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Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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-actinin, generate contractile force for retraction of the trailing edge; tropomyosin and -actinin seem to play a critical role in stabilizing stress fibers by protecting F-actin from cofilin-mediated disassembly and by cross-linking F-actin into bundles, respectively (Bernstein & Bamburg 1982; Nishida et al. 1985; DesMarais et al. 2002; Ono & Ono 2002; Otey & Carpen 2004). On the other hand, lamellipodia are highly dynamic actin structures that display a rapid turnover of actin filaments; and they grow from the barbed ends toward the plasma membrane to generate a protrusive force whilst undergoing simultaneously disassembly from their pointed ends by the action of cofilin (Cooper & Schafer 2000; Pollard & Borisy 2003).
Cofilin, a key regulator of actin filament dynamics, stimulates depolymerization and severing of actin filaments (Bamburg & Wiggan 2002). Cofilin is inactivated by phosphorylation of Ser-3 by LIM-kinase (LIMK) and testicular protein kinase (TESK) (Arber et al. 1998; Yang et al. 1998; Toshima et al. 2001). The Ser-3-phosphorylated cofilin (P-cofilin) is dephosphorylated and reactivated by the Slingshot (SSH) family of protein phosphatases, composed of SSH-1L, -2L and -3L in mammals (Niwa et al. 2002; Ohta et al. 2003), or chronophin (Gohla et al. 2005). A long isoform of SSH1 (SSH-1L; hereafter simply referred to as SSH1) is activated in response to a variety of extracellular stimuli, including neuregulin, insulin, stromal cell-derived factor-1 and calcium ionophore (Nishita et al. 2002, 2004, 2005; Nagata-Ohashi et al. 2004; Wang et al. 2005). SSH1 binds to F-actin, but not to globular actin (G-actin), through at least three F-actin binding sites (Niwa et al. 2002; Ohta et al. 2003; Nagata-Ohashi et al. 2004; Yamamoto et al. 2006). We previously showed that the cofilin-phosphatase activity of SSH1 is highly promoted by association with F-actin and that 14-3-3 proteins inhibit F-actin binding and F-actin-mediated activation of SSH1 (Nagata-Ohashi et al. 2004). Since SSH1 accumulates on to F-actin-rich lamellipodial protrusions after cell stimulation, it seems to be involved in local activation of cofilin and rapid turnover of actin filaments in the lamellipodium for cell migration (Nagata-Ohashi et al. 2004; Nishita et al. 2004, 2005). On the other hand, previous studies showed that over-expression of phosphatase-dead or wild-type (WT) SSH1 induced aberrantly thick actin bundles or F-actin accumulation in cultured cells (Niwa et al. 2002; Ohta et al. 2003; Soosairajah et al. 2005), which suggests that SSH1 has the potential to promote F-actin bundling or stabilization, irrespective of its cofilin-phosphatase activity. The existence of multiple F-actin binding sites in the SSH1 molecule (Yamamoto et al. 2006) further suggests that SSH1 may have F-actin-bundling activity.
In the present study, we provide direct evidence that SSH1 possesses F-actin-stabilizing and -bundling activities in cell-free assays. We also show that knockdown of SSH1 expression by small interfering RNA (siRNA) suppresses stress fiber formation in C2C12 cells, indicating that SSH1 is critically involved in the stability of stress fibers in cultured cells. Our results suggest a novel function of SSH1 in regulating actin filament dynamics through its F-actin-stabilizing and -bundling activities, in addition to its cofilin-phosphatase activity.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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We have previously shown that SSH1 binds to F-actin (Niwa et al. 2002; Ohta et al. 2003). To determine the affinity and stoichiometry of SSH1 binding to F-actin quantitatively, we carried out F-actin co-sedimentation assays, using various concentrations of F-actin. (Myc+His)-tagged SSH1 (WT) was expressed in Sf9 cells using the baculovirus expression system and purified through a nickel agarose column. Purified SSH1 (0.2 µM) was incubated with various concentrations of polymerized actin and ultracentrifuged at 100 000 g to precipitate F-actin. The amounts of SSH1 recovered in the pellet and the supernatant were measured by densitometric analysis of the Coomassie Brilliant Blue (CBB)-stained gel (Fig. 1A). SSH1 was detected in the pellet fraction after incubation with F-actin, but not in the absence of F-actin (Fig. 1A), which indicates that SSH1 specifically co-precipitated with F-actin. The percentages of the amount of SSH1 in the pellets (SSH1, % bound) were plotted as a function of the amounts of actin in the pellets (F-actin) (Fig. 1B). Binding of SSH1 to F-actin was saturated as the F-actin concentration increased. Based on the plot, the value of an apparent dissociation constant (Kd) and the stoichiometry of SSH1 binding to F-actin were estimated to be around 0.4 µM and one SSH1 molecule/~5 actin molecules, respectively.
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It has previously been demonstrated that over-expression of SSH1 often induced thick F-actin bundles in cultured cells (Niwa et al. 2002; Ohta et al. 2003; Soosairajah et al. 2005), which raises the possibility that SSH1 has F-actin-stabilizing and -bundling activities. To determine if SSH1 has the F-actin-stabilizing activity, we examined the effect of SSH1 on dilution-induced actin filament depolymerization, by measuring changes in fluorescence intensity of pyrene-labeled actin (Moriyama & Yahara 2002a). The fluorescence intensity of pyrene-labeled actin is known to decrease along with actin depolymerization (Kouyama & Mihashi 1981). Upon dilution of F-actin solution to a final concentration of 0.1 µM, a gradual decrease in pyrene-actin fluorescence was observed (Fig. 2), which indicates that dilution spontaneously induced F-actin depolymerization. When F-actin was preincubated with various concentrations of purified SSH1, the rate of dilution-induced actin depolymerization was significantly decreased in an SSH1 dose-dependent manner (Fig. 2). These results suggest that SSH1 has the potential to stabilize F-actin against spontaneous depolymerization, similar to other known F-actin-binding proteins, such as tropomyosin (Hitchcock-DeGregori et al. 1988) and WASP-interacting protein (WIP) (Martinez-Quiles et al. 2001).
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To further investigate the role of SSH1 on F-actin stability, we examined the effects of SSH1 on cofilin-induced actin depolymerization. While dilution of F-actin solution to a final concentration of 0.5 µM induced spontaneous depolymerization at a low speed, addition of 0.5 µM cofilin to 0.5 µM F-actin markedly accelerated the rate of actin depolymerization (Fig. 3A). When SSH1 was added to 0.5 µM F-actin at a final concentration of 0.05 µM, it significantly reduced the rate of cofilin-induced actin depolymerization, whereas no apparent effect was observed on spontaneous actin depolymerization at this concentration (Fig. 3A). The inhibitory effect of SSH1 on cofilin-induced actin depolymerization was dependent on the concentrations of SSH1; at 0.25 µM, SSH1 almost completely neutralized the accelerating effect of 0.5 µM cofilin on actin depolymerization (Fig. 3B). Conversely, the neutralizing effect of 0.25 µM SSH1 was overcome at higher cofilin concentrations; 4 µM cofilin induced rapid actin depolymerization even in the presence of 0.25 µM SSH1, to the extent observed by 0.5 µM cofilin without SSH1 (Fig. 3C). These results suggest that SSH1 has the potential to inhibit cofilin-induced actin depolymerization in a dose-dependent manner.
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Both the N- and C-terminal regions are required for F-actin-stabilizing activity of SSH1
SSH1 has N-terminal A, B, P (phosphatase) domains, conserved among members of the SSH family, and an S (serine-rich) domain, conserved between SSH1 and SSH2 (Niwa et al. 2002; Ohta et al. 2003) (Fig. 3E). We previously demonstrated that SSH1 binds to F-actin through at least three F-actin-binding sites located in different domains of the protein, as indicated in Fig. 3E (Yamamoto et al. 2006). To determine the regions required for the F-actin-stabilizing activity of SSH1, a set of deletion mutants of SSH1-(Myc+His) were expressed in the baculovirus system and purified (Fig. 3E, see also Fig. 5C, left). When F-actin was preincubated with full-length SSH1(WT)-(Myc+His) or its deletion mutants (at a final concentration of 0.05 µM) and then subjected to the cofilin-induced actin depolymerization assay, full-length SSH1 decreased the rate of cofilin-induced actin depolymerization, but neither the N-terminal fragments (NP, N461) nor the C-terminal fragments (C, PC) had any effect (Fig. 3F). These results suggest that both N- and C-terminal regions are required for the F-actin-stabilizing activity of SSH1.
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To examine the mechanism by which SSH1 suppresses cofilin-induced actin depolymerization, we analyzed the effect of SSH1 on cofilin-induced F-actin severing by microscopic analysis (Ichetovkin et al. 2000; Ono et al. 2004). Actin filaments partially labeled with AlexaFluor546 and biotin were tethered to a coverslip coated with anti-biotin antibody, and the flow cell was perfused with buffer with or without SSH1 or bovine serum albumin (BSA) (first perfusion), and then with buffer containing cofilin (second perfusion). The numbers of actin filaments in the same field at 30 s and 3 min after perfusion with cofilin were counted under fluorescence microscopy (Fig. 4A). In the control experiments perfused with buffer alone, actin filaments were stable for 3 min (Fig. 4A,b). In contrast, actin filaments were dramatically severed and disassembled after perfusion with cofilin for 3 min (Fig. 4A,d). When actin filaments were preincubated with SSH1, the cofilin-induced F-actin severing was remarkably suppressed (Fig. 4A,h). Preincubation with control BSA had no effect on cofilin-induced F-actin severing (Fig. 4A,l). Thus, SSH1 has the potential to inhibit cofilin-induced actin filament severing.
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F-actin-bundling activity of SSH1
Many proteins that contain multiple F-actin-binding sites have the potential to cross-link actin filaments into actin bundles. As SSH1 has multiple F-actin-binding sites, we next examined whether SSH1 has F-actin-bundling activity by employing low-speed centrifugation experiments that precipitate F-actin bundles but leave free F-actin in the supernatant. F-actin or SSH1 alone remained in the supernatants (Fig. 5A, lanes 1, 2). When actin monomers were polymerized in the presence of increasing concentrations of SSH1 and centrifuged at 10 000 g, F-actin was precipitated in a manner dependent on the concentration of SSH1 (Fig. 5A, lanes 3–7). At concentrations higher than 0.2 µM, SSH1 was also detected in the pellet (Fig. 5A, lanes 6, 7). These results suggest that SSH1 has F-actin-bundling activity in vitro. F-actin was also precipitated after incubation with 0.4 µM phosphatase-dead SSH1(CS), in which catalytic Cys-393 is replaced by Ser, thus indicating that SSH1 has F-actin-bundling activity, independent of the phosphatase activity (Fig. 5B).
To determine the regions required for the F-actin-bundling activity of SSH1, we next examined the potential of the deletion mutants of SSH1 to bundle F-actin in a similar experiment. Actin monomers were polymerized in the presence of equimolar amounts of SSH1 or its deletion mutants and subjected to low-speed centrifugation. As shown in Fig. 5C, F-actin was most effectively precipitated by full-length SSH1(WT). Deletion of the N-terminal or the C-terminal region of SSH1 significantly decreased the amount of F-actin in the precipitates; the C-terminal fragments (C, PC) more effectively induced F-actin precipitation than the N-terminal fragments (NP, N461, N698). These results suggest that both the N- and C-terminal regions are critically involved in the F-actin-bundling activity of SSH1.
To confirm the F-actin-bundling activity of SSH1, we analyzed the shapes of actin filaments by fluorescence and electron microscopy. To visualize F-actin bundles under fluorescence microscopy, 5% AlexaFluor546-labeled actin was added to the actin monomers. When 4 µM actin alone was polymerized, no actin bundles were observed. In contrast, when 4 µM actin was polymerized in the presence of 0.4 µM full-length SSH1(WT) or SSH1(CS), thick and massive actin bundles were produced (Fig. 6A). The C-terminal fragments (C, PC) of SSH1 also produced actin bundles, but unlike those of full-length SSH1, the bundles were not intertwined by the C-terminal fragments (Fig. 6A). The N-terminal fragments (NP, N461, N698) only faintly produced actin bundles. The relatively higher F-actin-bundling activity of the C-terminal fragments, compared to the N-terminal fragments, agrees with the results of the low-speed centrifugation experiments. Electron microscopy of a mixture of F-actin and SSH1(WT) revealed that SSH1(WT)-induced thick actin fibers are transverse F-actin bundles consisting of ordered actin filaments (Fig. 6B).
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While SSH1 can cross-link actin filaments as a monomer through its multiple F-actin-binding sites, it is also possible that SSH1 bundles F-actin as a dimer or a multimeric complex. We thus examined the possibility that SSH1 would form a dimer or a multimer by glutathione-S-transferase (GST) pull-down and chemical cross-linking assays. When purified SSH1-(Myc+His) was incubated with GST-SSH1(WT), GST-14-3-3, or GST immobilized on glutathione-Sepharose, SSH1-(Myc+His) co-precipitated with GST-14-3-3, but not with GST-SSH1 or GST (Supplementary Fig. S1). GST-14-3-3 was used as a control, because 14-3-3 proteins are known to bind SSH1 when it is phosphorylated on Ser-937 and Ser-978 (Nagata-Ohashi et al. 2004). Similarly, formation of a dimer or a multimer complex of SSH1-(Myc+His) was not detected by chemical cross-linking experiments, using the cross-linkers N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (data not shown). Thus, it is likely that SSH1 exists as a monomer and that it induces F-actin bundles through its multiple F-actin-binding sites.
SSH1 knockdown suppresses the formation of actin stress fibers in C2C12 cells
In vitro analyses showed that SSH1 has F-actin-stabilizing and -bundling activities. We next examined whether these activities of SSH1 are involved in actin cytoskeletal organization in cultured cells. When cyan fluorescent protein (CFP)-tagged SSH1 was expressed in C2C12 mouse myoblast cells, which bear well-formed stress fibers, CFP-SSH1 co-localized with actin filaments and accumulated on stress fibers (Fig. 7A), as reported in HeLa cells (Ohta et al. 2003; Yamamoto et al. 2006). To examine the role of SSH1 in F-actin stability in cells, expression of endogenous SSH1 in C2C12 cells was suppressed by transfection of an siRNA plasmid against mouse SSH1. Immunoblot analysis revealed that transfection of SSH1 siRNA significantly reduced the expression of endogenous SSH1, whereas a control siRNA or an empty vector had no apparent effect on SSH1 expression (Fig. 7B). C2C12 cells were co-transfected with the siRNA plasmid along with the plasmid for CFP or CFP-human SSH1 (WT or CS) and the plasmid for YFP at a ratio of 10 : 10 : 1, and stained with rhodamine–phalloidin. YFP fluorescence was used to monitor the expression of siRNA. Transfection of SSH1 siRNA, but not control siRNA, suppressed the formation of stress fibers in C2C12 cells (Fig. 7C). Quantitative analysis showed that stress fibers were detected in 50% of YFP-positive cells transfected with SSH1 siRNA, whereas they were detected in 78% of YFP-positive cells transfected with the control siRNA (Fig. 7D). Thus, knockdown of SSH1 expression significantly suppressed stress fiber formation in C2C12 cells. We next examined whether the co-expression of siRNA-resistant human SSH1 (WT or CS) could rescue the inhibitory effect of SSH1 siRNA on stress fiber formation. Co-transfection of CFP-human SSH1(CS) with SSH1 siRNA almost completely blocked the inhibitory effect of SSH1 siRNA on stress fiber formation (Fig. 7C, D). In contrast, co-transfection of CFP-human SSH1(WT) only partially recovered the effect of SSH1 siRNA (Fig. 7C,D). It is probably due to the cofilin-phosphatase activity of SSH1(WT), which stimulates cofilin activation and F-actin disassembly. Expression of CFP-SSH1(CS) frequently induced aberrantly thick actin bundles or F-actin accumulation in cells (Fig. 7C, arrows). Furthermore, in CFP-SSH1(CS)-expressing cells, stress fibers and thick actin bundles were resistant to cytochalasin B, an actin depolymerizing agent, under the conditions that stress fibers were destructed in control CFP-expressing cells (Supplementary Fig. S2). These results suggest that SSH1 is critically involved in stabilization of actin stress fibers in C2C2 cells and phosphatase-dead SSH1(CS) acts predominantly as an F-actin-stabilizing factor.
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Discussion |
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We and others have demonstrated that over-expression of the phosphatase-dead form of SSH1 frequently induced abnormal accumulation of F-actin bundles (Niwa et al. 2002; Ohta et al. 2003; Soosairajah et al. 2005), suggesting that SSH1 in a phosphatase-inactive state predominantly functions as an F-actin-bundling and -stabilizing factor. In response to various cell stimuli, SSH1 may change the mode of F-actin binding from one suitable for F-actin bundling to one leading to the activation of cofilin-phosphatase activity and reduction of F-actin-bundling activity. Interestingly, previous studies showed that the phosphatase activity of SSH1 is negatively regulated by phosphorylation (Kaji et al. 2003; Soosairajah et al. 2005). The dephosphorylated form of SSH1 is more activated by F-actin than the phosphorylated form (Soosairajah et al. 2005). Thus, phosphorylation/dephosphorylation of SSH1 may control the balance between F-actin bundling and cofilin-phosphatase activities. However, responsible kinase(s) and phosphatase(s) have remained to be characterized. Alternatively, binding of any modulator protein to SSH1 or F-actin could alter the F-actin binding modes and cofilin-phosphatase activity of SSH1. SSH1 may thus function as a molecular switch directing actin filament dynamics toward either depolymerization or stabilization via two opposite activities. Further studies on mechanisms of switching between F-actin-stabilizing and cofilin-phosphatase activities of SSH1 will provide new insights into regulation of actin filament dynamics. The binding of 14-3-3 proteins to SSH1, which is dependent on the phosphorylation of two Ser residues (Ser-937 and Ser-978) in its C-terminal Ser-rich region, inhibits both F-actin binding and F-actin-mediated activation of SSH1 (Nagata-Ohashi et al. 2004). Thus, it is unlikely that phosphorylation of SSH1 at Ser-937/Ser-978 contributes to the switch between SSH1 activities.
In C2C12 cells, SSH1 preferentially localized to stress fibers and siRNA knockdown of SSH1 significantly suppresses stress fiber formation. On the other hand, in MCF-7 carcinoma cells or Jurkat T-lymphoma cells, SSH1 was accumulated on the lamellipodium in response to cell stimulation, and knockdown of SSH1 suppressed stimulus-induced membrane protrusion and cell migration (Nagata-Ohashi et al. 2004; Nishita et al. 2004, 2005). Thus, it is possible that cells use the F-actin-bundling and cofilin-phosphatase activities of SSH1 according to their actin structure requirements; for instance, F-actin bundling activity could predominate in the stress fiber and cofilin-phosphatase activity could predominate in the lamellipodium. The protein components of each actin structure may play a role in the switching between these SSH1 activities.
F-actin-bundling proteins are known to cross-link actin filaments by using multiple F-actin binding sites in the molecule or by forming a homodimer complex when the protein has only a single F-actin binding site, like -actinin (Revenu et al. 2004). As the GST pull-down and chemical cross-linking assays indicated that SSH1 is monomeric, SSH1 probably cross-links actin filaments as a monomer by using its multiple F-actin-binding sites, at least three of which are located in the B and S domains and near the P domain (Fig. 3E) (Yamamoto et al. 2006). Deletion of N- or C-terminal regions significantly reduced the F-actin-bundling activity, indicating that both of these regions are critically involved in the bundling activity. On the other hand, both N- and C-terminal fragments exhibit weaker F-actin-bundling activity in both low-speed centrifugation and fluorescence microscopy assays, whereas NP (amino acids 1–456) and C (amino acids 509–1049) fragments contain only one known F-actin-binding site. This suggests that SSH1 has additional F-actin-binding sites in both of its N- and C-terminal regions. Consistent with this, SSH1 fragments mutated at known F-actin-binding sites still retained weak F-actin-binding ability (Yamamoto et al. 2006). Identification of these unidentified F-actin-binding sites is necessary to elucidate the precise mechanism of SSH1-mediated F-actin bundling. As the C-terminal fragments (PC and C) had relatively higher F-actin-bundling activity, compared to those of the N-terminal fragments (NP, N461 and N698), the C-terminal region appears to play a major role in SSH1 F-actin-bundling activity. On the other hand, the region around Trp-458 is indispensable for the F-actin-mediated activation of cofilin-phosphatase activity of SSH1 (Nishita et al. 2005). Thus, multiple F-actin-binding sites in the SSH1 molecule probably have distinct roles in the control of its F-actin-bundling activity and cofilin-phosphatase activity.
In addition to F-actin-bundling, SSH1 exhibited F-actin-stabilizing activity in vitro. Previous studies showed that tropomyosin competitively bind to F-actin with cofilin and suppresses cofilin-induced actin depolymerization (Bernstein & Bamburg 1982; Nishida et al. 1985; DesMarais et al. 2002; Ono & Ono 2002). Similarly, SSH1 reduced the rates of spontaneous and cofilin-induced actin depolymerization. We showed that SSH1 suppressed both cofilin(Y82F)- and cofilin(S94D)-induced actin depolymerization, which suggests that SSH1 has the potential to antagonize both severing and depolymerizing activities of cofilin. We also showed that SSH1 blocked F-actin binding and severing activities of cofilin. Together these results suggest that the F-actin-stabilizing activity of SSH1 is caused by protecting spontaneous and cofilin-mediated actin monomer release from the pointed ends of actin filaments as well as by inhibiting F-actin severing activity of cofilin by blocking cofilin binding to F-actin. It remains unclear whether the inhibitory effect of SSH1 on cofilin binding to F-actin is due to the competitive binding on actin filaments or the SSH1-induced conformational change of actin filaments to the form not accessible to cofilin. Since addition of SSH1 to F-actin in a 1 : 10 molar ratio induced F-actin bundling but did not significantly suppress spontaneous actin depolymerization, SSH1-mediated F-actin cross-linking is not mainly involved in the inhibition of spontaneous actin depolymerization. Further structural and functional analysis of SSH1 and its binding to F-actin will be important for understanding the mechanisms by which SSH1 regulates actin filament dynamics through its cofilin-phosphatase activity and F-actin bundling/stabilizing activity.
In conclusion, we obtained evidence that SSH1 possesses F-actin-stabilizing and -bundling activities in cell-free assays and in cultured cells. Our findings suggest a novel cellular function of SSH1 on the actin cytoskeletal reorganization through its F-actin-stabilizing and -bundling activities, in addition to its cofilin-phosphatase activity.
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Experimental procedures |
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Plasmids coding for C-terminally (Myc+His)-tagged (i.e., consisting of a Myc epitope peptide and six His residues) human SSH1 (WT or CS) and its deletion mutants, and CFP- or GST-tagged human SSH1(WT or CS) were constructed as previously described (Nishita et al. 2004; Yamamoto et al. 2006). Plasmids for C-terminally His6-tagged cofilin and its point mutants, cofilin(Y82F) and cofilin(S94D), were constructed as described (Moriyama & Yahara 1999, 2002b). For protein expression in the baculovirus system, cDNA inserts were subcloned into pFastBac1 vector (Invitrogen). The pSUPER vector for siRNA was provided by Dr R. Agami (the Netherlands Cancer Institute) (Brummelkamp et al. 2002). The siRNA plasmid that targets the mouse SSH1 mRNA sequence (5'-GAGGAGCTGTCCCGATGAC-3') and the control siRNA plasmid for mutated human SSH1 (5'-TCTTCCCCCAAGAAAGATA-3') were constructed as previously described (Brummelkamp et al. 2002; Nishita et al. 2004).
Proteins
SSH1-(Myc+His), cofilin-His6, and their mutants were expressed in Sf9 cells, using the Bac-to-Bac baculovirus expression system (Invitrogen), according to the manufacturer's instructions. Recombinant baculovirus-infected cells were lyzed in lysis buffer (40 mM Tris–HCl, pH 8.0, 100 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, 1 mM PMSF, 10 µg/mL leupeptin, 1 mM 2-mercaptoethanol). Cell lysates were clarified by centrifugation, charged on to a nickel–nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) column, and eluted with 0.2 M imidazole buffer. Actin was purified from rabbit skeletal muscle, and monomeric actin was isolated by gel filtration on Sephacryl S-200 (GE Healthcare) in G-buffer (2 mM Tris–HCl, pH 8.0, 0.2 mM ATP, 0.1 mM CaCl2, 10 µM dithiothreitol). G-actin was labeled with N-(1-pyrene)iodoacetamide or AlexaFluor546 C5-maleimide, according to the manufacturer's protocols (Molecular Probes). Pyrene- or AlexaFluor546-labeled G-actin was polymerized by addition of 100 mM KCl and 2 mM MgCl2. In the case of AlexaFluor546-actin, an equimolar amount of unlabeled G-actin was mixed before polymerization. Polymerized actin was collected by centrifugation at 100 000 g for 2 h, and the pellet resuspended in G-buffer and dialyzed against G-buffer for 24 h to depolymerize actin filaments. The sample was clarified by centrifugation at 100 000 g for 1 h to remove residual F-actin and the supernatant containing G-actin was aliquoted, frozen in liquid nitrogen, and stored at –80 °C. Gelsolin was purified as previously described (Kurokawa et al. 1990).
F-actin cosedimentation assay
Actin was polymerized for 1 h at room temperature by addition of 10x polymerization buffer (0.2 M Tris–HCl, pH 7.5, 1 M KCl, 20 mM MgCl2, 1 mM dithiothreitol). Various concentrations of F-actin were incubated with 0.2 µM purified SSH1-(Myc+His) for 1 h at room temperature in 1x polymerization buffer containing 0.01% Nonidet P-40. The mixtures were ultracentrifuged at 100 000 g for 30 min. Equal portions of precipitates and supernatants were subjected to SDS-PAGE and amounts of each protein were quantified by densitometric analysis of the CBB-stained gel. To examine the effect of SSH1 on F-actin binding of cofilin, 2 µM actin was polymerized and preincubated with or without SSH1-(Myc +His) for 30 min, and then incubated with 2 µM cofilin for 30 min. The mixtures were ultracentrifuged at 100 000 g for 30 min, and the resulting precipitates and supernatants were analyzed as above.
Actin depolymerization assay
Actin depolymerization assay was performed as previously described (Moriyama & Yahara 2002a) with slight modification. Pyrenyl-G-actin (4–10 µM, containing 5%–15% pyrene-labeled actin) was polymerized in the presence of 40 nM gelsolin for 6 h. Pyrenyl-F-actin was incubated with SSH1-(Myc+His) or its deletion mutants for 10 min, and depolymerization initiated by 20- to 40-fold dilution with dilution buffer (20 mM Tris–HCl, pH 7.5, 2 mM MgCl2, 100 mM KCl, 0.5 mM EGTA, 0.1 mM dithiothreitol, 2 nM gelsolin–actin complex) with or without cofilin. Immediately after dilution, fluorescence intensity was monitored at 407 nm with excitation at 365 nm by a fluorescence spectrophotometer (Hitachi, F-2500).
Microscopic analysis of actin filament severing
F-actin severing assay was performed as previously described (Ono et al. 2004). Unlabeled-actin (1.4 µM), AlexaFluor546-labeled actin (0.4 µM) and biotin-labeled actin (0.2 µM, Cytoskeleton) were copolymerized for 1 h in ISAP buffer (50 mM KCl, 20 mM HEPES–KOH, 5 mM EGTA, 2 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol, pH 7.2). A coverslip was coated with 3 µg/mL antibiotin antibody 2F5 (Molecular Probes) in ISAP buffer for 3 min, and washed with five volumes of 1 mg/mL BSA in ISAP buffer. AlexaFluor546/biotin-labeled F-actin was diluted to 0.05 µM in ISAP buffer, and perfused into the flow cell. After 3 min, unbound filaments were washed away with one volume of anti-bleaching buffer (ISAP buffer containing 36 µg/mL catalase, 20 µg/mL glucose oxidase, 6 mg/mL glucose, and 100 mM dithiothreitol), and then two volumes of anti-bleaching buffer with or without 50 nM SSH1 or BSA were perfused and incubated for 3 min. The flow cell was set on a fluorescence microscope (model DMIRBE, Leica Microsystems) equipped with an Apochromat 63x oil immersion objective lens and a Photometrics Cascade II CCD camera (Roper Scientific). Cofilin (50 nM) in anti-bleaching buffer was perfused into the flow cell, and fluorescence images were obtained 30 s and 3 min after perfusion with cofilin.
F-actin-bundling assay
F-actin bundling assay was performed according to the published protocol (Kato & Takenawa 2005). SSH1-(Myc+His) or its deletion mutants were incubated with 4 µM G-actin in XO buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.2 mM ATP, 1 mM dithiothreitol) for 30 min at room temperature and centrifuged at 10 000 g for 20 min. Equal portions of the precipitates and supernatants were subjected to SDS-PAGE and analyzed by CBB staining.
Fluorescence and electron microscopic analysis of F-actin bundles
For fluorescence microscopy, 4 µM G-actin containing 5% AlexaFluor546-labeled G-actin was incubated with or without 0.4 µM SSH1-(Myc+His) or its deletion mutants under the conditions described in the F-actin-bundling assay. Reaction mixtures were diluted 20-fold in XO buffer containing 20 µM phalloidin and 0.5% methylcellulose, put between two BSA-coated coverslips, and analyzed by fluorescence microscopy. For electron microscopy, 4 µM G-actin was incubated with 0.4 µM SSH1-(Myc+His) under the conditions described in the F-actin bundling assay and the sample was then stained with 2% uranyl acetate.
Cell culture, transfection and staining
C2C12 mouse myoblast cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. For RNA interference, siRNA plasmids were electroporated into C2C12 cells; 2 x 106 cells were mixed with 20 µg plasmids in 400 µL of Opti-MEM and electroporated at 325 V and 975 µF, using a Gene Pulser II (Bio-rad). After a 48-h culture period, expression of endogenous SSH1 was analyzed by immunoblotting with rabbit anti-mouse SSH1 antibody, raised against the C-terminal peptide of mouse SSH1 (GKSAPEHLKSPSRVNKS). For cell staining, C2C12 cells were grown on fibronectin-coated coverslips and transfected with plasmids using Lipofectamine 2000 (Invitrogen). For RNA interference, cells were co-transfected with plasmid for YFP, siRNA plasmid for control or mouse SSH1, and plasmid for CFP or CFP-human SSH1 (CFP-hSSH1) at the ratio of 1 : 10 : 10. The siRNA-expressing cells were monitored by YFP fluorescence. After a 48-h culture period, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then stained with rhodamine-phalloidin. Fluorescence images were obtained using a fluorescence microscope (model DMIRBE, Leica Microsystems) equipped with an Apochromat 63x oil objective lens.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kmizuno{at}biology.tohoku.ac.jp
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References |
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Received: 18 November 2006
Accepted: 18 February 2007
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