Membrane Ruffles in Cell Migration: Indicators of Inefficient Lamellipodia Adhesion and Compartments of Actin Filament Reorganization
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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 actin 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 filopodia 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