti-Rac and mouse monoclonal anti-Rho anti- area of interest (sum of all gray levels) marking membrane bodies (Cytoskeleton) and appropriate secondary HRP- ruffles or lamellipodia was measured and normalized to the conjugated antibodies. Staining was detected using size of the respective area. On this basis, the fluorescence enhanced chemiluminescence and densitometry has been ratios of membrane ruffles versus lamellipodia from five done using Optiquant (Packard Instruments, Meridan, individual cells were calculated for actin/filamin and for USA). actin/ezrin. Statistical analysis Correlative light and electron microscopy Statistical evaluation of SACED-derived motility data Cells grown on CELLocatek cover slips were labeled was performed with SPSS software (SPSS Inc., Chicago, against actin filaments using Alexa546-Phalloidin (Mole- USA). Results were expressed as mean values with bars cular Probes) as described above. After light microscopic representing 95% confidence interval of mean values. analysis, the cells were treated as described below for Statistical significance between data groups was determined scanning EM. by Whitney U test and considered to be significantly different at values P b 0.01. Data were tested for linear Scanning electron microscopy regression and rank correlation was determined according to Spearman and considered to have statistical significance at For scanning EM, cells were fixed in 2% glutaraldehyde values P b 0.01. in 0.1 M sodium cacodylate, pH 7.3, for 20 min, transferred to 0.1% aqueous tannic acid, and rinsed with distilled water. For scanning EM of the cytoskeleton, the cell membrane Results was removed by detergent lysis. In detail, cells were incubated in PEG-GTX (10 mM PIPES, 50 mM EDTA, Ruffles are flat membrane folds on the lamella surface 10 mM KOH, 27 mM KCl, 0.1% (vol/vol) Triton X-100, 4% (vol/vol) polyethylenglycol (MW 6000), 10% (vol/vol) In phase contrast microscopy of motile keratinocytes, glycerin, pH 7.2) for 5 min, washed briefly in PBS, and membrane ruffles were clearly visible as waves of dark fixed with 4% (vol/vol) glutaraldehyde in PBS. All speci- contrast that move centripetally from the leading edge of mens were dehydrated through a graded series of ethanol the organelle free cell lamella toward the organelle and critical point dried from CO2 in 10 cycles according containing main cell body where they finally disappeared. to Ref. [29] using a Balzers CPD 030 (BAL-TEC, Cells grown on not precoated glass surfaces migrated Schalksmqhlen, Germany). Dried specimens were mounted inefficiently (about 0.2 Am/min; see below) and exhibited on aluminum sample holders and coated with a 2-nm layer numerous ruffles evenly distributed over the cell lamella of platinum/palladium in a HR 208 sputter coating device (Fig. 2a, arrows), whereas cells on FN-coated surfaces (Cressington, Watford, UK). SEM was performed at an migrated efficiently (about 0.5 Am/min; see below) and acceleration voltage of 3 kV using a XL 30 SFEG (Philips, bore only few if any ruffles (Fig. 2b). Scanning EM Eindhoven, Netherlands) equipped with a through lens revealed a narrow, fold-bearing lamella for cells grown on secondary electron detector. not precoated glass surfaces (Fig. 2c) and an extended, smooth lamella for cells grown on FN (Fig. 2d). At higher Rac1 and rhoA activity assays magnifications, ruffles appeared as flat membrane folds that protruded from the lamella resembling waves on a Many Rho family effector proteins specifically recog- water surface (Fig. 2e, arrows). Underneath these mem- nize the activated, GTP-bound form of RhoA and Rac1. brane folds newly formed membrane protrusions were We used PAK-1- and Rhotekin-coupled agarose beads observed (Fig. 2e, arrowheads). Time-lapse studies of the (Cytoskeleton) to precipitate PAK-1/RacGTP- and Rhote- cell lamella allowed showing that lamellipodia detachment kin/RhoGTP complexes that can be identified in Western and their subsequent retraction toward the cell body are blots using anti-Rac and anti-Rho antibodies. In brief, the main steps of ruffle formation (Figs. 3a and b). keratinocytes grown on 10-cm culture plates or FN-coated Analysis of cells grown on microgrid cover slips revealed 10-cm culture plates were stimulated with 50 nM EGF for that the dark waves on the cell lamella visualized by phase B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 87

and b, circle) and ezrin (Figs. 4c and d, circle), which were only sparsely distributed in the underlying lamella. Fluorescence quantification revealed a 50% higher density of actin filaments within membrane ruffles as compared to lamellipodia. This is consistent with the dense packaging of actin filaments observed by SEM (Fig. 3f). An even higher increase in the concentration of ezrin by 150% and of filamin by 100% was measured. This corresponds to an about 2-fold increase in the filamin/actin and an about 3- fold increase in the ezrin/actin ratio in membrane ruffles as compared to lamellipodia and points to a specific recruit- ment of both proteins during membrane ruffle formation and concomitant actin filament reorganization. Intriguingly, the actin filament severing protein ADF/cofilin was found in ruffles exclusively in its phosphorylated, that is, inactive, form (Figs. 3e and f, circle), whereas the

Fig. 2. Phase contrast (a and b) and scanning EM (c–e) of motile keratinocytes. Keratinocytes grown on not precoated glass surfaces showed numerous ruffles (a, arrows) evenly distributed over the cell lamella, which appeared narrow and fold bearing (c). On 2.5 Ag/cm2 FN, only few if any ruffles were observed (b) on a broad and smooth lamella (d). Higher magnification revealed ruffles as flat membrane folds protruding from the lamella surface (e, arrows). At the cell edge, newly formed membrane protrusions were observed (e, arrowheads). Scale bars: (a–d) 10 Am; (e) 1 Am. contrast microscopy (Fig. 3c, arrows) indeed corresponded to the extended membrane folds observed by scanning EM (Fig. 3d, arrows).

Membrane ruffles contain densely packed actin filaments with a characteristic set of associated proteins

Scanning EM of the keratinocyte lamella after removal of the plasma membrane by detergents revealed that ruffles contained densely packed arrays of thin filaments (Fig. 3f, Fig. 3. Time lapse sequences (25 s) of the cell lamella provided evidence arrows), which were identified as actin filaments by that ruffle formation results from lamellipodia detachment and their subsequent retraction toward the cell body (a and b). Using microgrid phalloidin staining (Fig. 3e, arrows) or immunogold cover slips allowed to show that the wave-like regions of dark contrast that labeling (not shown). These actin filaments were located were visible in phase contrast (c) correspond to the membrane folds upon a more loosely arranged network of actin filaments observed by scanning EM (d) and contain high amounts of actin filaments that were regularly distributed across the cell lamella (Fig. as shown by phalloidin staining (e). Scanning EM of the cell lamella after 3f). Immunofluorescence studies on the composition of removal of the plasma membrane (f) revealed that ruffles contain densely packed arrays of actin filaments that lie upon a more loosely arranged actin membrane ruffles revealed a specific set of actin-associated filament network (inset in f). Arrowheads in c: the edges of microgrids proteins that differed from the underlying lamella by a became visible due to light refraction. Scale bars: (a, b, and f) 1 Am; (c–e) clear enrichment of the actin cross-linkers filamin (Figs. 4a 10 Am; (inset in f) 200 nm. 88 B. Borm et al. / Experimental Cell Research 302 (2005) 83–95

mined by exploiting the clear halo, which represents the border between the main cell body and the lamella. Marking this halo (Fig. 5a, dashed black line) allowed to calculate the cell body displacement in relation to the substrate that is a common definition of cell translocation [25].

High ruffle frequency indicates low lamellipodia persistence and inefficient cell migration

Evaluation of the SACED-derived motility data showed that migration velocity reached a minimum of about 0.21 (F0.18) Am/min on not precoated glass surfaces (Fig. 5b), whereas significantly higher velocities occurred on FN- coated surfaces with a maximum of about 0.53 (F0.3) Am/ min at 2.5 Ag/cm2 FN. With increasing FN concentrations, the migration velocity dropped in a linear fashion to only 0.24 Am/min at 10 Ag/cm2 FN. Lamellipodia persistence reached lowest values on not precoated glass surfaces (Fig. 5c) with an average life time of about 0.37 (F0.15) min, whereas at 2.5 Ag/cm2 FN, a nearly 10-fold higher persistence of about 3.34 (F1.33) min was determined. As for migration velocity, the persistence of lamellipodia decreased in a linear relation with increasing FN concen- trations dropping to about 0.6 min at 10 Ag/cm2 FN. Ruffle frequencies reached maximum values on not precoated glass surfaces with about 0.2 (F0.08) ruffles/min (Fig. 5d), whereas lowest ruffle frequencies were measured with about 0.06 (F0.04)/min at 2.5 Ag/cm2 FN. With increasing Fig. 4. Immunocytochemical (a, c, e, and g) and phase contrast studies (b, FN concentrations, a linear rise in ruffle frequencies d, f, and h) on the composition of membrane ruffles revealed a specific occurred up to 0.14 (F0.06)/min at 10 Ag/cm2. All data set of actin-associated proteins that differ from the underlying lamella by point to a clear correlation between migration velocity, that increased filamin/actin (a, circle) and ezrin/actin ratios (c, circle). The actin filament severing protein ADF/cofilin was found in ruffles is, the efficiency of migration, the persistence of lamelli- exclusively in its phosphorylated, that is, inactive form (e, circle), podia, and the frequency of membrane ruffle formation. whereas nonphospho cofilin was more evenly distributed over the lamella Under substrate conditions that supported the formation of (g). Scale bars: 10 Am. highly persistent lamellipodia, efficient migration occurred and ruffling became less frequent. Under conditions activated form was evenly distributed across the lamella favoring short-lived lamellipodia, migration was slow, that region (Figs. 3g and h). is, inefficient, and ruffle formation was more frequent. Hence, membrane ruffles might be considered as indicators The SACED motility assay allows to quantify the dynamics of inefficient migration due to reduced lamellipodia of ruffles and lamellipodia in relation to migration velocity persistence.

The SACED cell motility assay-derived stroboscopic Lamellipodia persistence and ruffle formation depend on time space plots allowed to simultaneously visualize the the interaction of integrin a5b1 with its ligand FN dynamics of membrane ruffles, which appeared as dark descending lines (Figs. 5a and e, black lines), and of Newly formed lamellipodia are known to be stabilized lamellipodia, which represented linear ascending contours by the interaction of integrins with their ECM ligands [16]. (Figs. 5a and e, white lines). The slope of these lines After addition of antibodies blocking the interaction of the corresponded to the velocity of ruffle and lamellipodia integrins a5 and h1 with FN, we observed a significantly movement and projections of these lines along the x-axis reduced migration velocity of keratinocytes dropping by (time) allowed to calculate the persistence of lamellipodia, 75% to about 0.08 (F0.04) Am/min for integrin h1(Fig. that is, the period a protrusion lasts before its retraction 5f). Simultaneously, lamellipodia persistence decreased to begins (Fig. 5a, dashed white line). The number of lines per 0.037 (F0.15) min thus reaching only 1% of control time was counted to determine the frequency of ruffle values (Fig. 5g), whereas ruffle formation became dramati- formation. In addition, cell migration velocity was deter- cally more frequent reaching 5-fold values of controls (Fig. B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 89

Fig. 5. SACED-derived time space plots of motile keratinocytes visualized the dynamics of membrane ruffles (a, e, black lines) and of lamellipodia (a, e, white lines). Time space plots from keratinocytes grown on not precoated glass surfaces showed short-lived lamellipodia and high ruffling frequencies (a, top). On 2.5 Ag/cm2 FN, the cells produced more persistent lamellipodia with reduced ruffling frequencies (a, middle). On 10 Ag/cm2 FN, lamellipodia persistence decreased whereas ruffling frequency increased (a, bottom). Statistical evaluation of migration velocity (b), lamellipodia persistence (c), and ruffle frequency (d) showed a clear correlation between migration, persistence of lamellipodia, and the frequency of ruffles formation depending on substrate adhesiveness. High lamellipodia persistence due to appropriate adhesiveness is a prerequisite for efficient cell migration that is indicated by low ruffling frequencies. Keratinocytes grown on 2.5 Ag/cm2 FN after addition of antibodies inhibiting the interaction between FN and integrin a5 (e, top) and h1 (e, bottom) showed significantly reduced lamellipodia persistence (g) that is correlated to decreased migration efficiency (f) and is marked by clearly increased ruffle frequencies (h). Error bars: 95% confidence interval of mean values.

5h). Incubation with 1% BSA had no effect on cell the formation of ruffles is apparently a consequence of a motility (not shown). Using function-blocking antibodies suboptimal interaction between integrin a5h1 and its against various other integrins expressed by migrating ligand FN. keratinocytes caused no significant effects on the motility parameters described above (not shown). Our data indicate Efficient migration depends on low and high rho activity that integrin a5h1 interaction with FN is essential for the attachment of lamellipodia leading to high lamellipodia The small GTPases Rac1 and RhoA belonging to the persistence and allowing efficient cell migration. Hence, Rho-family are known to be involved in the downstream 90 B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 signaling of ligand-bound integrins. We applied Rac1 and Formation of stress fibers and focal adhesions are RhoA activity assays to determine the relationship between characteristics of inefficiently migrating cells GTPase activity and cell migration efficiency. Rac1 activity was low for efficiently migrating keratinocytes exhibiting Downstream effectors of the Rho GTPase are various low ruffle frequencies and highly persistent lamellipodia at actin-associated proteins that modulate the formation of 2.5 Ag/cm2 FN (Fig. 6a). Rac1 activity was found to be contractile acto-myosin fibers and the clustering of integrin significantly increased when cells were grown on high FN h1 [17] in the form of FC at the front of lamellipodia that concentrations or on not precoated glass surfaces, that is, on are required to convert contraction into traction forces substrates that allowed only reduced migration efficiency necessary for cell migration [18]. Cells grown on not accompanied by diminished lamellipodia persistence and precoated glass surfaces, which have been shown to contain frequent ruffle formation. Highest Rac1 activity was found low levels of activated RhoA and high levels of Rac1, were at 10 Ag/cm2 reaching about 3-fold higher levels as enriched with numerous densely packed bundles of actin compared to 2.5 Ag/cm2 FN. About 2.5-fold higher levels filaments (Figs. 7b and c, arrows) and showed a nearly even of Rac1 activity were also measured for cells grown on not distribution of integrin h1 but no obvious clustering at the precoated glass surfaces. Contrary to Rac1, RhoA showed lamella region (Fig. 7a). Cells grown on 2.5 Ag/cm2 FN, highest activity levels in cells grown on 2.5 Ag/cm2 which exhibited the highest levels of RhoA activity and low dropping significantly on higher FN concentrations to only levels of Rac1 activity, contained only few and significantly 40% on 10 Ag/cm2 and to 60% for cells grown on not smaller bundles of actin filaments (Figs. 7e and f), and precoated glass surfaces (Fig. 6b). Hence, high RhoA and integrin h1 was clustered at the cell lamella in the form of low Rac1 activities coincided with efficient cell migration, distinct patches (Fig. 7d, circles) with a size of about 2 Am that is, fast cell movement, high lamellipodia persistence, that is typical for FCs [30]. Very fine and short bundles of and low ruffling frequencies. actin filaments were observed that were orientated along the direction of cell migration (Fig. 7e, circles) and were linked to the putative FC sites (Fig. 7f, circles). With increasing FN concentrations, we observed significantly more and thicker bundles of actin filaments representing stress fibers (Fig. 7h, arrows) and larger but less distinct integrin h1 patches (Fig. 7g).

Discussion

The protrusion of lamellipodia at the cell front is regarded as the initial step of a cyclic process that underlies cell migration [1,2]. For efficient migration to occur, lamellipodia have to be stabilized by the formation of cell substrate adhesions that represent molecular bridges by the heterodimeric transmembrane receptors of the integrin family connecting the cytoskeleton and the ECM [16]. Depending on the adhesiveness of the substrate, the formation of adhesions can fail, and it has been speculated that lamellipodia without appropriate anchorage become detached and are retracted from the leading lamella centripetally toward the main cell body [17], thereby forming characteristic structures on the cell surface that Fig. 6. Rac1 (a) and RhoA (b) activities were determined in relation to are generally described as membrane ruffles [20,21]. substrate adhesiveness by quantitative Western blot analysis of cell lysates To validate the assumed relationship between lamellipo- and displayed in relation to the amounts of total Rac1 (d, black columns) dia adhesion and ruffle formation, we applied the high- and total RhoA (d, grey columns), respectively. Rac1 activity, stimulating the formation of lamellipodia and ruffles, was found to be low for resolution SACED cell motility assay [20,22,23] that allows efficiently migrating keratinocytes on 2.5 Ag/cm2, whereas high Rac1 to acquire and quantify various parameters of cell migration activity was measured for inefficiently migrating keratinocytes on high FN and motility simultaneously, and has been successfully used concentrations or on not precoated glass surfaces (a). RhoA activity to characterize the motogenic activity of growth factors on stimulating the formation of contractile acto-myosin complexes and focal epidermal keratinocytes and melanocytes [22,31]. To learn adhesions was high in efficiently migrating cells on 2.5 Ag/cm2 but was reduced in inefficiently migrating cells on higher FN concentrations or on more about the signals and mechanisms involved in ruffle not precoated glass surfaces (b). Actin blots of cell lysates are shown to formation, we investigated the structural organization and demonstrate equal loading of all samples (c). the protein composition of membrane ruffles, and analyzed B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 91

Fig. 7. Immunofluorescence labeling against Integrin h1 (red) and actin filaments (green) of keratinocytes grown on not precoated glass surfaces (a–c), 2.5 Ag/cm2 FN (d–f), or 10 Ag/cm2 FN (g–i). Cells on not precoated glass surfaces were enriched with massive bundles of actin filaments (b, arrows) showing the characteristic appearance of stress fibers. Integrin h1 was almost evenly distributed across the cell lamella without any obvious clustering (a). Cells on 2.5 Ag/cm2 FN exhibited only few and significantly smaller bundles of actin filaments (e). Very fine and short bundles of actin filaments were observed (e, f, circles) that were oriented along the direction of cell migration and inserted in the cell lamella at sites of integrin h1 clusters with a size of about 2.5 Am (d, circles). At 10 Ag/cm2 FN, the number of large actin filament bundles increased (h, arrows), and larger but less distinct integrin h1 patches were observed (g). Scale bars: 20 Am. the activities of the small GTPases Rac and Rho that are dered to be a consequence of insufficient detachment of known as downstream effectors of integrin signaling and as adhesion sites at the cell rear [35]. mediators of cytoskeletal and adhesive structures formation. A clear relationship between the application of different motogenic growth factors, the frequency of ruffle formation, Ruffling is a function of lamellipodia persistence and and cell migration velocity has been reported [20,23]. directly related to migration efficiency Accordingly, the formation of membrane ruffles is usually considered as a sign of increased lamella dynamics and of FN is one of the major constituents of the dermal ECM elevated migration levels [36]. To address the question and has been shown to represent the preferred migration whether membrane ruffles result from lamellipodia that have substrate for keratinocytes during the process of epidermal failed to form firm attachments, we measured the persis- wound healing [32]. Surfaces coated with FN have been tence of lamellipodia, that is, the period a observed to be appropriate for efficient migration of exists before it is retracted, which we found to be a keratinocytes in vitro. In the absence of motogenic growth sensitive, rate-limiting, and adhesion-dependent motility factors, a saturable increase in migration velocity has been parameter. Highly persistent lamellipodia were found to be shown in relation to FN concentration for keratinocytes characteristic of efficiently, that is, fast moving, cells on [23], whereas a biphasic relation between migration velocity appropriate FN substrates and to be correlated in all cases and the adhesiveness to substrates has been described for with minimal ruffle frequencies. Reciprocal proportions CHO [33]. In contrast, this report shows that keratinocytes were observed for inefficiently migrating cells that gener- exhibit this biphasic relation only after EGF stimulation ated only short-lived lamellipodia accompanied by high with maximal values occurring at intermediate FN levels, ruffling frequencies. A correlation between lamellipodia which has also been reported for NR6 fibroblasts [34]. persistence and cell migration efficiency has also been Lower FN concentrations failed to support efficient cell reported recently for fibroblasts [24] without addressing migration, which might be due to insufficient cell–matrix directly the relationship with ruffle formation. Our findings adhesion at the cell front that in turn is essential for the provide for the first time clear evidence that ruffles originate generation of traction forces during cell migration [18].On in lamellipodia that fail to properly attach to the substrate FN concentrations above 5 Ag/cm2,weobserveda and are retracted toward the main cell body. Thus, significant decrease of migration velocity, which is consi- membrane ruffle formation can be looked upon as a specific 92 B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 but indirect criterion for the evaluation of lamellipodia stability of which decides whether lamellipodia are stabi- persistence, which depends on substrate adhesiveness and is lized and, therefore, contribute to efficient cell migration or directly related to cell migration efficiency (Fig. 8). whether they are retracted as ruffles.

Lamellipodia persistence depends on the specific Ruffle formation is regulated by the small GTPases interaction between integrin a5b1 and FN rac and rho

Cell-substrate adhesion is mainly accomplished by the Besides acting as molecular linkers between FN and the interaction of ECM receptors from the integrin family and is actin filament system, integrin h1 can activate GTPases of rate limiting for efficient cell migration [1],andany the Rho family following ligand binding [42,43]. Adhesion disturbance of the ECM–integrin interaction has inhibitory of keratinocytes to laminin 5 via the integrins a3h1 and a6h4 effects on cell migration [37]. Keratinocytes migrating on leads to increased levels of activated Rho [44], and the FN use integrin a5h1 for the interaction with the substrate, adhesion of untransformed NIH-3T3 cells on FN specifi- whereas integrin a3h1 is responsible for dynamic inter- cally enhances RhoA activity [45]. Reexpression of integrin actions with laminin 5 [38]. Antibodies that block the h1inh1-deficient epithelial cells triggered the activation of interaction of the integrins a5 and h1 have been shown both RhoA and Rac1, which raises the possibility that recently to significantly inhibit keratinocyte migration integrin h1 might regulate the motility of epithelial cells by velocity on FN [37,39], whereas inhibition of integrin a3 the use of this small GTPases [46]. In mice, loss of integrin had measurable effects on migration of mammary carci- h1 caused a severe defect in epidermal wound healing and noma cells on laminin 5 [40]. Our SACED assay-derived h1-null keratinocytes showed impaired migration [47].An data on keratinocytes show that inhibition of the FN increase in RhoA activity occurred after prolonged contact receptor components integrin a5 and h1 causes a significant to FN, although an initial decrease was observed upon decrease in lamellipodia persistence and migration velocity, plating NIH3T3-fibroblasts on FN [48]. RhoA activity, whereas ruffle formation becomes dramatically more which has been found to antagonize lamellipodia formation frequent. The FN-binding integrin a5h1 has been shown [49,50] also, stimulates cellular contractility as a prere- previously to be essential for the formation of FC at the quisite for cell migration [51]. RhoA activity, therefore, front of newly generated lamellipodia [41], which, besides might be a prerequisite for sustained cell migration under anchoring the lamellipodia, are indispensable for the trans- appropriate substrate conditions by regulating cellular formation of cellular contraction forces into traction forces, contractility and focal adhesions. Our findings on keratino- that is, cell body translocation during migration [18]. Our cytes show that high Rho activity coincides with the findings show that small but very distinct integrin clusters formation of highly persistent lamellipodia, efficient cell characteristic of FC are formed at the lamellipodia front of migration, and low ruffling rates. Under these conditions, efficiently migrating cells, but absent from the lamella of we observed the formation of very faint bundles of actin inefficiently migrating cells. The interaction of integrin filaments terminating in FC sites. It has been demonstrated a5h1 with FN can be considered the molecular anchor, the that 3–10 actin filaments are sufficient to support the forces applied to individual FC [52]. Hence, these bundles of actin filaments might represent the contractile structures respon- sible for cell body contraction during keratinocyte migration (see Figs. 7d–f). Upon activation by ligand-bound integrins and growth factor receptors, the GTPase Rac1 triggers the activation of the Arp2/3 complex, resulting in increased assembly of actin filaments, thereby promoting lamellipodia protrusion [10,16,17]. Expression of a constitutively active form of Rac1 showed that activation of this GTPase induces the formation of lamellipodia and ruffling but is not sufficient to propagate cell movement [53,54]. Accordingly, Rac1- deficient macrophages show normal migration and chemo- taxis but reduced membrane ruffling in response to colony stimulating factor-1 [55]. Our findings that highest Rac1 activity is observed under conditions of inefficient migra- Fig. 8. Schematic illustration summarizing the correlation between tion, low lamellipodia persistence, and high ruffling rates adhesion-dependent lamellipodia persistence, migration efficiency, and support the view that Rac1 triggers the protrusion of ruffling rates. High ruffling rates indicate high Rac-activity, suboptimal adhesion, and low persistence of lamellipodia that results in inefficient lamellipodia. Without appropriate adhesion these lamellipo- migration. Low ruffling rates point to high Rho activity, adequate adhesion, dia are not able to support forward movement and are and highly persistent lamellipodia that result in efficient cell migration. quickly retracted as membrane ruffles. Membrane ruffles are B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 93 apparently an appropriate structural indicator for Rac1 suggest that membrane ruffles might act as a compartment activity pointing to suboptimal lamellipodia adhesion and involved in the highly regulated reorganization of actin inefficient cell migration (Fig. 8). filaments that become necessary when lamellipodia are retracted due to impaired adhesion. Filamin and ezrin might act as organizers and stabilizers of membrane ruffles Acknowledgments The actin-associated protein filamin is known as an actin filament cross-linker that is able to form either three- The authors wish to thank Prof. J. R. Bamburg, Colorado dimensional orthogonal networks when the filamin/actin State University, USA, for providing the anti-phospho ADF/ ratio is about 1:150 or parallel bundles when the ratio is cofilin antibody, Prof. W. Alt, University of Bonn, about 1:10 [56]. Our observations showing that the filamin/ Germany, for excellent discussions, and Dr. W. Neumqller actin ratio in ruffles is about 2-fold higher than in for critical reading of the manuscript. We greatly appreciate lamellipodia point to filamin as the bundling protein the technical assistance of C. Hennes and N. Florin. responsible for the dense packaging of actin filaments observed in membrane ruffles. These results imply that membrane ruffle formation is accompanied by compaction References of actin filaments. Besides its cross-linking function, filamin has been shown to be mechanically linked to integrins [57], [1] D.A. Lauffenburger, A.F. Horwitz, Cell migration: a physically thereby providing a link between the plasma membrane and integrated molecular process, Cell 84 (1996) 359–369. the actin filament system that might be necessary for the [2] A.J. Ridley, M.A. Schwartz, K. Burridge, R.A. Firtel, M.H. Ginsberg, stability of membrane ruffles during their retraction. G. Borisy, J.T. Parsons, A.R. Horwitz, Cell migration: integrating Accordingly, filamin-deficient human melanoma cells are signals from front to back, Science 302 (2003) 1704–1709. [3] T.D. Pollard, G.G. Borisy, Cellular motility driven by assembly and still capable to form protrusions upon serum stimulation, but disassembly of actin filaments, Cell 112 (2003) 453–465. the protrusions form blebs and ruffling is not observed [4] A.Y. Chan, S. Raft, M. Bailly, J.B. Wyckoff, J.E. Segall, J.S. [58,59]. Finally, filamin has been shown to be integrated in Condeelis, EGF stimulates an increase in actin nucleation and filament cellular signaling pathways that use Rho GTPases as number at the leading edge of the lamellipod in mammary mediators and lead to a phosphorylation-dependent modu- adenocarcinoma cells, J. Cell Sci. 111 (1998) 199–211. [5] C.H. Damsky, D. Ilic, Integrin signaling: it’s where the action is, Curr. lation of the binding capacity of filamin for actin [60]. The Opin. Cell Biol. 14 (2002) 594–602. activity levels of Rac1 and RhoA observed in migrating [6] R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, keratinocytes might represent a direct switch for filamin to Cell 110 (2002) 673–687. preferentially stabilize actin filament bundles as found in [7] A. Wells, EGF receptor, Int. J. Biochem. Cell Biol. 31 (1999) membrane ruffles. Ezrin is another prominent actin binding 637–643. [8] K.A. DeMali, K. Wennerberg, K. Burridge, Integrin signaling to the protein known to tether actin filaments to the plasma actin cytoskeleton, Curr. Opin. Cell Biol. 15 (2003) 572–582. membrane by its interaction with the hyaluron receptor [9] P. Aspenstrom, Effectors for the Rho GTPases, Curr. Opin. Cell Biol. CD 44 [61]. Functional ablation of ezrin has been shown to 11 (1999) 95–102. block ruffling and migration of fibroblasts [62]. Our finding [10] T. Takenawa, H. Miki, WASP and WAVE family proteins: key that the ezrin/actin ratio in ruffles is 3-fold higher than in molecules for rapid rearrangement of cortical actin filaments and cell movement, J. Cell Sci. 114 (2001) 1801–1809. lamellipodia provides further support for its suggested role [11] M.D. Welch, A.H. DePace, S. Verma, A. Iwamatsu, T.J. Mitchison, during the formation and stabilization of membrane ruffles. The human Arp2/3 complex is composed of evolutionarily conserved The actin filament severing protein ADF/cofilin has been subunits and is localized to cellular regions of dynamic actin filament shown to produce free actin monomers that are necessary assembly, J. Cell Biol. 138 (1997) 375–384. for the Arp2/3-dependent assembly of actin filaments, [12] A.M. Weaver, M.E. Young, W.L. Lee, J.A. Cooper, Integration of signals to the Arp2/3 complex, Curr. Opin. Cell Biol. 15 (2003) which power lamellipodia protrusion [3]. Recent findings 23–30. additionally point to a positive engagement of cofilin in [13] J.R. Bamburg, A. McGough, S. Ono, Putting a new twist on actin: actin assembly by generating free barbed ends that are a ADF/cofilins modulate actin dynamics, Trends Cell Biol. 9 (1999) prerequisite for filament elongation by the addition of actin 364–370. monomers [15]. [14] N. Yang, O. Higuchi, K. Ohashi, K. Nagata, A. Wada, K. Kangawa, E. Nishida, K. Mizuno, Cofilin phosphorylation by LIM-kinase 1 and Upon phosphorylation by the Rho effector kinase LIM, its role in Rac-mediated actin reorganization, Nature 393 (1998) ADF/cofilin becomes inactivated [63]. Our observation that 809–812. ruffles contain high amounts of phosphorylated cofilin [15] M. Ghosh, X. Song, G. Mouneimne, M. Sidani, D.S. Lawrence, J.S. points to ruffles as a compartment where the Arp2/3- and Condeelis, Cofilin promotes actin polymerization and defines the Cofilin-dependent actin filament turnover that powers direction of cell motility, Science 304 (2004) 743–746. [16] K.A. DeMali, K. Burridge, Coupling membrane protrusion and cell lamellipodia protrusion becomes inactivated during mem- adhesion, J. Cell Sci. 116 (2003) 2389–2397. brane ruffle formation. The specific protein composition of [17] K. Burridge, K. Wennerberg, Rho and Rac take center stage, Cell 116 ruffles and their characteristic arrays of actin filaments (2004) 167–179. 94 B. Borm et al. / Experimental Cell Research 302 (2005) 83–95

[18] K.A. Beningo, M. Dembo, I. Kaverina, J.V. Small, Y.L. Wang, unique roles of RGD site and integrins, J. Cell. Physiol. 151 (1992) Nascent focal adhesions are responsible for the generation of strong 443–450. propulsive forces in migrating fibroblasts, J. Cell Biol. 153 (2001) [40] R.B. Lichtner, A.R. Howlett, M. Lerch, J.A. Xuan, J. Brink, B. 881–888. Langton-Webster, M.R. Schneider, Negative cooperativity between [19] N.Q. Balaban, U.S. Schwarz, D. Riveline, P. Goichberg, G. Tzur, I. alpha 3 beta 1 and alpha 2 beta 1 integrins in human mammary Sabanay, D. Mahalu, S. Safran, A. Bershadsky, L. Addadi, B. carcinoma MDA MB 231 cells, Exp. Cell Res. 240 (1998) 368–376. Geiger, Force and focal adhesion assembly: a close relationship [41] B.J. Dzamba, H. Bultmann, S.K. Akiyama, D.M. Peters, Substrate- studied using elastic micropatterned substrates, Nat. Cell Biol. 3 specific binding of the amino terminus of fibronectin to an integrin (2001) 466–472. complex in focal adhesions, J. Biol. Chem. 269 (1994) 19646–19652. [20] B. Hinz, W. Alt, C. Johnen, V. Herzog, H.W. Kaiser, Quantifying [42] H. Miao, S. Li, Y.L. Hu, S. Yuan, Y. Zhao, B.P. Chen, W. Puzon- lamella dynamics of cultured cells by SACED, a new computer- McLaughlin, T. Tarui, J.Y. Shyy, Y. Takada, S. Usami, S. Chien, assisted motion analysis, Exp. Cell Res. 251 (1999) 234–243. Differential regulation of Rho GTPases by beta1 and beta3 integrins: [21] M. Abercrombie, The crawling movement of metazoan cells, Proc. R. the role of an extracellular domain of integrin in intracellular Soc. Lond., Ser. B 207 (1980) 129–147. signaling, J. Cell Sci. 115 (2002) 2199–2206. [22] G. Kirfel, B. Borm, A. Rigort, V. Herzog, The secretory beta-amyloid [43] A.L. Berrier, R. Martinez, G.M. Bokoch, S.E. LaFlamme, The integrin precursor protein is a motogen for human epidermal keratinocytes, beta tail is required and sufficient to regulate adhesion signaling to Eur. J. Cell Biol. 81 (2002) 664–676. Rac1, J. Cell Sci. 115 (2002) 4285–4291. [23] T. Libotte, H.W. Kaiser, W. Alt, T. Bretschneider, Polarity, protrusion- [44] B.P. Nguyen, X.D. Ren, M.A. Schwartz, W.G. Carter, Ligation of retraction dynamics and their interplay during keratinocyte cell integrin alpha 3beta 1 by laminin 5 at the wound edge activates Rho- migration, Exp. Cell Res. 270 (2001) 129–137. dependent adhesion of leading keratinocytes on collagen, J. Biol. [24] J.E. Bear, T.M. Svitkina, M. Krause, D.A. Schafer, J.J. Loureiro, G.A. Chem. 276 (2001) 43860–43870. Strasser, I.V. Maly, O.Y. Chaga, J.A. Cooper, G.G. Borisy, F.B. [45] E.H. Danen, P. Sonneveld, A. Sonnenberg, K.M. Yamada, Dual Gertler, Antagonism between Ena/VASP proteins and actin filament stimulation of Ras/mitogen-activated protein kinase and RhoA by cell capping regulates fibroblast motility, Cell 109 (2002) 509–521. adhesion to fibronectin supports growth factor-stimulated cell cycle [25] D.R. Soll, The use of computers in understanding how animal cells progression, J. Cell. Biol. 151 (2000) 1413–1422. crawl, Int. Rev. Cytol. 163 (1995) 43–104. [46] C. Gimond, A. van Der Flier, S. van Delft, C. Brakebusch, I. [26] J.V. Small, Organization of actin in the leading edge of cultured cells: Kuikman, J.G. Collard, R. Fassler, A. Sonnenberg, Induction of cell influence of osmium tetroxide and dehydration on the ultrastructure of scattering by expression of beta1 integrins in beta1-deficient epithelial actin meshworks, J. Cell Biol. 91 (1981) 695–705. cells requires activation of members of the rho family of GTPases and [27] A.Y. Chan, M. Bailly, N. Zebda, J.E. Segall, J.S. Condeelis, Role of downregulation of cadherin and catenin function, J. Cell Biol. 147 cofilin in epidermal growth factor-stimulated actin polymerization and (1999) 1325–1340. lamellipod protrusion, J. Cell Biol. 148 (2000) 531–542. [47] R. Grose, C. Hutter, W. Bloch, I. Thorey, F.M. Watt, R. Fassler, C. [28] P.J. Meberg, S. Ono, L.S. Minamide, M. Takahashi, J.R. Bamburg, Brakebusch, S. Werner, A crucial role of beta 1 integrins for Actin depolymerizing factor and cofilin phosphorylation dynamics: keratinocyte migration in vitro and during cutaneous wound repair, response to signals that regulate neurite extension, Cell Motil. Development 129 (2002) 2303–2315. Cytoskeleton 39 (1998) 172–190. [48] X.D. Ren, W.B. Kiosses, M.A. Schwartz, Regulation of the small [29] T.M. Svitkina, A.A. Shevelev, A.D. Bershadsky, V.I. Gelfand, GTP-binding protein Rho by cell adhesion and the cytoskeleton, Cytoskeleton of mouse embryo fibroblasts. Electron microscopy of EMBO J. 18 (1999) 578–585. platinum replicas, Eur. J. Cell Biol. 34 (1984) 64–74. [49] W.T. Arthur, K. Burridge, RhoA inactivation by p190RhoGAP [30] B. Geiger, A. Bershadsky, R. Pankov, K.M. Yamada, Transmembrane regulates cell spreading and migration by promoting membrane crosstalk between the extracellular matrix-cytoskeleton crosstalk, Nat. protrusion and polarity, Mol. Biol. Cell 12 (2001) 2711–2720. Rev., Mol. Cell Biol. 2 (2001) 793–805. [50] R.A. Worthylake, K. Burridge, RhoA and ROCK promote migration [31] T. Quast, S. Wehner, G. Kirfel, K. Jaeger, M. De Luca, V. Herzog, by limiting membrane protrusions, J. Biol. Chem. 278 (2003) sAPP as a regulator of dendrite motility and melanin release in 13578–13584. epidermal melanocytes and melanoma cells, FASEB J. 17 (2003) [51] M. Chrzanowska-Wodnicka, K. Burridge, Rho-stimulated contractility 1739–1741. drives the formation of stress fibers and focal adhesions, J. Cell Biol. [32] R.O. Hynes, Fibronectins, Springer-Verlag, New York, 1990. 133 (1996) 1403–1415. [33] S.P. Palecek, J.C. Loftus, M.H. Ginsberg, D.A. Lauffenburger, A.F. [52] A. Kishino, T. Yanagida, Force measurements by micromanipulation Horwitz, Integrin-ligand binding properties govern cell migration of a single actin filament by glass needles, Nature 334 (1988) 74–76. speed through cell-substratum adhesiveness, Nature 385 (1997) [53] A.J. Ridley, H.F. Paterson, C.L. Johnston, D. Diekmann, A. Hall, The 537–540. small GTP-binding protein rac regulates growth factor-induced [34] G. Maheshwari, A. Wells, L.G. Griffith, D.A. Lauffenburger, membrane ruffling, Cell 70 (1992) 401–410. Biophysical integration of effects of epidermal growth factor and [54] D.G. Stupack, S.Y. Cho, R.L. Klemke, Molecular signaling fibronectin on fibroblast migration, Biophys. J. 76 (1999) 2814–2823. mechanisms of cell migration and invasion, Immunol. Res. 21 [35] S.P. Palecek, A. Huttenlocher, A.F. Horwitz, D.A. Lauffenburger, (2000) 83–88. Physical and biochemical regulation of integrin release during rear [55] C.M. Wells, M. Walmsley, S. Ooi, V. Tybulewicz, A.J. Ridley, Rac1- detachment of migrating cells, J. Cell Sci. 111 (1998) 929–940. deficient macrophages exhibit defects in cell spreading and membrane [36] J.V. Small, T. Stradal, E. Vignal, K. Rottner, The lamellipodium: ruffling but not migration, J. Cell Sci. 117 (2004) 1259–1268. where motility begins, Trends Cell Biol. 12 (2002) 112–120. [56] B.M. Jockusch, P. Bubeck, K. Giehl, M. Kroemker, J. Moschner, M. [37] F. Decline, P. Rousselle, Keratinocyte migration requires alpha2beta1 Rothkegel, M. Rudiger, K. Schluter, G. Stanke, J. Winkler, The integrin-mediated interaction with the laminin 5 gamma2 chain, J. Cell molecular architecture of focal adhesions, Annu. Rev. Cell Dev. Biol. Sci. 114 (2001) 811–823. 11 (1995) 379–416. [38] F.M. Watt, M.D. Hertle, Keratinocyte integrins, in: I. Leigh, E.B. [57] M. Pfaff, S. Liu, D.J. Erle, M.H. Ginsberg, Integrin beta cytoplasmic Lane, M. Watt (Eds.), The Keratinocyte Handbook, Cambridge Univ. domains differentially bind to cytoskeletal proteins, J. Biol. Chem. Press, Cambridge, England, 1994. 273 (1998) 6104–6109. [39] J.P. Kim, K. Zhang, J.D. Chen, K.C. Wynn, R.H. Kramer, D.T. [58] L.A. Flanagan, J. Chou, H. Falet, R. Neujahr, J.H. Hartwig, T.P. Woodley, Mechanism of human keratinocyte migration on fibronectin: Stossel, Filamin A, the Arp2/3 complex, and the morphology and B. Borm et al. / Experimental Cell Research 302 (2005) 83–95 95

function of cortical actin filaments in human melanoma cells, J. Cell tein CD44 and actin-based , J. Cell Biol. 126 (1994) Biol. 155 (2001) 511–517. 391–401. [59] R.K. Vadlamudi, F. Li, L. Adam, D. Nguyen, Y. Ohta, T.P. Stossel, R. [62] R.F. Lamb, B.W. Ozanne, C. Roy, L. McGarry, C. Stipp, P. Mangeat, Kumar, Filamin is essential in actin cytoskeletal assembly mediated by D.G. Jay, Essential functions of ezrin in maintenance of cell shape and p21-activated kinase 1, Nat. Cell Biol. 4 (2002) 681–690. lamellipodial extension in normal and transformed fibroblasts, Curr. [60] J.M. Bellanger, C. Astier, C. Sardet, Y. Ohta, T.P. Stossel, A. Debant, Biol. 7 (1997) 682–688. The Rac1- and RhoG-specific GEF domain of Trio targets filamin to [63] K. Ohashi, K. Nagata, M. Maekawa, T. Ishizaki, S. Narumiya, K. remodel cytoskeletal actin, Nat. Cell Biol. 2 (2000) 888–892. Mizuno, Rho-associated kinase ROCK activates LIM-kinase 1 by [61] S. Tsukita, K. Oishi, N. Sato, J. Sagara, A. Kawai, ERM family phosphorylation at threonine 508 within the activation loop, J. Biol. members as molecular linkers between the cell surface glycopro- Chem. 275 (2000) 3577–3582.