Correspondence to Kensaku Mizuno: kmizuno{at}biology.tohoku.ac.jp
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Abbreviations used in this paper: LIMK, LIM kinase; P-cofilin, Ser-3phosphorylated cofilin; PI3K, phosphatidylinositol-3 kinase; SDF-1
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Introduction |
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Cofilin is a potent regulator of actin filament dynamics and mediates the rapid turnover of actin filaments by severing actin filaments and by stimulating actin filament disassembly near the pointed ends (Moon and Drubin, 1995; Theriot, 1997; Bamburg, 1999; Pantaloni et al., 2001; Bamburg and Wiggan, 2002; Hotulainen et al., 2005). Cofilin appears to play a key role in maintaining and extending the lamellipodial protrusion at the leading edge of migrating cells (Bailly and Jones, 2003; Dawe et al., 2003). Cofilin is inactivated by LIM kinase (LIMK) or related testicular kinase (TESK) through the phosphorylation of Ser-3 (Arber et al., 1998; Yang et al., 1998; Toshima et al., 2001). This phosphorylation inhibits the interactions of cofilin with actin filaments and actin monomers. The overexpression of LIMK1, which inactivates cellular cofilin, impairs the formation of polarized cell morphology and directional cell migration, which suggests that cofilin plays a crucial role in cell polarity formation and directional cell migration (Dawe et al., 2003). Notably, although LIMK1 inhibits cofilin, it is activated in Jurkat T cells in response to stromal cellderived factor-1 (SDF-1
), which is a member of the Cys-X-Cystype chemokine family, and the inhibition of LIMK1 activity by a cell-permeable cofilin-derived peptide blocks SDF-1
induced chemotaxis of Jurkat cells (Nishita et al., 2002). These observations suggest that LIMK1 is required for chemotaxis, but the mechanism involved remains unclear.
The inactive Ser-3phosphorylated cofilin (P-cofilin) is dephosphorylated and reactivated by Slingshot (SSH) family cofilin phosphatases, including SSH1L, -2L, and -3L (Niwa et al., 2002; Ohta et al., 2003) or chronophin, which is a member of haloacid dehalogenases (Gohla et al., 2005). We have shown that SSH1L is highly activated by its association with F-actin in cell-free assays and that it accumulates in the F-actinrich lamellipodium after stimulation of carcinoma cells with neuregulin or insulin (Nagata-Ohashi et al., 2004; Nishita et al., 2004). This suggests that SSH1L is locally activated in the lamellipodium in response to cell stimulation and may be involved in the spatial regulation of cofilin activity. However, it remains unclear whether F-actinmediated SSH1L activation is required for directional cell migration.
In this study, we investigated the role cofilin phosphoregulation plays in the chemotactic migration of Jurkat cells in response to SDF-1. We show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in Jurkat T cells that are stimulated with SDF-1
. Knocking down LIMK1, SSH1L, or cofilin expression by small interfering RNA (siRNA) suppresses chemokine-induced polarized F-actin assembly and chemotactic responses of Jurkat cells. We provide evidence that LIMK1 is required for cell migration by stimulating lamellipodium formation in the early stages of cell response. Furthermore, SSH1L is critically involved in directional cell migration by restricting the membrane protrusion to one direction in the early stages of cell response and then locally stimulating cofilin activity in the lamellipodium at the leading edge of the migrating cell. In addition, we identified Trp-458 as a critical residue for SSH1L to be activated by F-actin. By using an F-actininsensitive SSH1L mutant in which Trp-458 is mutated, we show that F-actinmediated SSH1L activation is required for the chemotactic response of Jurkat cells. Therefore, we propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity plays a critical role in chemokine-induced polarized cell migration.
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Results |
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Trp-458 is critical for the F-actinmediated activation of SSH1L
As SSH1L is highly activated by its association with F-actin (Nagata-Ohashi et al., 2004), we hypothesized that the F-actinmediated activation of SSH1L is critical for polarized cell migration. To identify the sites of SSH1L that are required for F-actinmediated activation, a series of COOH-terminally deleted mutants of SSH1L were constructed. The truncated proteins were subjected to in vitro cofilin phosphatase assays in the absence or presence of F-actin by using P-cofilin as a substrate (Fig. 7 A). As previously reported (Nagata-Ohashi et al., 2004), wild-type (WT) SSH1L was activated by F-actin, but the COOH-terminally deleted mutant NP (composed of amino acid residues 1456) was not. Unexpectedly, the other COOH-terminally deleted mutants (N960, N698, N486, and N461) were all activated by F-actin. This indicates that the region corresponding to amino acids 457461 is required for the F-actinmediated activation of SSH1L. To further define the residues that are essential for activation, various point mutants of N461 were constructed and subjected to cofilin phosphatase assays with or without F-actin (Fig. 7 B). The assays revealed that W458A (Trp-458 replaced by Ala) and 3A (Leu-457, Trp-458, and Arg-459 replaced by Ala) mutants of N461 were only weakly activated in the presence of F-actin, whereas other point mutants were remarkably activated by F-actin to a level similar to that of WT N461. Furthermore, the full-length SSH1L(W458A) mutant was poorly activated by F-actin (Fig. 7 B). Thus, Trp-458 is a critical residue for SSH1L activation by F-actin. Kinetic analysis revealed that although WT SSH1L was 10-fold activated by F-actin, SSH1L(W458A) was only approximately twofold activated by F-actin (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). However, SSH1L(W458A) retained the basal cofilin phosphatase activity (activity in the absence of F-actin) to a level similar to that of WT SSH1L (Fig. S2 B), which indicates that Trp-458 is specifically involved in the process of F-actinmediated activation.
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Discussion |
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The mechanism that induces the conversion of multiple protrusions to the single lamellipodium to form polarized F-actin assembly during 15 min after cell stimulation is not well understood. It is also not known whether the formation of multiple protrusions is the generally occurring event in the first stage of cell stimulation regardless of the conditions of uniform or gradient concentrations of SDF-1. SSH1L siRNA cells, in marked contrast to control cells, retained multiple protrusions for up to 20 min after SDF-1
stimulation, which strongly suggests that SSH1L is required for the formation of the single lamellipodium and polarized cell morphology. In SSH1L knockdown cells, only LIMK1 is activated after SDF-1
stimulation, and, as a result, cofilin-phosphorylating activity dominates cofilin phosphatase activity throughout the cell. This leads to the less dynamic nature of the lamellipodia and the failure of actin filament remodeling and holds on multiple membrane protrusions around the cell. This probably causes the loss of directionality in the migration of SSH1L siRNA cells. Consistent with this observation, the overexpression of LIMK1 induced multiple protrusions in random directions and impaired the formation of polarized morphology and directional migration of fibroblasts (Dawe et al., 2003).
On the other hand, phosphatidylinositol-3 kinase (PI3K) and its lipid products are known to be essential for the formation of polarized membrane protrusion in the chemotactic responses of neutrophils and Dictyostelium discoideum cells (Rickert et al., 2000; Merlot and Firtel, 2003; Van Haastert and Devreotes, 2004). Once one of the protrusions is selected stochastically or dependently on the SDF-1 gradient, the amplification mechanism by a positive feedback loop between PI3K, Rac, and F-actin (Wang et al., 2002; Weiner et al., 2002) may induce polarized lamellipodium formation in one direction. As LIMK1 siRNA cells continuously exhibit faint and multiple protrusions for 120 min after cell stimulation, LIMK1 (as one of the effectors of Rac) may also play a role in the conversion of multiple protrusions to a single lamellipodium. Thus, both excessive (as seen in SSH1L siRNA cells) and insufficient cofilin phosphorylation (as seen in LIMK1 siRNA cells) result in the sustained formation of multiple protrusions in random directions and the loss of cell polarity, although the protrusions are significantly weak in the latter case. It is likely that the precise control of cofilin phosphorylation and dephosphorylation activities is required for polarized F-actin assembly. In addition, the inability of the W458A or NP mutant of SSH1L to rescue the phenotypes of SSH1L siRNA cells suggests that SSH1L translocation to the lamellipodium and its activation by F-actin are essential to the construction of polarized cell morphology and directional cell migration.
In the later stages of the cell response (after polarity is formed), both LIMK1 and SSH1L are activated. As LIMK1 is diffusely distributed in the cytoplasm and SSH1L is recruited to the lamellipodium, it appears that both LIMK1 and SSH1L are active in the front, but only LIMK1 is active in the rear of the cell (Fig. 9). This spatially distinct distribution of LIMK1 and SSH1L could restrict cofilin activity to the front of migrating cells, and the local activation of cofilin in the front can stimulate actin filament turnover for lamellipodium extension. Previous studies have shown that the temporal and spatial regulation of PI3K activity and its antagonistic lipid phosphatase PTEN generates the polarized accumulation of PI(3,4,5)P3 in the anterior region of migrating cells (Funamoto et al., 2002; Iijima and Devreotes, 2002), and a local excitationglobal inhibition model was proposed to explain the polarity formation and maintenance in the chemotactic response (Devreotes and Janetopoulos, 2003). A similar mechanism, which consisted of the local excitation of cofilin by F-actinassociated SSH1L and its global inhibition by diffusely distributed LIMK1, might be responsible for the local activation of cofilin in the leading edge of the cell. The activation of both LIMK1 and SSH1L in the lamellipodium could cooperatively accelerate the recycling of cofilin and actin via the LIMK1-stimulated dissociation of cofilinactin complexes that are released from the pointed ends of actin filaments (Rosenblatt and Mitchison, 1998). Alternatively, SSH1L might down-regulate LIMK1 activity by dephosphorylation, as reported by Soosairajah et al. (2005), and, thereby, further enhance the cofilin dephosphorylation/activation in the front of the cell.
Although both cofilin and SSH1L siRNA cells protrude multiple lamellipodia, cofilin siRNA cells almost completely lose cell migration activity, whereas SSH1L siRNA cells are competent for cell migration but only lose directionality. Why do these cells exhibit such distinct phenotypes in migration? In time-lapse analyses in Fig. 6 A and Videos 5 and 7, cofilin siRNA appears to depress the motility of lamellipodia, whereas SSH1L siRNA cells retain the dynamic nature of protrusions. Thus, it is likely that actin filament dynamics differ between cofilin and SSH1L knockdown cells. In contrast to cofilin siRNA cells, where cofilin expression is directly suppressed, SSH1L siRNA cells may retain some populations of cofilin in active state by global dephosphorylation of P-cofilin by other cofilin phosphatases such as SSH2, SSH3, or chronophin (Niwa et al., 2002; Gohla et al., 2005).
Recent studies on EGF-stimulated carcinoma cells suggested that cofilin may have a role in initiating the actin filament polymerization by severing actin filaments and exposing free barbed ends for polymerization (Ghosh et al., 2004). This could occur under conditions in which polymerization-ready actin monomers are abundant (DesMarais et al., 2005). However, in most cases, the mutation or knockdown of cofilin expression in yeast, Drosophila melanogaster, and mammalian cells resulted in excessive F-actin assembly (Gunsalus et al., 1995; Lappalainen and Drubin, 1997; Chen et al., 2001; Rogers et al., 2003; Hotulainen et al., 2005), which indicates that at least in the steady state of these cells, cofilin regulates actin filament dynamics by accelerating actin filament disassembly. Consistent with these observations, we showed in this study that cofilin siRNA induced abnormal F-actin accumulation in Jurkat cells before and after SDF-1 stimulation; LIMK1 siRNA suppressed lamellipodium formation, and SSH1L siRNA induced aberrant F-actin assembly after SDF-1
stimulation. These results suggest that in Jurkat cells, cofilin regulates actin turnover by stimulating actin filament disassembly, and LIMK1 and SSH1L regulate actin filament dynamics through inactivating and activating cofilin, respectively.
In summary, our data indicate that phosphoregulation of cofilin activity by LIMK1 and SSH1L is essential for SDF-1induced T cell migration and chemotaxis. LIMK1 regulates cell movement by producing a stimulus-dependent membrane protrusion, and the subsequent activation of SSH1L in the lamellipodium controls the directionality of cell migration by stimulating cofilin in this region. This LIMK1- and SSH1L-mediated spatial and temporal regulation of cofilin activity appears to be important for establishing and maintaining a polarized cell morphology and chemotactic response. As SDF-1
is a potent chemotactic factor for various cells (Zlotnik and Yoshie, 2000; Muller et al., 2001; Zhu et al., 2002; Arakawa et al., 2003), future studies will shed light on the roles of cofilin phosphoregulation in various pathophysiologically important processes, including immune responses, neuronal development, and tumor metastasis.
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Materials and methods |
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Plasmid construction
Plasmids encoding YFP- or CFP-tagged LIMK1, SSH1L, and actin were constructed as described previously (Endo et al., 2003; Nishita et al., 2004). Plasmids for phosphatase-dead SSH1L(C393S), NP(N456), S3E-cofilin, and chick LIMK1 were constructed as described previously (Niwa et al., 2002; Endo et al., 2003; Nagata-Ohashi et al., 2004). The expression plasmid for COOH-terminally (myc + His) tagged human SSH1L was constructed by using the pcDNA3.1/myc-His(+) vector (Invitrogen). The plasmids encoding COOH-terminally deleted and/or point-mutated (myc + His) SSH1L proteins were generated by PCR amplification and subcloned into the pcDNA3.1/myc-His(+) vector. Oligonucleotides were annealed and subcloned into pSUPER vector plasmids (provided by R. Agami, Netherlands Cancer Institute, Amsterdam, Netherlands) as described previously (Brummelkamp et al., 2002) to generate siRNA plasmids against human LIMK1, SSH1L, cofilin, and GFP. Oligonucleotides were composed of pairs of the following 19-base target sequences plus a nine-base spacer: LIMK1 (nucleotide positions from the start ATG codon, 209237; GAAGGACTACTGGGCCCGC), SSH1L (nucleotide positions 2,3292,347; TCGTCACCCAAGAAAGATA), cofilin (nucleotide positions 288306; GGAGGATCTGGTGTTTATC), and GFP (GCAGCACGACTTCTTCAAG). To construct expression plasmids encoding the sr-SSH1L (WT, C393S, or W458A) protein, two bases in the 19-base target sequence in the corresponding (myc + His)-SSH1L cDNA were mutated (TCTTCCCCCAAGAAAGATA) by PCR amplification and subcloning.
Cell culture and transfection
Jurkat human leukemic T cells were obtained from T. Kudo (Cell Resource Center for Biomedical Research, Tohoku University, Sendai, Japan) and were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 9% FCS. For transfection, 107 cells were mixed with plasmids in 400 µl of electroporation medium (RPMI 1640 medium containing 20% FCS and 25 mM Hepes, pH 7.4) and were electroporated at 280 V and 975 µF using a Gene Pulser II (Bio-Rad Laboratories). After electroporation, the cells were cultured for 1860 h in RPMI 1640 medium supplemented with 10% FCS. The transfection efficiency was >90%, as assessed by the transfection of YFP cDNA plasmids.
Immunoprecipitation and immunoblotting
Whole Jurkat cell lysates were prepared as described previously (Nishita et al., 2002). After centrifugation, the lysates were subjected to SDS-PAGE and immunoblotted with antibodies against P-cofilin or cofilin as described previously (Toshima et al., 2001). To analyze endogenous LIMK1 or SSH1L, the cell lysates were immunoprecipitated and immunoblotted with anti-LIMK1 or anti-SSH1L antibodies.
Cell staining
Jurkat cells that were suspended in RPMI 1640 medium containing 25 mM Hepes, pH 7.4, and 0.2% BSA were incubated for 20 min at 37°C. The cells were then plated on coverslips and allowed to attach for 5 min at 37°C. The attached cells were stimulated with 5 nM SDF-1 for 020 min, fixed with 4% PFA for 20 min and cold 100% methanol for 10 min at 20°C, blocked with 2% FCS for 30 min, and costained with anticofilin or antiP-cofilin rabbit pAbs and antiß-actin mouse mAb. The FITC-conjugated antirabbit and rhodamine-conjugated antimouse IgG antibodies were used as second antibodies. To stain F-actin by rhodamine-phalloidin, the methanol fixation step was omitted. Stacked optical sections were obtained and merged by using a laser scanning confocal imaging system (LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with a plan Apo NA 1.4 63x oil immersion objective lens (Carl Zeiss MicroImaging, Inc.).
Time-lapse fluorescence analysis
For time-lapse imaging, Jurkat cells were transfected with plasmids for YFP- and/or CFP-fused protein. Images of stacked optical sections were collected every 30 s for up to 1020 min after SDF-1 stimulation by using the aforementioned laser scanning confocal microscope and objective lens.
Cell migration assays
For the cell migration assays using Transwell culture chambers (5-µm pore size; Costar), the lower wells were filled with 600 µl of medium (RPMI 1640 containing 0.5% BSA and 25 mM Hepes, pH 7.4) with or without 5 nM SDF-1. Jurkat cells (2 x 105 cells) suspended in 100 µl of medium were loaded into the upper wells. 5 nM SDF-1
was added only to the lower well (for chemotaxis assays), to both the lower and upper wells (for chemokinesis assays), or to neither well (control). After incubation for 3 h at 37°C, the cells that had migrated into the lower wells were counted and shown as percentages of the input cells. Chemotaxis was also assessed by directly observing the migrating cells in an SDF-1
gradient using the Dunn chamber (Weber Scientific International; Allen et al., 1998). In these assays, Jurkat cells that were suspended in RPMI 1640 medium containing 9% FBS were loaded into both the inner and outer wells of the chamber, and the wells were covered with a coverslip. The medium in the outer well was replaced with the same medium containing 5 nM SDF-1
. After incubation for 3 h at 37°C, cells migrating on the bridge between the inner and outer wells were observed by using an inverted microscope (DMIRBE; Leica). Time-lapse images were digitally captured every 30 s for 50 min with a cooled CCD camera (CoolSNAP HQ; Roper Scientific). To track migration paths, a series of images were analyzed using IPLab image analysis software (Scanalytics), and the data were plotted with Microsoft Excel. Net translocation distance was determined as the straight distance between the start and the end points during a 50-min period. Migration speed was calculated from the total length of the migration path during a 50-min period. To visualize the directionality of cell migration, we constructed circular histograms showing the percentage of cells whose final position relative to a common origin was within one of 18 20° sectors.
In vitro cofilin phosphatase assay
(Myc + His)-tagged SSH1L and its mutants expressed in 293T cells were immunoprecipitated with anti-myc antibody and were incubated with 100 ng cofilin-(His)6 for 1 h at 30°C in 20 µl lysis buffer in the absence or presence of 5 µg F-actin as described previously (Nagata-Ohashi et al., 2004). The reaction mixtures were then subjected to SDS-PAGE. P-cofilin was analyzed by staining with the Pro-Q Diamond phosphoprotein gel stain kit (Invitrogen), whereas cofilin and actin were analyzed by Coomassie brilliant blue staining. SSH1L and its mutants were analyzed by immunoblotting with an anti-myc antibody.
Online supplemental material
Fig. S1 shows changes in the total F-actin content in Jurkat cells before and after SDF-1 stimulation. Fig. S2 shows the kinetic analyses of cofilin phosphatase activity of WT, W458A, and C393S mutants of SSH1L with or without F-actin. Fig. S3 shows the effects of chick LIMK1 or S3E-cofilin expression on the phenotypes of LIMK1 siRNA cells. Video 1 is the three-dimensional projection of the cell that was stimulated for 5 min with SDF-1
in Fig. 2 A. Video 2 shows the time-lapse fluorescence of Jurkat cell coexpressing CFP-LIMK1 and YFP-SSH1L. Video 3 shows the time-lapse fluorescence of a Jurkat cell coexpressing YFP-actin and CFP-SSH1L. Videos 47 are the time-lapse fluorescence videos of YFP-actinexpressing control and various knockdown Jurkat cells (corresponds to Fig. 6 A), which are listed as follows: Video 4, control cell; Video 5, cofilin siRNA cell; Video 6, LIMK1 siRNA cell; and Video 7, SSH1L siRNA cell. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1.
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Acknowledgments |
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This work was supported by a grant-in-aid for Creative Scientific Research and by the 21st Century Centers of Excellence Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Submitted: 6 April 2005
Accepted: 20 September 2005
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