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1 Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo, Japan
2 CREST, Japan Science and Technology Corporation
3 Department of Biological Resources, Integrated Center for Sciences, Ehime University, Ehime, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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To generate the locomotive force, the protrusive membrane must adhere to the substrate. The molecular interaction underlying this adhesion remains unknown. Recently, interaction between the Arp2/3 complex and vinculin was found to be essential for lamellipodium formation (DeMali et al. 2002). Vinculin recruits Arp2/3 complex to focal complexes and nearby Arp2/3 activators at the leading edges. Formation of a vinculin-Arp2/3 complex might physically couple newly polymerized actin to sites of cell-substrate adhesion. These findings suggest the presence of mechanisms that coordinate actin reorganization and cell-substrate adhesion.
Giannone et al. (2004) proposed an attractive model to explain extension and adhesion of lamellipodia. In this model, a rearward actin wave (retrograde flow) arising at the back of the lamellipodial edge conveys the contractile signal, which involves myosin light chain kinase (MLCK), from the tip to the base of the lamellipodium. MLCK activates myosin-based contractility and causes contraction of actin structures in the lamellipodial protrusion. The rearward force applied to the tip of the lamellipodium enhances integrin binding to the extracellular matrix and initiates integrin-mediated signaling, thereby stabilizing the points of adhesion. Activated integrin signaling causes another round of MLCK release at the front of the lamellipodium. This positive feedback loop causes periodic interruptions in the process of extension of the membrane. However, it is still unclear how actin reorganization is regulated in such complex processes.
WASP family Verproline-homologous proteins (WAVEs) are crucial for Rac-induced membrane ruffling (Miki et al. 1998). Three WAVE proteins (WAVE1-3) have been identified in mammals (Suetsugu et al. 1999). These WAVEs have distinct tissue distributions, and their actin polymerization activities are regulated differentially by associations with various molecules (Miki et al. 2000; Westphal et al. 2000; Eden et al. 2002; Miki & Takenawa 2002; Soderling et al. 2002; Blagg et al. 2003; Bogdan & Klambt 2003). In a previous study, we established cultures of mouse embryonic fibroblasts (MEFs) from WAVE1 knockout (KO) and WAVE2 KO embryos and showed that WAVE1 and WAVE2 have distinct roles in different membrane ruffling structures in response to platelet-derived growth factor (PDGF) (Suetsugu et al. 2003). WAVE1 is essential for formation of membrane protrusions at the dorsal cell surface (dorsal ruffles) and is localized to the upper part of membrane ruffles. WAVE2 is required for formation of membrane ruffles at the cell periphery (peripheral ruffles) and is localized to the extreme edge of such ruffles; however, WAVE1-deficiency causes no defect during such processes. Thus, WAVE1 and WAVE2 appear to induce actin polymerization in different directions according to their localization and to promote formation of different membrane structures.
Here we show a novel function of WAVE1 in lamellipodia. WAVE1-deficiency increases the extension speed of lamellipodial protrusions, causing deformation transient cell-substrate adhesive structures in lamellipodia. It is suggested that WAVE1 is required for formation of stable lamellipodial protrusions with adhesions to a substrate.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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To investigate the relation between membrane-protrusive actin reorganization and the formation of cell-substrate adhesions in lamellipodia, we prepared WAVE-deficient MEFs as previously described (Fig. 1A) (Suetsugu et al. 2003). Of the WAVE family of proteins, only WAVE1 and WAVE2 are expressed in wild-type (WT) MEFs. To observe the process of coordination of membrane extension and cell-substrate adhesion, we observed cell spreading on glasses coated with fibronectin (FN). FN induces integrin-mediated activation of Rac and Cdc42 and subsequent formation of membrane protrusions (Price et al. 1998).
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WAVE1 KO MEFs also showed smooth, sheet-like protrusions at the cell periphery similar to those in WT MEFs. However, the spreading edges tended to curl up and retract, followed by formation of new protrusions (Fig. 1C, arrows; Supplemental Fig. S2). These processes occurred repeatedly even after the cells had spread fully. Such curling up of the spreading edges was not observed in WT MEFs, though some WT MEFs had ruffled edges that did not curl up (Fig. 1I, ruffle). When observed by scanning electron microscopy, curling up of the spreading edges was confirmed in WAVE1 KO MEFs (Fig. 2B).
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We next examined whether expression of exogeneous WAVE1 and WAVE2 could restore formation of normal lamellipodial protrusions in WAVE1 and WAVE2 KO MEFs, respectively. WAVE1 KO MEFs expressing only vector showed features similar to those of untransfected WAVE1 KO MEFs (Fig. 1D). However, WAVE1 KO MEFs expressing exogeneous WAVE1 (rescued WAVE1 KO MEFs) generated lamellipodial membrane protrusions similar to those in WT MEFs (Fig. 1E). The percentage of WAVE1 KO MEFs with lamellipodia after expression of exogeneous WAVE1 (74.1 ± 7.9%) was similar to that in WT MEFs (81.3 ± 5.0%) (Fig. 1I). Thus, WAVE1 is not essential for formation of lamellipodial membrane protrusions but is required for their stabilization.
WAVE2KO MEFs expressing only vector showed features similar to those of untransfected WAVE2 KO MEFs (Fig. 1G). Uniform cell spreading with lamellipodia was partially restored in WAVE2 KO MEFs expressing exogeneousWAVE2 (rescued WAVE2 KO MEFs) (55.2 ± 11.2%) compared with WT MEFs (88.6 ± 3.2%) (Fig. 1H,J). This partial restoration may reflect differential levels and/or regulation of exogeneous WAVE2 expression in WAVE2 KO MEFs. Interestingly, some rescued WAVE2 KO MEFs (10.0 ± 4.2%) showed curling up of the spreading edges observed in WAVE1 KO MEFs (Fig. 1J). Thus, in contrast to WAVE1, WAVE2 is essential for formation of lamellipodial structures.
Distribution of actin filaments in WAVE-deficient MEFs
To examine whether WAVE-deficiency affects distribution of actin filaments at the spreading edges, we examined spreading MEFs with phalloidin staining. Lamellipodial protrusions contain a highly branched actin-filament meshwork (Svitkina & Borisy 1999). During spreading on FN, lamellipodial protrusions were supported by high-density actin filaments at the spreading edges of WT MEFs (Fig. 3A). Actin structures at the peripheral areas in WT MEFs could be divided into two zones: a first actin-dense zone at the extreme periphery of the cells (indicated by arrows in Fig. 3B) and a second central zone of lower actin density. However, in WAVE1 KO MEFs with curling edges, density of actin filaments at the extreme periphery decreased and a boundary between two zones was ambiguous as compared with WT MEFs (compare Fig. 3A,B with I, J, respectively). Thus, actin filaments at the spreading edges were not properly organized in WAVE1 KO MEFs. Because expression of exogeneous WAVE1 in WAVE1 KO MEFs corrected the distribution of actin filaments, WAVE1 appears to be essential for formation of normal actin structures in lamellipodia (Fig. 3R).
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Localization of WAVEs during cell spreading on FN
To define the role of WAVEs at the spreading edges, we investigated the localization of these proteins in spreading WT MEFs. When stimulated by PDGF, WAVE1 was scarcely detected at the tip of peripheral ruffles (Suetsugu et al. 2003). However, while spreading on FN, WAVE1 showed a punctate pattern throughout the cytoplasm, and some WAVE1 was localized at the edges of lamellipodial protrusions (Fig. 3C,D). This localization of WAVE1 at the lamellipodial edge often appeared discontinuous (Fig. 3C, arrowheads). Exogeneously expressed WAVE1 showed similar localization in rescued WAVE1 KO MEFs (Fig. 3Q). Thus, WAVE1 was localized at the tips of spreading edges different from peripheral ruffles induced by PDGF. In contrast, WAVE2 was clearly and continuously localized at the extreme edges of lamellipodial protrusions (Fig. 3G,H). Thus, WAVE1 and WAVE2 show different localization patterns in lamellipodia.
Localization of WAVE1 at the web-like protrusions in WAVE2 KO MEFs was continuous and clearer than that at the lamellipodial edges in WT MEFs (compare Fig. 3C,D with 3O, P, respectively). Despite strong WAVE1 expression, defective lamellipodium formation was not compensated in WAVE2 KO MEFs. Similarly, at the spreading edges in WAVE1 KO MEFs, the localization of WAVE2 was confirmed. These results suggest that WAVE1 and WAVE2 cannot compensate for each other's deficiencies.
Cell-substrate adhesion in WAVE1 KO MEFs
To investigate the role of WAVE1 in stabilization of lamellipodia, we examined whether cell-substrate adhesions formed properly at the spreading edges in WAVE1 KO MEFs. At the spreading edges in WT MEFs, focal complexes were localized just behind the extreme edges in the lamellipodial protrusions (Fig. 4A–D). WAVE1 and WAVE2 were localized at more forward position in the lamellipodial protrusions compared with focal complexes (Fig. 4D,I). Intensity profiles reveal that there was no co-localization of vinculin and WAVEs (Fig. 4J,K). In contrast, Arp2 and vinculin were partially co-localized at the spreading edges (Fig. 4L,M).
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Membrane dynamics of WAVE1KO MEFs during spreading on FN
Recently, it was shown that there was a close relation between membrane extension and adhesion to a substrate in lamellipodia during cell spreading on FN (Giannone et al. 2004). To better understand the membrane dynamics of WAVE1 KO MEFs, we made kymographs of WT and WAVE1 KO MEFs during cell spreading on FN. Kymographs show membrane activity at spreading edges (Bear et al. 2002). When WT MEFs spread, periodic interruptions of membrane extension were observed at the spreading edges (Fig. 5A, boxed region in the far right panel). The extension speed of the spreading edges decreased gradually when periodic interruptions were observed (compare the neighboring phases in Fig. 5A). In contrast, there was no interruption of membrane extension in WAVE1 KO MEFs until the spreading edges curled up (Fig. 5B, boxed region in the right most panel). The speed of membrane extension in WAVE1 KO MEFs before curling was relatively constant and was approximately 2.5 times faster than that of WT MEFs before periodic interruptions (Fig. 5C). When exogeneous WAVE1 was expressed in WAVE1 KO MEFs, membrane extension speed decreased to that observed in WT MEFs. Thus, WAVE1 KO MEFs showed different membrane dynamics from WT MEFs before curling up of spreading edges.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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WAVE1 KO and WAVE2 KO MEFs showed different phenotypes for lamellipodium formation and WAVE1 and WAVE2 can not compensate for each other's deficiencies. We further speculate the function of each WAVE from phenotypes of WAVE KO MEFs. In the case of WAVE2 KO MEFs, lamellipodial actin structure is absent. Also in the other situations (stimulation by growth factors) and cell types (endothelial cells), it was shown that WAVE2 is essential for lamellipodial structure formation (Suetsugu et al. 2003; Yamazaki et al. 2003). Thus, WAVE2 mainly forms membrane protrusive structures when membrane protrudes parallel to a substrate.
In the case of WAVE1 KO MEFs, lamellipodial membrane protrusions develop but are not stabilized by adhesive structures. WAVE1 may function directly or indirectly in the formation of cell-substrate adhesions.
One possibility is that WAVE1 indirectly affects the formation of adhesive structures in the lamellipodium by regulating actin reorganization. Recently, a model concerning the formation of adhesive structures in lamellipodium was proposed (Giannone et al. 2004) (see Introduction). This model is very attractive, but it is unclear how the rearward actin wave is generated. According to this model, the lack of a rearward actin wave in lamellipodia of WAVE1 KO MEFs appears to cause defects in formation of adhesive structures. If WAVE1 is involved in the generation of the rearward actin wave, we propose two possible roles of WAVE1 in the process. One possible role is that in response to lamellipodial contraction, WAVE1 directly creates a new actin structure by inducing actin polymerization at the back of the lamellipodium, which generates the rearward actin wave. During spreading on FN, contact with FN activates Rac and Cdc42 via integrin signaling (Price et al. 1998). Activated integrin signaling by lamellipodial contraction may induce WAVE1-mediated actin reorganization. The other possible role is that WAVE1 influences the highly organized mesh-like structure of actin filaments by modifying the actin framework structure formed by WAVE2. Normal actin structures may be required to produce the rearward actin wave. In cells treated with cytochalasin D, which decreases the concentration of actin filaments in lamellipodia, periodic interruptions of extension of the membrane were rare (Giannone et al. 2004). In WAVE1 KO MEFs with curling spreading edges, the accumulation of actin filaments was not uniform and decreased at the spreading edges. These two possibilities are not exclusive, and WAVE1 may be involved in both processes.
Another possibility is that WAVE1 directly recruits adhesion proteins such as vinculin to sites of cell-substrate adhesion. However, we did not observe localization of WAVE proteins at cell-substrate adhesion sites in MEFs in contrast to Arp2/3 complex. In addition, we did not observe any interaction between WAVE proteins and vinculin (data not shown). On the basis of these findings, we believe that WAVEs are not involved directly in adhesion of the cell membrane to a substrate. However, in a previous study, WAVE1 was shown to be localized at focal adhesions in NIH3T3 cells (Westphal et al. 2000); this discrepancy may be due to the differences in the types of cells used for the experiments.
At the lamellipodial edges, WAVE1 and WAVE2 induce actin polymerization in different directions and the balance of these activities influences the direction and the extension speed of membrane protrusions. When relative activity of WAVE1 increases as compared with that of WAVE2 (WAVE2 KO MEFs), strong localization of WAVE1 at the peripheral area causes upward protrusions of the membrane. In contrast, when relative activity of WAVE2 is high (WAVE1 KO MEFs), membrane extension speed parallel to a substrate increases. Interestingly, some of rescued WAVE2 KO MEFs showed membrane dynamics similar to WAVE1 KO MEFs (Fig. 1J). In these cells, excessive WAVE2 activity may break the balance of actin polymerization.
The difference between the directions of WAVE1- and WAVE2-mediated actin polymerization is thought to be the result of their distinct localization patterns. WAVE1 was shown to localize to the dorsal part of membrane protrusions in dorsal ruffles (Suetsugu et al. 2003). WAVE1 may function at the back of a lamellipodium. Because lamellipodia are thin structures and can be observed only within a single phase, we could not confirm the apical distribution of WAVE1 in lamellipodia (data not shown). Further analysis with immuno-electron microscopy should resolve this problem. In contrast to WAVE1, WAVE2 is clearly localized at the tips of lamellipodial protrusions and therefore induces actin polymerization parallel to the substrate during lamellipodium formation. The mechanism underlying localization of WAVEs is not known. WAVE1 and WAVE2 interact with distinct molecules, and such molecules and interactions may influence localization (Stradal et al. 2004; Vartiainen & Machesky 2004).
In the present study, we observed a novel relation between WAVEs-dependent actin polymerization and cell-substrate adhesion during lamellipodium formation. Formation of cell-substrate adhesions is a critical step in cell migration. Further study of the regulation of WAVEs will bring new insights into our understanding of cell migration.
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Experimental procedures |
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All cells in this study were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal calf serum (FCS). WT, WAVE1 KO and WAVE2 KO MEFs were established from embryonic day (E) 9.5 embryos from heterozygous crosses (Suetsugu et al. 2003). E9.5 embryos were incubated in 0.25% trypsin and 0.5 mM EDTA in phosphate-buffered saline (PBS) for 30 min at 37 °C and then dispersed with a micropipette. Cells were cultured in DMEM supplemented with 10% FCS for 3 months and became spontaneously immortalized lines. Experiments were performed with two independently established lines.
Expression of exogeneous WAVEs
Full-length WAVE1- and WAVE2-expression plasmids were constructed in pEF-BOS plasmid vectors (Miki et al. 1998; Suetsugu et al. 1999). Recombinant plasmids and pTK-Hyg, which contains the hygromycin-resistance gene, were co-transfected into MEFs with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Single colonies were isolated after 1 month of hygromycin selection (50 µg/mL). Protein expression was confirmed by immunoblotting of cell lysates with appropriate antibodies. Stable rescued cell lines were used in the spreading assays.
Video microscopy
Sub-confluent cells were trypsinized, centrifuged, and dispersed as single-cell suspensions in DMEM supplemented with 10% FCS. Glass-bottomed culture dishes were coated with FN (25 µg/mL; Sigma, St. Louis, MO, USA) for 1 h at room temperature. Single-cell suspensions (50 cells/µL) were plated on FN-coated culture dishes, and observed with phase-contrast microscopy at 2-s intervals.
Phenotypes of WAVE1 KO MEFs in Fig. 1I were classified on the basis of sequential images as follows. Lamellipodia, cells with lamellipodial protrusions across 75% of the cell periphery. Curl up, cells with curling membranes across over (L) or below (S) 10% of the cell periphery. Ruffle, cells with ruffled membranes that did not curl up. Others, other cells.
Phenotypes of WAVE2 KO MEFs in Fig. 1J were classified on the basis of sequential images as follows. Lamellipodia, cells with lamellipodial protrusions (including ruffled membranes) across 75% of the cell periphery. Curl up, cells with curling membranes across over 10% of the cell periphery. Irregular, cells spreading irregularly with finger-like projections. Others, other cells.
For kymography, phase-contrast time-lapse sequences were obtained using IP labs software using 40x objective. Video clips were 10 min long with frames taken every 2 s. Kymographs were produced and analyzed with ImageJ software (the United States National Institutes of Health). For quantitative analysis, kymographs illustrating the lamellipodial activity during 10-min time-lapse videos were made at four places around the edge of the cells. Straight lines were drawn on kymographs before periodic interruptions (in the case of cells with lamellipodia) or curling up (in the case of cells with curling membranes). Slopes of these lines were used to calculate the membrane extension speed. The speed of membrane extension was calculated with each kymograph of 100 s.
Antibodies
Polyclonal anti-WAVE1 and anti-WAVE2 antibodies were prepared as previously described (Yamazaki et al. 2003). Polyclonal anti-Arp2 antibody was generated in rabbits immunized with the CEKGVRVLEKLGVTVR peptide and affinity-purified with CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. Monoclonal anti-vinculin antibody was purchased from Sigma. Monoclonal anti-actin antibody was from Chemicon (Temecula, CA, USA). Rhodamine- and Alexa 647-conjugated phalloidin were from Molecular Probes (Eugene, OR, USA).
Immunofluorescence microscopy
Sub-confluent cells were trypsinized, centrifuged, and then resuspended as single-cell suspensions in DMEM supplemented with 10% FCS. Coverslips were coated with FN (25 µg/mL) for 1 h at room temperature. Suspended cells (100 cells/µL) were replated on FN-coated coverslips at 37 °C. Every 5 min, cells were fixed with 3.7% formaldehyde in PBS for 10 min. Cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min and then incubated with primary antibodies at 1 : 100 dilution for 1 h. Cells were then incubated for 30 min with secondary antibodies conjugated to Alexa 488 or Alexa 568 (Molecular Probes). Coverslips were mounted on glass slides and observed by confocal microscopy. Intensity profiles in Fig. 4 were produced and analyzed with ImageJ software.
Scanning electron microscopy
Spreading MEFs were fixed with 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.3. Observation with a scanning electron microscope was performed as previously described (Yamazaki et al. 2003).
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Supplementary material |
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http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC845/GTC845.htm.
Figure S1 Video of spreading WT MEF in Figs 1B and 5A,1. Time series acquired at the interval of 2 s are shown.
Figure S2 Video of WAVE1 KO MEF in Figs 1C and 5B,2. Time series acquired at the interval of 2 s are shown.
Figure S3 Video of WAVE2 KO MEF in Fig. 1F,3 on FN are shown, respectively. Time series acquired at the interval of 2 s are shown.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: takenawa{at}ims.u-tokyo.ac.jp
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References |
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Received: 26 December 2004
Accepted: 17 January 2005
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