Article |
Address correspondence to Pamela J. Woodring, The Salk Institute, 10010 North Torrey Pines Rd., La Jolla, CA 92037-1099. Tel.: (858) 453-4100. Fax: (858) 457-4765. E-mail: woodring{at}salk.edu
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Abstract |
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Key Words: Abl-/- Arg-/- fibroblasts; latrunculin; STI571; fibronectin cell spreading; F-actin microspikes
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Introduction |
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The c-Abl gene encodes a nonreceptor tyrosine kinase that contains a binding site for F-actin (McWhirter and Wang, 1993; Van Etten et al., 1994; Woodring et al., 2001). Cells deficient for c-Abl show a reduced and delayed ruffling response to PDGF (Plattner et al., 1999). Inhibition of c-Abl kinase activity with signal transduction inhibitor 571 (STI571) can increase cell migration measured in Boyden chamber assays (Frasca et al., 2001; Kain and Klemke, 2001). The abl gene is conserved in invertebrates, and the Drosophila Abl (dAbl) functions in axon guidance (Lanier and Gertler, 2000). The c-Abl tyrosine kinase can phosphorylate proteins such as Mena, paxillin, SH3-SH2 containing adaptor proteins (e.g., Crk, p130Cas, disabled), and Cdk5 that are involved in the regulation of focal adhesions and F-actin structure and dynamics (Van Etten, 1999). c-Abl can also associate with WAVE1 (Westphal et al., 2000), an activator of the Arp2/3 complex. Furthermore, some extracellular signals that activate c-Abl also cause alterations in the F-actin cytoskeleton. For example, clustering of integrins by ECM proteins activates c-Abl (Lewis et al., 1996; Lewis and Schwartz, 1998; Woodring et al., 2001) and promotes cell attachment and spreading. Conversely, c-Abl mutated in the F-actin binding domain (FABD) is active independent of integrins. Indeed, F-actin itself is an inhibitor of the c-Abl activity in vitro (Woodring et al., 2001). Altogether, these studies point toward a reciprocal regulation between c-Abl and F-actin in which c-Abl regulates and is regulated by the F-actin cytoskeleton.
We report here that F-actin surface protrusions are stimulated by c-Abl tyrosine kinase in spreading fibroblasts and along growing axons. In both systems, increased c-Abl activity correlates with increased quantities of F-actin microspike protrusions. The positive effect of c-Abl on the formation of F-actin microspikes may be balanced by the negative effect of F-actin on c-Abl kinase activity to limit the extent and duration of these F-actin protrusions. Thus, our results suggest a self-limiting mechanism where F-actin and c-Abl exert rapid effects on each other to regulate the dynamics of cell morphology and motility.
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Results |
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In vivo association of c-Abl with F-actin is affected by cell adhesion
We have shown previously that F-actin binds to and inhibits the c-Abl tyrosine kinase. Moreover, deletion of the F-actin binding domain of c-Abl increases kinase activity in detached cells (Woodring et al., 2001). These results would predict that c-Abl is associated with F-actin in detached cells. Indeed, we observed a time-dependent increase in the amount of actin found in anti-Abl immunoprecipitates as cells were held in suspension (Fig. 2 A). After 40 min in suspension, the amount of actin in the anti-Abl immunoprecipitates was 2.7 ± 0.7-fold higher than that at 5 min after detachment (Fig. 2 A, lanes 3 and 5). The increased actin in the anti-Abl precipitates corresponded with a decrease in c-Abl kinase activity (Fig. 2 B). Coimmunoprecipitation of actin and c-Abl was also observed to a limited extent in attached cells. However, a majority of this coprecipitation was also observed in anti-Abl immunoprecipitates prepared from Abl-/- cells, suggesting a nonspecific trapping of actin (Fig. 2 C, lanes 1 and 2).
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To further explore the role of c-Abl in neurite branching and to address the specificity of STI571 for c-Abl, we cultured dissociated cortical neurons from E17 mouse embryos which were abl+/+, abl+/- and abl-/- for Abl expression (see Materials and methods). We observed a twofold reduction in the number of F-actin branches on the neurites on cells from Abl-/- embryos (Table I). STI571 had no to little affect on the already low number of branches on Abl-/- mouse embryo neurons, whereas it significantly decreased branching of Abl+/- and Abl+/+ mouse embryo neurons. Consistently, as the dosage of Abl decreased, we observed significant reduction in neurite branching (Table I, compare control-treated neurons for each genotype). These data strongly imply a role for c-Abl in processes that involve dynamic changes in the F-actin cytoskeleton of neurons.
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Discussion |
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Regulation of the F-actin cytoskeleton by c-Abl
Our data, together with those from other laboratories, imply a role for c-Abl in the dynamic organization of the F-actin cytoskeleton. With multiple approaches, we have obtained direct evidence that c-Abl kinase activity positively regulates the F-actin cytoskeleton. Using a small molecule inhibitor for c-Abl kinase, we demonstrated that inhibition of endogenous c-Abl activity not only blocked formation of F-actin microspikes in spreading fibroblasts, but also reduced F-actin branching in embryonic cortical neurons. Conversely, expression of activated c-Abl increased the quantity of microspikes in fibroblasts and increased branching of neurons. Moreover, neurons derived from mouse embryos lacking c-Abl exhibited decreased branching relative to littermate controls.
The stimulatory effect of c-Abl on branching of murine and rat embryonic cortical neurons reported in this study is consistent with the proposed positive regulatory role of dAbl in growth cone dynamics. dAbl is involved in axon outgrowth/guidance (Gertler et al., 1989; Henkemeyer et al., 1990; Gertler et al., 1995; Comer et al., 1998; Wills et al., 1999a; Bashaw et al., 2000; Bear et al., 2001) and specific motor neurons in dAbl-/- Drosophila undergo growth cone arrest similar to the phenotype observed in profilin-/- Drosophila (Wills et al., 1999b). Genetic evidence has established a connection between dAbl and molecules known to regulate F-actin dynamics of neurons. This is supported by the dosage-sensitive effects between dAbl and profilin (Wills et al., 1999b), trio (Rac/Rho GEF) (Liebl et al., 2000), disabled (dab) (Gertler et al., 1989), failed axon connections (fax) (Hill et al., 1995), enabled (ena) (Gertler et al., 1990, 1995; Comer et al., 1998; Wills et al., 1999a; Bashaw et al., 2000) and Dlar (Wills et al., 1999a). Murine c-Abl has also been implicated in the regulation axon outgrowth. Overexpression of activated c-Abl can stimulate neurite outgrowth (Zukerberg et al., 2000), whereas Abl-/-Arg-/- mouse embryos die at E11 due to defects in neurulation associated with gross actin cytoskeletal abnormalities in the neuroepithelium (Koleske et al., 1998). In addition, murine disabled (mDAB) is essential in the layering of cortical neurons (Howell et al., 1999) and the murine enabled (Mena) is essential for proper axon guidance during development (Gertler et al., 1996; Lanier et al., 1999). Further evidence that murine c-Abl is involved in processes that require regulated F-actin dynamics has been reported in fibroblasts and epithelial cells where c-Abl can affect membrane ruffling (Plattner et al., 1999) and cell motility (Frasca et al., 2001; Kain and Klemke, 2001). Here, we provide evidence linking c-Abl kinase activity directly to the formation of peripheral F-actin microspikes. F-actin microspikes may be stabilized through F-actin bundling to form filopodia, highly dynamic structures with exploratory/sensory function, which are precursors to dendritic spines (Kozma et al., 1995). Indeed, c-Abl activity correlated with increased F-actin microspikes in processes that involve exploring and sensing environmental cues: fibroblasts spreading onto fibronectin and in neurons extending neurites.
Consistent with our observation that cortical neurons grown in the presence of STI571 decreased overall neurite length by 20%, overexpression of
SH3Abl caused extension of neurites on neurons transfected after 2 d in culture (Zukerberg et al., 2000). In addition to this effect, we have found that overexpression of
F-actin c-Abl and Abl-PP caused increased branching in our analysis in which neurons were transfected immediately following attachment. The increased branching was revealed by phalloidin staining of fixed cortical neurons. Neurofilament staining did not readily detect these short F-actincontaining branches (Fig. 8 B). In live neurons, GFP-actin has been used to detect actin-rich filopodia and dendritic spines, extremely small neuronal branchlike structures along neurites that form postsynaptic contact sites (Fischer et al., 1998; Colicos et al., 2001). The actin in filopodia and dendritic spines is highly dynamic and drives rapid and continuous changes in cytoskeletal morphology (Fischer et al., 1998; Colicos et al., 2001), which are essential for neuronal pathfinding and synaptogenesis. Furthermore, the stimulatory effect of c-Abl on branch formation in neurons is consistent with a role for c-Abl in regulating growth cone dynamics, as sites of branch formation along axon shafts exhibit many of the dynamic properties of growth cones (Sato et al., 1994; Bastmeyer and O'Leary, 1996; Szebenyi et al., 1998), including a requirement for the rapid regulation of F-actin (Kalil et al., 2000).
Since c-Abl kinase activity is essential for increased F-actin microspike formation, it is likely that a substrate of c-Abl is also essential. It is noteworthy that a number of Abl substrates or Abl-binding proteins, including Crk (Nakashima et al., 1999; Escalante et al., 2000; Kain and Klemke, 2001), p130Cas (Mayer et al., 1995; Klemke et al., 1998; O'Neill et al., 2000), paxillin (Nakamura et al., 2000; Turner, 2000), Abl interactor proteins (Abi) (Stradal et al., 2001), enabled (Wills et al., 1999a; Bashaw et al., 2000; Bear et al., 2000), LAR phosphatase (Wills et al., 1999a), Scar/WAVE (Westphal et al., 2000), p190 RhoGAP (Brouns et al., 2001), Cdk5, and cables (Zukerberg et al., 2000), have been implicated in pathways which involve rearrangement of the F-actin cytoskeleton (Lanier and Gertler, 2000). For example, like c-Abl, Mena negatively regulates fibroblast motility and potentiates outgrowth of actin-rich structures in cultured murine fibroblasts (Bear et al., 2000). Additionally, PDGF-stimulated membrane ruffling requires active c-Abl (Plattner et al., 1999) and causes c-Abl to colocalize with Scar/WAVE at the ruffling cell membrane (Westphal et al., 2000).
Interestingly and unexpectedly, the effect of c-Abl on microspike formation can occur in the presence of Toxin B and dominant negative RhoA, Rac1, or Cdc42. Although these results do not rule out the possibility that c-Abl may affect F-actin morphology through GTPase-dependent mechanisms, it is noteworthy that GTPase-independent mechanisms for modulating cytoskeleton have been reported. For example, vaccinia virus stimulates actin motility in a Cdc42-independent manner (Moreau et al., 2000) and actin capping or severing proteins do not require GTPases to regulate polymerized actin (Pollard et al., 2000). Also, cortactin and WASp/Scar/WAVE proteins can stimulate the nucleating activity of Arp2/3 complex in vitro in the absence of GTPases (Yarar et al., 1999; Higgs and Pollard, 2001; Uruno et al., 2001; Weed and Parsons, 2001). Altogether, these observations suggest that c-Abl may affect multiple targets that can regulate the F-actin cytoskeleton and that the downstream targets of c-Abl may be dependent on the cell context.
Reciprocal regulation between F-actin and c-Abl tyrosine kinase
Current evidence supports a role for c-Abl in physiological responses that entail the organization and reorganization of the actin cytoskeleton. Modulation of F-actin dynamics is crucial in physiological processes such as neurulation, axonal pathfinding, branching, membrane ruffling, cell spreading, and cell migration, all of which have been shown to involve c-Abl. We have found that c-Abl can stimulate F-actin microspike formation through its kinase activity, and interestingly, F-actin can in turn inhibit the kinase activity of c-Abl. This reciprocal regulation between F-actin and c-Abl provides a dynamic mechanism for modulating the F-actin structure. Signals that override the inhibitory effect of F-actin on c-Abl can stimulate the formation of microspikes. The increase in the local concentration of F-actin might then inhibit the c-Abl kinase and thus limit the life span of these Abl-dependent F-actin protrusions. Understanding how extracellular signals modulate the reciprocal regulation between F-actin and c-Abl will provide important insights on the regulation of F-actin dynamics.
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Materials and methods |
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Cell culture
Stable cell lines.
The stable Abl-deficient fibroblast cell line Abl-/- was obtained from Dr. Rubio Ren (Brandeis University, Waltham, MA) and the stable Abl-/-Arg-/- (double knock out, DKO) cell line from Dr. Anthony Koleske (Yale University, New Haven, CT). The stable Src-deficient cell line Src-/- and Src reexpressing cells were obtained from Dr. Martin A. Broome (SUGEN, San Francisco, CA). Cells were maintained in high glucose DME supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics. The DKO cell lines were plated 20 h before experiments in complete media without antibiotics. For detachment, growing cells were trypsinized at 75% confluency and held in suspension in DME supplemented with 1 mg/ml BSA and 0.5 mg/ml soybean trypsin inhibitor. For reattachment, suspension cells were replated onto 10 µg/ml fibronectin. For Latrunculin A (Molecular Probes) pretreatment, growth media was replaced with serum-free media containing 1 µM latrunculin for a 2 h before cell detachment. Control cells were incubated under the same conditions except with DMSO (the vehicle for the STI571). After detachment, cells were held in suspension in the same media for an additional 40 min. STI571 was added to a final concentration of 5 µM for fibroblasts or 13 µM for cortical neuronal cells. For fibroblasts, an 8-h pretreatment was used, and for cortical cells, STI571 was continually present in growth media from time of cell plating. Control cells were incubated with DMSO. For Toxin B pretreatment, growth media was replaced with serum-free media containing either vehicle or 4 ng/ml Toxin B (Calbiochem) for 8 h. Cells were detached and replated as described above except in the continuous presence of Toxin B.
Cortical neuronal cell culture.
The cerebral cortices from E18 rats or E17 mice were dissected in L-15. Tissue was dissociated by treatment for 20 min at 27°C with 1% papain (Worthington) in HBSS (GIBCO BRL) containing 0.45% glucose, 0.2 mg/ml BSA, 0.2 mg/ml L-cysteine, 0.005% DNase I, 0.01 mg/ml APV, and 0.2 mg/ml kynurenic acid. Tissue was then washed three times in HBSS containing 0.1 mg/ml trypsin inhibitor, 0.45% glucose, 0.2 mg/ml BSA, 0.01 mg/ml APV, and 0.2 mg/ml kynurenic acid. Tissue was rinsed one time in growth medium (Neurobasal, 4% B-27, 0.5 mM glutamine, 50 U-µg/ml penicillin-streptomycin) containing 0.2 mg/ml BSA, 0.01 mg/ml APV, and 0.2 mg/ml kynurenic acid, and then triturated 20 times in 1 ml of the same medium using a fire-polished glass pipet. Dissociated cells were counted using trypan blue. Cells were plated on laminin-coated coverslips in growth medium at a density of 40,000 cells/cm2 for transfections, and 4,000 cells/cm2 for all other experiments. Transient transfections of neurons were performed with Lipofectamine 2000 reagent (GIBCO BRL) according to the manufacturer's protocol.
Biochemistry
Fibroblast experiments involving cell detachment, fibronectin stimulation, immunoprecipitation, and kinase assay were performed as described previously (Woodring et al., 2001). Antibodies used for immunoprecipitation and immunoblotting are indicated in figure legends.
Immunofluorescence staining and cell spreading
Tissue culture dishes containing glass coverslips were coated with 10 µg/ml purified fibronectin or 1 mg/ml poly-L-lysine as described previously (Schlaepfer et al., 1997). Confluent cells were detached by trypsinization and held in suspension for 40 min to inactivate c-Abl. For adherent cells, cells were plated onto washed fibronectin-coated coverslips and incubated at 37°C for the indicated times. To stop cell spreading immediately or to fix detached cells and neurons formaldehyde was added directly to the media to a final concentration of 3.7%. Fixed cells were washed gently with PBS/3.7% formaldehyde and then prepared for immunofluorescence staining as described previously (Lewis et al., 1996). Detached cells were applied to a charged surface either by allowing cells to settle to the surface or by gentle centrifugation using cryospin. FITC- or TRITC-conjugated phalloidin was used to detect F-actin; anti-8E9 and Texas red or Cy-2conjugated goat antimouse were used to detect c-Abl; and Hoechst 33258 was used to detect nuclei. Microscopy was performed using the 60x objective on an Olympus microscope unless indicated otherwise. DeltaVision (Applied Precision software) was used to deconvolve z-series images and also to measure neurite length and assess neuronal branching. For cell-spreading analysis we scored cells as either spreading with rounded or sharp cell perimeters. Antivinculin antibodies were from Sigma-Aldrich, and anti-Src monoclonal antibodies (clone 327.5) were a gift from Suzanne Simon (Salk Institute).
Online supplemental material
A supplemental figure is available at http://www.jcb.org/cgi/content/full/jcb.200110014/DC1. To examine the localization of c-Abl with the F-actin cytoskeleton in detached cells at various optical planes along the vertical z-axis, cells were stained as described in Fig. 3 B. Top, individual cells visualized with deconvolution microscopy. Bottom, field of cells visualized with confocal microscopy.
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Footnotes |
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* Abbreviations used in this paper: CTD, COOH-terminal repeat domain; DKO, double knock out; ECM, extracellular matrix; FABD, F-actin binding domain; GST, glutathione S-transferase; STI571, signal transduction inhibitor 571.
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Acknowledgments |
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This work was supported by a postdoctoral fellowship from the National Cancer Institute (NIH CA76710 to P.J. Woodring) and National Institutes of Health grants HL57900 (to J.Y.J. Wang), CA82863 (to T. Hunter), and NS31558 (to D. O'Leary). J.Y.J. Wang is the Herbert Stern Endowed Chair of Biology, University of California at San Diego. T. Hunter is a Frank and Else Schilling American Cancer Society Research Professor.
Submitted: 2 October 2001
Revised: 22 January 2002
Accepted: 22 January 2002
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References |
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