A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability
© Jayo et al; licensee BioMed Central Ltd. 2012
Received: 10 July 2012
Accepted: 10 August 2012
Published: 10 August 2012
Fascin-1 is an actin crosslinking protein that is important for the assembly of cell protrusions in neurons, skeletal and smooth muscle, fibroblasts, and dendritic cells. Although absent from most normal adult epithelia, fascin-1 is upregulated in many human carcinomas, and is associated with poor prognosis because of its promotion of carcinoma cell migration, invasion, and metastasis. Rac and Cdc42 small guanine triphosphatases have been identified as upstream regulators of the association of fascin-1 with actin, but the possible role of Rho has remained obscure. Additionally, experiments have been hampered by the inability to measure the fascin-1/actin interaction directly in intact cells. We investigated the hypothesis that fascin-1 is a functional target of Rho in normal and carcinoma cells, using experimental approaches that included a novel fluorescence resonance energy transfer (FRET)/fluorescence lifetime imaging (FLIM) method to measure the interaction of fascin-1 with actin.
Rho activity modulates the interaction of fascin-1 with actin, as detected by a novel FRET method, in skeletal myoblasts and human colon carcinoma cells. Mechanistically, Rho regulation depends on Rho kinase activity, is independent of the status of myosin II activity, and is not mediated by promotion of the fascin/PKC complex. The p-Lin-11/Isl-1/Mec-3 kinases (LIMK), LIMK1 and LIMK2, act downstream of Rho kinases as novel binding partners of fascin-1, and this complex regulates the stability of filopodia.
We have identified a novel activity of Rho in promoting a complex between fascin-1 and LIMK1/2 that modulates the interaction of fascin-1 with actin. These data provide new mechanistic insight into the intracellular coordination of contractile and protrusive actin-based structures. During the course of the study, we developed a novel FRET method for analysis of the fascin-1/actin interaction, with potential general applicability for analyzing the activities of actin-binding proteins in intact cells.
Cell protrusions are dynamic and morphologically varied extensions of the plasma membrane, supported by the actin cytoskeleton, that are essential for cell migration. Fascin-1 is a prominent actin-bundling protein that characterizes the filopodia, microspikes, and dendrites of mesenchymal, neuronal, and dendritic cells, respectively, and also contributes to filopodia, podosomes, and invadopodia in migratory vascular smooth muscle cells and cancer cells [1–4]. Fascin-1 is absent from most normal adult epithelia, yet is upregulated in human carcinomas arising from a number of tissues. There is evidence that fascin-1 supports the migratory and metastatic capacities of carcinomas [3–7]. Fascin-1 is an independent indicator of poor prognosis in non-small-cell lung carcinomas and colorectal, breast, and other carcinomas [4, 8–11]. In colon, breast, or prostate carcinomas, fascin-1 protein correlates with increased frequency of metastasis [7, 10, 11]. Fascin-1 is thought to be the target of macroketone, which is under investigation as an anti-cancer agent . For these reasons, identification of the signaling pathways that regulate fascin-1 in carcinoma cells has become an important focus of research.
Actin bundling has been shown in vitro to be a conserved activity of fascins [12–15]. In filopodia, fascin-1 molecules crosslink actin filaments into parallel bundles, yet also move dynamically in and out of the bundle, which may allow for bundle turning and bending . F-actin cross linking by fascin-1 involves the N-terminal and C- terminal domains of fascin-1, and a major mechanism that inhibits the actin-bundling activity of fascin-1 is the phosphorylation of an N-terminal motif (S39 in human fascin-1) by conventional isoforms of protein kinase C (cPKC) [17–19]. cPKC phosphorylation of S39 inhibits actin binding and drives the formation of a complex between phosphorylated fascin-1 and active cPKC, resulting in a diffuse cytoplasmic distribution of fascin-1 [18, 20]. In migrating carcinoma cells, fascin-1 and cPKC associate dynamically in filopodia and at cell edges, and the cycling of phosphorylated fascin-1 is necessary for directional cell migration and experimental metastasis [5, 19]. Rac1 is a major upstream regulator of both these activities of fascin-1; it promotes the bundling of F-actin by fascin-1 in lamellipodia , and drives the formation of a complex between phosphorylated fascin-1 and active cPKC, through a pathway involving group I p21-activated kinases .
Effective cell migration depends on integration of the F-actin cytoskeleton of protrusions with the contractile actomyosin stress fibers in the cell body . The molecular basis of this integration is not well understood, but fascin-1 is known to associate with stress fibers under conditions associated with moderate extracellular matrix (ECM) adhesion, such as on mixed thrombospondin-1/fibronectin (FN) surfaces or under conditions of partial impairment of cell spreading on FN caused by a function-pertubing antibody to α5 integrin [20, 23, 24]. In fish keratocyes, fascin-containing filopodia contribute actin filament bundles into myosin II-containing stress fibers or fold back to incorporate into lamellipodial F-actin arcs . The small guanine triphosphatase (GTPase) Rho is a major regulator of cell contractility that acts antagonistically to Rac in several cellular pathways . but whether Rho regulates fascin-1 is unknown. Several lines of evidence indicate functional links between fascin-1 protrusions and the contractile focal adhesions that are promoted by active Rho; the phosphofascin-1/cPKC complex regulates the balance between protrusions and focal adhesions in mesenchymal cells, and depletion of fascin-1 from colon carcinoma cells inhibits focal adhesion disassembly and prevents filopodia formation [5, 18]. Whether Rho participates in these processes is unknown. Although overexpression of constitutively active Rho alters fascin-1 localization in quiescent fibroblasts, dominant-negative Rho does not inhibit the long-lived fascin-1 protrusions of cells adherent on thrombospondin-1 . Tenascin-C, another ECM glycoprotein that activates assembly of fascin-1 protrusions, suppresses Rho activity in fibroblasts by a syndecan-4 dependent pathway [27–30].
In this study, we investigated the hypothesis that fascin-1 is a functional target of Rho and identified a pathway from Rho via Rho kinases to p-Lin-11/Isl-1/Mec-3 kinases (LIMK)1 and LIMK2. We found that LIMK1/2 is a novel positive regulator of the fascin-1/actin interaction and is a novel interaction partner of fascin-1. These data have important implications for consideration of the role of fascin-1 in carcinoma metastasis.
RhoA supports the interaction of fascin-1 with actin in migrating cells
FN-adherent C2C12 cells have significant levels of endogenous active Rho-guanine triphosphate (GTP) relative to cells adherent to thrombospondin-1 (Figure 1B). Under conditions of Rho inhibition by C3 exotoxin, C2C12 cells adherent to FN have irregular shapes, with increased fascin-1 bundles at cell edges (Figure 1A). These observations were confirmed by scoring the numbers of peripheral fascin-containing bundles in adherent cells. BIM or C3 treatments increased the number of bundles, but did not alter the lengths of bundles containing fascin-1 (Figure 1C,D). Increased association of fascin-1 with microfilament bundles within the cell body was also seen in many C3-treated cells (Figure 1A). The effects of BIM and C3 were confirmed in SW480 colon carcinoma cells undergoing Rac-dependent migration on laminin (LN) by mechanisms previously identified to depend functionally on fascin-1-dependent filopodia, dynamic fascin/PKC complexing, and focal adhesion turnover [5, 15]. BIM treatment of SW480 cells on LN resulted in more irregular morphologies with non-polarized formation of fascin-1-positive protrusions at cell margins (Figure 1E). SW480 typically contain relatively few stress fibers, and the effects of C3 on fascin-1 relocalization to cell edges was less pronounced in these cells (Figure 1E, insets). Rho activity in migrating SW480 cells and its effective inhibition by C3 exotoxin was confirmed by measurement of RhoA activity under the different experimental conditions (see Additional file 1, Figure S1A). Together, these data implicate Rho activity in regulation of the dynamic balance of fascin-1 interactions with F-actin.
As expected from the initial experiments (Figure 1), treatment with BIM or C3 resulted in altered cell morphologies (Figure 2A,B). C3-treated C2C12 cells typically showed reduction of actin stress fibers within cell bodies (Figure 2A). BIM treatment did not prevent the fascin-1S39A/lifeact FRET/FLIM interaction, confirming the independence of this interaction from cPKC activity (Figure 2). In both cell types, the interaction between GFP-lifeact and mRFP-fascin-1S39A was strongly dependent on Rho activity (Figure 2A-C (C shows quantification from multiple cells)). These FRET data confirm that the direct interaction of fascin-1 with actin can be imaged using GFP-lifeact as a probe for F-actin, and that the interaction occurs preferentially with non-phosphorylated fascin-1 in intact cells. They also reveal that Rho acts in intact, ECM-adherent cells to promote the interaction of fascin-1 with actin.
Rho inhibition does not alter levels of the fascin-1/cPKC complex
Modulation of the fascin-1/actin interaction by Rho depends on Rho kinases but not on myosin-based contractility
The effects of Y27632 treatment were analyzed further by confocal time-lapse imaging of live SW480 cells co-expressing GFP-fascin-1 and mRFP-lifeact, in order to enable clear visualization of fascin-positive filopodia. In control cells, all filopodia contained both fascin-1 and lifeact (Figure 4C; for single-channel images, see Additional file 2, Figure S2C). Individual filopodia initiated, extended, and retracted over 1 to 3 minutes (Figure 4C, arrows; also see Additional file 4, movie 2). Line-scan analysis of fluorescence intensities for GFP-fascin-1 and mRFP-lifeact along the length of individual filopodia showed a strong fascin-1 signal along the entire length of the shaft of each filopodium, and a progressive reduction in the lifeact signal towards the tip (Figure 4D). The filopodia of Y27632-treated cells were less linear, remained extended over a longer timescale (Figure 4E shows filopodium at timepoints i to iii (arrow); see Additional file 5, movie 3), and had reduced fascin-1 intensity along the length of each filopodium (Figure 4E; see Additional file 2, Figure S2C for single-channel images). Thus, the Y27632-induced bending and altered dynamics of filopodia are probably due to alterations in organization of the core actin bundle of each filopodium and to the expected alteration in cell-body contractility caused by reductions in contractile stress fibers.
Another major mediator of cell contraction is myosin light chain kinase, (MLCK) . To establish whether either MLCK or myosin activity act to inhibit actin bundling by fascin-1, FN-adherent C2C12 cells were treated with ML-7 as an inhibitor of MLCK, or 2,3-butanedione monoxime (BDM) as a broad-spectrum inhibitor of actomyosin, which has also been reported to act as a chemical phosphatase . In contrast to the Y27632 treatment, no enhancement of endogenous fascin-1 in peripheral bundles was detected, indicating that the inhibitory activity of Rho kinases is not mediated by myosin-based contractility (Figure 5A; Figure 5C shows quantification of multiple cells). Similarly, expression of a dominant-negative truncated caldesmon that blocks stress-fiber assembly  did not promote peripheral fascin-1/actin bundles (Figure 5B; Figure 5C shows quantification of multiple cells). The possible roles of MLCK and myosin ATPase were also examined by FRET/FLIM analysis of SW480 cells co-expressing GFP-lifeact and mRFP-fascin-1S39A. Neither ML-7 nor blebbistatin (the latter tested as a specific inhibitor of myosin II ATPase) inhibited the interaction between fascin-1 and actin (Figure 5D; Figure 5E shows quantification of multiple cells). Thus, under native conditions, Rho activity promotes the interaction of fascin-1 with actin through a Rho kinase-dependent, myosin II-independent mechanism.
Fascin-1/actin binding is promoted by interaction of fascin-1 with LIM kinases
Having identified from the above experiments that a Rho/Rho kinase/fascin-1 pathway is active in two distinct cell types, our further experiments focused on SW480 cells migrating on LN, for which signaling regulation of fascin-1 has been studied extensively [5, 19]. Because the activity of Rho kinases on fascin-1 is not mediated by myosin-based contractility, we first investigated if fascin-1 might interact with a Rho kinase. SW480 express Rho kinases I and II (see Additional file 2, Figure S2A). However, using FLIM analysis, there was no FRET seen between mRFP-fascin-1S39A or mRFP-fascin-1S39D with either GFP-Rho kinase I or GFP-Rho kinase II. Furthermore, neither Rho kinase I nor Rho kinase II co-immunoprecipitated with either endogenous or overexpressed fascin-1 in SW480 cells, or with purified hexahistidine-tagged fascin-1, and fascin-1 was not a substrate in Rho kinase assays in vitro (data not shown). We conclude that fascin-1 is not a direct binding partner of Rho kinase I or II.
To confirm the novel fascin-1/LIMK1/2 interaction by an independent biochemical method, and to investigate whether the S39 phosphorylation site of fascin-1 has a role in the interaction, hexahistidine (6His)-tagged fascin-1 (wild-type), or 6His-fascin-1 with point mutations of S39 were expressed in Escherichia coli, purified with metal-affinity beads, and matched amounts loaded onto fresh metal-affinity beads. Equal protein loadings of SW480 cell lysate were passed over the beads, and bound candidate proteins were identified by immunoblotting after extensive washing. Relative to a control bead matrix, LIMK1 was found to bind to all three forms of fascin-1, indicating that S39 phosphorylation of fascin-1 does not control this interaction (Figure 6D; Figure 6F shows quantification from multiple cells). However, some enrichment of LIMK1 on fascin-1S39A matrix was detected across multiple experiments (Figure 6F). Active LIMK1/2 bound to both the wild-type and mutant fascin-1 proteins, as detected with an antibody to T508/T505-phosphorylated LIMK1/2 (Figure 6D). Very low levels of LIMK2 were detected in SW480 cells, and LIMK2 binding to 6His-fascin-1 was not detected (Figure 6D). The LIMK1 interaction with fascin-1 was specific, because binding of Rho kinase I or II was not detected (Figure 6D) in agreement with the previous FRET analyses of possible Rho kinase binding to fascin-1. Also in agreement with the FRET data, lysates from cells treated with C3 exotoxin or Y27632 showed reduced binding of LIMK1 to 6His-fascin-1 (Figure 6E). This result again indicates the importance of LIMK1 activation for its binding to fascin-1. The combined FRET and biochemical data show that Rho-dependent regulation of the fascin-1/actin interaction is achieved by activation of LIMK1/2 and an unsuspected direct interaction of fascin-1 with LIMK1/2.
The fascin-1/LIMK1/2 interaction depends on LIMK1/2 activation and modulates filopodia dynamics
Several lines of indirect evidence have linked the formation of fascin-containing cell protrusions with the status of actomyosin contractility or focal adhesions, but the processes involved, in particular the role of Rho GTPase, have remained obscure. In this study, we established, with multiple lines of evidence, that Rho activity modulates the ability of fascin-1 to interact with actin in both normal and carcinoma-derived cells. The discovery of this novel function of Rho was advanced by the development of a novel assay to measure the fascin-1/actin interaction by FRET/FLIM microscopy. In this assay, a small, actin-binding peptide, lifeact, was adopted as the FRET donor. Lifeact binds reversibly to F-actin, and thus FRET with fascin-1 takes place only when both molecules are in close proximity (<10 nm) and bound to filamentous actin. The specificity of the interaction measured was established by: 1) the lack of FRET interaction of lifeact with the non-actin-bundling form of fascin-1, fascin-1S39D; 2) the independence of fascin-1/lifeact FRET from PKC kinase activity, and 3) the correspondence of lifeact distribution and fascin-1/lifeact FRET with a subset of the F-actin structures to which phalloidin binds.
Using the combined approaches of immunofluorescence microscopy, time-lapse imaging of cells expressing GFP-fascin-1, and the novel FRET assay, we found that inhibition of either Rho or its effectors Rho kinase I and II resulted in inhibition of the fascin-1/lifeact interaction as measured by FRET/FLIM. According to the indirect immunofluorescence of endogenous F-actin or fascin-1 and the imaging of live cells expressing GFP-fascin-1, inhibition of either Rho or Rho kinases also altered cell morphology and led to the formation of more protrusions containing fascin-1 at cell edges. When analyzed in detail by confocal time-lapse microscopy, the filopodial activity of Y27632-treated cells was seen to occur within regions of increased membrane ruffling. These filopodia had distinct characteristics: GFP-fascin-1 was not localized strongly along the filopodial shaft; the filopodia were less straight, probably as a result of loss of actin-bundle rigidity caused by decreased localization of fascin-1 towards filopodial tips (Figure 4), and the lifetimes of these filopodia were longer (Figure 4; see Additional file 3, movie 1; see Additional file 4, movie 2; see Additional file 5, movie 3).
The FRET assay studies a specific molecular interaction within the overall assembly/disassembly dynamics of protrusions. Because Rho kinase inhibition of SW480 migration on LN is fascin-1-independent , it is likely that control of protrusion number has a multifactorial basis, for example, it may be linked to both actomyosin-based cell tension and chemical signaling. Inhibition of Rho or Rho kinases is well known to inhibit stress-fiber microfilament bundles within cell bodies [43, 44]; however, in our experiments, many cells treated with these inhibitors displayed increased association of fascin-1 with cell-body microfilament bundles. Fascin-1 and tropomyosin act as antagonists for actin binding, and previous studies of ECM-adherent cells have indicated preferential association of fascin-1 with cell-body microfilament bundles in cells under conditions of reduced focal adhesion assembly and contractility [4, 20, 24]. Treatments with C3 toxin or Y27632 are likely to lead to similar conditions of reduced cell contractility in adherent cells.
By combining the new lifeact FRET assay with biochemical analyses of cell extracts, we have identified Rho-dependent regulation as a novel signaling process that affects both fascin-1 and the fascin-1/actin interaction. Importantly, the mechanistic basis for the pathway downstream of Rho and Rho kinases resides in a previously unidentified process, the interaction of LIMK1/2 with fascin-1. Both of the LIMK isoforms are expressed in many tissues, although specificity of expression patterns and subcellular localizations has been described [37, 45, 46]. In this study, we identified the interaction of fascin-1 with active LIMK1/2 in SW480 cells by biochemical pull-down from cell extracts and by FRET/FLIM in intact, migrating cells. As established by the immunoblots, fascin-1 pull-down experiments, confocal microscopy, and FRET/FLIM analysis for LIMK1, the mechanism of the interaction depends on LIMK1 activation by Rho kinase phosphorylation, but is independent of the status of S39-phosphorylation of fascin-1. Both S39-phosphorylated and non-phosphorylated fascin-1 are needed for efficient migration of SW480 cells , and these new data suggest that the LIMK1/fascin-1 interaction is a possible mechanism to retain S39-phosphorylated fascin-1 in proximity to actin structures at cell edges. We found that fascin-1 localized in filopodia in cells expressing wild-type or T508A mutant forms of LIMK1; however, only wild-type LIMK1 was competent to stabilize filopodia. LIMK1-T508A, which did not interact with fascin-1 in the FRET assay, did not increase filopodial persistence. Thus the LIMK1/fascin-1 interaction contributes to the stability of filopodia. As reported previously [41, 42], expression of kinase-dead LIMK1 reduced in decreased numbers of filopodia, and the remaining filopodia were not stabilized.
Similar to fascin-1, LIMK1 and LIMK2 contribute functionally to cancer cell invasion and metastasis, and are therefore of interest as therapeutic targets [36, 47, 48]. Thus, fascin-1 and LIMK1/2 might co-operate to promote protrusions and cell motility if they are co-expressed in tumors. For example, LIMK1/2 overexpression is well known to increase F-actin, protrusions, and stress fibers in various cells, and correspondingly, LIMK1/2 knockdown or expression of kinase-dead LIMK1/2 decreases F-actin stability, filopodia/lamellipodia, and cell migration [41, 42, 48]. We propose that the cell protrusion phenotypes observed after LIMK1/2 inhibition may, in part, be mediated by reduced actin binding and bundling by fascin-1. Interestingly, LIMK1/2 are also downstream effectors of p21-activated kinases (PAK), which, as we have previously shown regulate formation of the fascin-1/cPKC complex in colon carcinoma cells, and which also correlate clinically with metastatic progression [19, 49]. Thus, LIMK1/2 might represent a nexus between several pathways that regulate the ability of fascin-1 to participate in different protein complexes.
A well-characterized substrate of LIMK1/2 is cofilin, an actin-binding protein that acts to depolymerise F-actin by its actin-severing activity. Phosphorylation of cofilin on ser-3 by LIMK1/2 inhibits its severing activity, resulting in the aforementioned stabilization or reorganization of actin structures [50–52]. Similar to fascin-1, the cofilin pathway has been linked to breast-cancer metastasis . It was recently shown that cofilin associates transiently with retracting filopodia, and that its actin-severing activity is enhanced on actin bundles crosslinked by fascin-1 . The relationship between the fascin-1/LIMK1/2 complex and cofilin in the dynamics of filopodia are questions of future interest. It will be important to establish if fascin-1 competes with cofilin for LIMK1 kinase availability, thereby modulating actin dynamics directly, or whether LIMK1/2 act directly or indirectly as a scaffold to maintain fascin-1 in proximity to F-actin. Alternatively, fascin-1 binding might inhibit the kinase activity of LIMK1/2, thereby affecting actin dynamics indirectly, as previously shown for nischarin . LIMK1 contributes to the transcriptional activity of serum response factor , and it will be interesting to determine if this activity is modulated by fascin-1.
In this study, we have identified a novel activity of the small GTPase Rho in promoting the interaction of fascin-1 with actin in ECM-adherent, migratory cells. Rho, Rho kinases, and LIMK1/2 were identified as key effectors, and activated LIMK1 or LIMK2 act as novel binding partners of fascin-1. These findings have many implications for our understanding of how fascin-1 protein complexes and actin dynamics are coordinated in contractile and protrusive actin-based structures in intact cells, and may be particularly relevant to carcinoma cell migration and metastasis. In the course of the study, we developed a novel method for analysis of the fascin-1/actin interaction by FRET/FLIM. This method has potential general applicability for analyzing the activities of actin-binding proteins in intact cells.
Cell lines and materials
C2C12 mouse skeletal myoblast and SW480 human colon carcinoma cells were from the American Type Culture Collection (ATCC). GFP-fascin-1 and PKCγ-mRFP constructs were prepared as previously described [15, 17]. PKCα-mRFP  was a gift from T. Ng, and the GFP-tagged forms of LIMK1, LIMK2 , ROCKI, and ROCKII were gifts from G. Jones (both King's College London, London, UK). GFP-caldesmon and the deletion mutant GFP-caldesmon-445  were a gift from Alexander Bershadsky (Weizman Institute of Science). The Ras-binding domain-glutathione S-transferase (RBD-GST) plasmid was the gift of M. Schwartz (Yale University) . GFP-lifeact  was a gift from R. Wedlich-Soldner (MPI, Munich, Germany).
Primer and oligonucleotide pairs used for PCR, cloning and site-directed mutagenesis
Subclonings into pcDNA3.mRFP(N)
Subcloning into pCR-Blunt
For bacterial expression of 6His-tagged fascin-1, cDNAs encoding wild-type, S39A, or S39D human fascin-1 from the pEGFP-fascin-1 plasmids were cloned between the NotI and XhoI restriction sites of pET-30a(+) (Novagen). The reading frame was adjusted by subsequent digestion and blunting of the NotI site. BIM, Y27632, ML7, BDM and blebbistatin were obtained from Calbiochem (San Diego, CA, USA). C3 endotoxin and RhoA G-LISA kit were from Cytoskeleton Inc (). Antibodies used included mouse monoclonal antibody to fascin-1 (clone 55k-2; Dako, Glostrop, Denmark); to LIMK1, LIMK2 and phosphoLIMK1/2 (Cell Signaling Technology, Beverly, MA, USA) and to Rho, ROCK I and II (BD Transduction Labs).
Cell-extracellular matrix adhesion and Immunofluorescence
C2C12 cells were maintained in DMEM containing 20% fetal calf serum (FCS), and SW480 cells were maintained in DMEM with 10% FCS. For transient transfections, cells were plated at 30% to 40% confluency, and transfected with transfection reagents (Polyfect (Qiagen Inc., Valencia, CA, USA or Lipofectamine 2000 (Invitrogen Corp.)) in accordance with the manufacturer's instructions. At 48 hours post-transfection, cells were re-plated as single cell suspensions onto glass surfaces coated with 50 nmol/L FN (Calbiochem) or Engelbreth-Holm-Swarm (EHS) laminin (Sigma-Aldrich, St Louis, MO, USA) under serum-free conditions, as described previously [5, 18]. In experiments involving pharmacological inhibitors, cells were pretreated with the agent for 30 minutes prior to the adhesion assays being set up, and the agent was maintained in the medium throughout the assay. The concentrations of inhibitors to be used were established in pilot experiments, and the lowest concentrations that affected cell morphology and actin organization reproducibly without evidence of cytotoxicity, as determined by Trypan blue exclusion, were chosen for the experiments. After 1 (C2C12) or 2 (SW480) hours at 37°C to allow adhesion and initiation of random cell migration, non-adherent cells were removed by rinsing in Tris-buffered saline (TBS) and adherent cells were fixed in 2% paraformaldehyde (EMS) and permeabilized in TBS with 0.5% Triton-X100 for staining with tetramethyl rhodamine isothiocyanate-phalloidin (Sigma), phalloidin-633, or antibodies to vinculin (clone h-VIN1; Sigma) or phosphotyrosine (clone 4G10; Millipore Corp., Billerica, MA, USA). For fascin-1 staining, cells were fixed in absolute methanol, and for LIMK1/2 staining, they were fixed in 4% paraformaldehyde. Digital images were taken at room temperature under the ×63 objective of a laser scanning confocal microscope using confocal acquisition software (Leica, Heerbrugg, Switzerland). Morphometric analysis of adherent C2C12 cells was carried out by measuring cell areas and the numbers and lengths of individual peripheral fascin or F-actin bundles from calibrated digital images using the software Improvision Openlab (Perkin Elmer, Waltham, MA, USA).
Fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy
FLIM was performed cells transfected with specified constructs, with fixation and data analyzed as described previously . Details of time-domain FLIM performed with a multiphoton microscope system have been described previously . FLIM capability was provided by time-correlated single-photon-counting electronics (SPC 700; Becker & Hickl, Berlin, Germany). Widefield acceptor (mRFP) images were acquired using a charge-coupled device camera (Hamamatsu Photonics, Hamamatsu, Japan) at exposure times of <100 ms. Data were analyzed using TRI2 software (developed by Dr P. Barber). All histogram data were plotted as mean FRET efficiency from >10 cells per sample. Lifetime images of exemplary cells are presented using a pseudocolor scale, with blue depicting normal GFP lifetime (that is, no FRET) and red depicting reduced GFP lifetime (areas of FRET). Each experiment was repeated at least three times.
Live cell imaging
SW480 cells were transfected with GFP-fascin-1, and 24 hours later, were plated onto glass-bottomed imaging chambers (LabTek; Nunc) coated with 15 nmol/l laminin for 6 hours before treatment with DMSO (solvent control) or 10 μmol/l Y27632 for 30 minutes. Live confocal microscopy imaging was performed on a microscope (A1R; Nikon, Tokyo, Japan) equipped with a 40× oil objective lens (CFI Plan Fluor; Nikon) at 37°C. Images were captured and exported with NIS Elements software (Nikon). Movies were acquired as one image per 2 seconds over 60 to 150 frames, and are presented at playback rates of 15 frames/second. Morphometric analyses of filopodia formation in SW480 cells were performed by measuring the number and length of GFP-tagged or RFP-tagged fascin-positive filopodia, and by line-scan analysis of fluorescence intensity along the length of single, randomly selected filopodia. To analyze the dynamics of an individual filopodium, kymographs of 2 × 10 μm areas encompassing one filopodium were selected, and the montage was performed in the X-axis. The mRFP-fascin-1 signal was thresholded, the front of the filopodium outlined, and the resulting coordinates used to calculate several dynamic values including maximum filopodia displacement. All image analyses were performed with ImageJ software (http://rsb.info.nih.gov/ij/download.html). Each experimental condition was tested in at least three independent experiments.
Immunoblotting and fascin-1 pull-downs
Adherent cells for immunoblotting were extracted on ice in 1% Triton X-100 in TBS containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) for 12 minutes. SDS-PAGE and immunoblotting with selected antibodies and enhanced chemiluminescence (ECL) detection was carried out as described previously . Recombinant 6His-tagged fascin-1 was expressed in BL21 E. coli by induction with 1 mmol/l isopropyl β-D-1-hiogalactopyranoside (IPTG; Sigma-Aldrich) for 5 hours at 25°C. Bacterial pellets were resuspended in buffer(300 mmol/l NaCl, 10 mmol/l imidazole and 50 mmol/l Na2HPO4 pH 8), then sonicated and clarified. Lysates were passed over nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen Inc.) to allow fascin-1 binding, beads were washed (300 mmol/l NaCl, 25 mmol/l imidazole and 50 mmol/l Na2HPO4 pH 8), and fascin-1 was eluted (300 mmol/l NaCl, 250 mmol/l imidazole in 50 mmol/l Na2HPO4 (pH 8), and dialyzed against buffer (150 mmol/l NaCl, 10 mmol/l imidazole and 50 mmol/l Na2HPO4, pH 8). For pull-down experiments, the wild-type or mutant fascin-1 proteins were immobilized on Ni-NTA beads in disposable polystyrene chromatography columns (Thermo Fisher Scientific Inc., Rockford, IL, USA ). SW480 cell lysates were extracted (1% Triton, 150 mmol/l NaCl, 2 mmol/l MgCl2 and 10 mmol/l imidazole pH 7.5), then passed through the columns, washed extensively (1% Triton, 150 mmol/l NaCl, 2 mmol/l MgCl2 and 25 mmol/l imidazole, pH 7.5), and the bound proteins were eluted (1% Triton, 150 mmol/l NaCl, 2 mmol/l MgCl2 and 250 mmol/l imidazole pH 7.5). The eluted proteins were mixed with SDS-sample buffer and subjected to SDS-PAGE and immunoblotting as described previously .
Rho activity assays
Rho-GTP pull-down assays were carried out on extracts from C2C12 cells adherent on 30 nmol/l FN for 1 hour with a GST fusion protein containing the effector domain of rhotekin (RBD-GST) as described previously . All extracts contained equivalent total Rho protein, as measured by immunoblotting with anti-Rho (Transduction Labs) and ECL detection. Rho-GTP levels were compared with Rho status in C2C12 cells adherent on 30 mmol/l thrombospondin-1 for 1 hour or suspended for 90 minutess over BSA-blocked dishes. RhoA-GTP levels were also quantified with a G-LISA kit used in accordance with manufacturer's instructions (Cytoskeleton Inc). Lysates were prepared from SW480 cells plated on 15 nmol/l laminin for 2 hours, either after 4 hours of treatment with DMSO (control) or 1 μmol/l C3, or 30 minutes of pre-treatment with 1 μmol/l BIM.
Data were analyzed by descriptive statistics. P values were calculated using a two-tailed Student's t-test for unpaired samples Triplicate samples were scored in each experiment, and three to four independent experiments were carried out for each experimental condition. ANOVA was used to test statistical significance between different populations of data. Statistical analysis was performed with normalized data by one-way ANOVA and subsequent multiple range test between conditions. P < 0.05 was considered significant.
List of abbreviations
Bovine serum albumin
fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy
green fluorescent protein
p-Lin-11/Isl-1/Mec-3 (LIM) kinase
myosin light chain kinase
monomeric red fluorescent protein
protein kinase C
- Rho kinase:
Rho-associated coiled-coil-forming kinase
We thank all the colleagues who contributed gifts of plasmids used in these experiments and Martin Schwartz for advice on the Rho-GTP pull-down assays. JCA thanks the NIH and AICR for financial support. MP is funded by the Royal Society (University Research Fellowship) and Medical Research Council MR/J000647/1 and AJ by the Basque Government, Spain (Research Staff Training Fellowship) and MR/J0000647/1.
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