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Actin filament nucleation and turnover are interdependent processes in migrating cells, but the mechanisms coordinating these events are unknown. Coronin 1B influences motility, lamellipodial dynamics and actin filament architecture at the leading edge of Rat2 cells in a manner consistent with a role in coordinating filament formation and turnover. Coronin 1B interacts simultaneously with both Arp2/3 complex and Slingshot (SSH1L) phosphatase, two regulators of actin filament formation and turnover, respectively. Coronin 1B inhibits filament nucleation by Arp2/3 complex and this inhibition is attenuated by phosphorylation of Coronin 1B on Serine 2, a site targeted by SSH1L. Coronin 1B directs SSH1L to lamellipodia where it likely regulates Cofilin. Accordingly, depleting Coronin 1B increases phospho-Cofilin levels and expressing activated Cofilin partially suppresses the effects on lamellipodia dynamics of Coronin 1B depletion. Thus, Coronin 1B coordinates filament nucleation via Arp2/3 complex and turnover by Cofilin at the leading edge of migrating cells.
Coronins are highly-conserved F-actin-binding proteins (Uetrecht and Bear, 2006). Functional studies in Dicytostelium amoeba, fibroblasts and thymocytes indicate that Coronins play an important role in lamellipodial protrusion, whole cell motility and chemotaxis (Cai et al., 2005; de Hostos et al., 1993; Foger et al., 2006; Mishima and Nishida, 1999), but the mechanism(s) by which Coronins influence motility are unknown. In yeast, one mechanism may be through inhibition of Arp2/3 complex (Humphries et al., 2002; Rodal et al., 2005), but the effects of mammalian Coronins on actin nucleation activity by Arp 2/3 complex are unknown. Mammalian Coronin 1B is ubiquitously expressed and localizes to the leading edge of migrating fibroblasts (Cai et al., 2005). The interactions of Coronin 1B or Coronin 1A with Arp2/3 complex are regulated by phosphorylation of Serine 2 via PKC, where phosphorylation of Ser2 reduced the interaction with Arp2/3 and diminished cell motility (Cai et al., 2005; Foger et al., 2006).
ADF/Cofilin proteins (hereafter referred to as Cofilin) control actin filament turnover at the leading edge and at other cellular locations (Bamburg, 1999). Mechanistically, Cofilin severs and potentially enhances depolymerization of filaments by cooperatively binding along the sides of actin filaments and inducing conformational changes in filament structure (Bamburg et al., 1999). In vivo, Cofilin regulates the dynamics of actin-based structures such as stress fibers, dendritic spines and lamellipodia (Dawe et al., 2003; Hotulainen et al., 2005; Zhou et al., 2004). The activity of Cofilin is regulated in a variety of ways including phosphorylation, PIP2 binding, intracellular pH changes and interactions with binding partners such as AIP1 (Bamburg, 1999).
Phosphorylation of Cofilin-serine 3 by LIM Kinase or TESK leads to decreased F-actin binding and inactivation of Cofilin (Stanyon and Bernard, 1999). Dephosphorylation of Serine 3 on Cofilin enhances F-binding and activates its severing/depolymerization activity (Agnew et al., 1995). Two classes of phosphatases act on Cofilin - the Slingshots and Chronophin (Huang et al., 2006). Slingshot is a family of atypical Serine/Threonine protein phosphatases that in mammals includes Slingshot-1, -2 and –3; Slingshot-1 and –2 exist as long and short isoforms (Niwa et al., 2002; Ohta et al., 2003). The long isoform of Slingshot-1 (SSH1L) functions during chemotaxis of hematopoeitic cells (Nishita et al., 2005). Regulation of SSH1L activity may occur via phosphorylation of serine residues in its C-terminus and binding of inhibitory 14-3-3 proteins (Nagata-Ohashi et al., 2004). In addition, SSH1L activity is greatly enhanced by its interaction with F-actin (Nagata-Ohashi et al., 2004; Soosairajah et al., 2005). The other known Cofilin phosphatase, Chronophin, is a HAD-type serine phosphatase that plays an important role in cytokinesis (Gohla et al., 2005).
Considerable evidence suggests that the activities of Arp2/3 complex and Cofilin are coordinately regulated at the leading edge of motile cells. Studies using correlative microscopy (Svitkina and Borisy, 1999) suggest an array treadmilling model for actin assembly and turnover within lamellipodia in which Arp2/3-dependent filament nucleation at the front of the lamellipodia was balanced by Cofilin-dependent depolymerization of filaments at the rear. Arp2/3 complex and Cofilin also work together to promote lamellipodial protrusion in quiescent adenocarcinoma cells stimulated with growth factors, albeit via a different mechanism (DesMarais et al., 2005). MTLn3 cells stimulated with EGF initiate protrusion when Cofilin severs filaments creating new barbed ends that are preferred sites for Arp2/3-dependent filament nucleation (Ichetovkin et al., 2002). Together, filament severing and filament nucleation synergize to produce a burst of new barbed ends at the leading edge that support lamellipodial protrusion (DesMarais et al., 2004). Finally, studies using quantitative fluorescent speckle microscopy in epithelial cells suggest that Arp2/3 complex and Cofilin activities within lamellipodia may be coupled (Ponti et al., 2005), but very little is known about how this occurs. In this work, we describe a molecular connection between the Arp2/3 complex and Cofilin activities at the leading edge of motile cells that involves Coronin 1B.
To test the role of Coronin 1B in cellular motility, we depleted Coronin 1B in Rat2 cells and monitored the effects on whole cell migration and lamellipodial dynamics. An shRNA that selectively targets mouse and rat, but not human, Coronin 1B (Fig. 1A, B) decreased the level of Coronin 1B in mouse or rat cells (Fig. 1C, ,4J)4J) (Rubinson et al., 2003). Depletion of Coronin 1B leads to ~33% decrease in cell speed relative to uninfected cells, cells infected with a control shRNA (NS) or cells expressing the Coronin 1B shRNA and human Coronin 1B-GFP that is refractory to the shRNA (Fig. 1D). Since the decreased rate of cell motility was rescued by expressing human Coronin 1B-GFP, this effect is specifically due to loss of Coronin 1B and not due to off-target silencing. Thus, Coronin 1B is required for normal whole cell motility.
Since Coronin 1B is concentrated at the leading edge (Fig. S1), we reasoned that the effects of Coronin 1B depletion on whole cell motility might arise from defects in lamellipodial dynamics. We used kymography to quantify the effects of Coronin 1B depletion on lamellipodial dynamics (Fig. 1E). Three parameters of lamellipodial behavior were analyzed: protrusion rate, protrusion persistence and protrusion distance (Hinz et al., 1999). Depletion of Coronin 1B increased the protrusion rate and decreased the protrusion persistence and the distance protruded (Fig. 1F). Thus, Coronin 1B modulates lamellipodial dynamics, but is not absolutely required for protrusions to form.
The assembly and disassembly of actin filament networks underlie the dynamic behavior of lamellipodia. Since Coronin 1B depletion affects lamellipodia and Coronins bind F-actin, we tested if Coronin 1B depletion affects actin dynamics at the leading edge. The network of actin filaments assembling at the cell margin moves rearwards towards the cell body via retrograde flow. Using kymography of cells expressing GFP-actin to visualize actin, the rate of retrograde actin flow in Coronin 1B-depleted cells was reduced to ~50% the rate in control cells (Fig. 2A, B), supporting the idea that Coronin 1B modulates the dynamics of the actin network at the leading edge.
A zone of rapidly growing filament barbed ends is a hallmark of the actin network at the leading edge (Condeelis et al., 1988). To measure the distribution and density of barbed ends in Coronin 1B depleted cells, we used an established assay to monitor barbed ends in situ (Symons and Mitchison, 1991). Depletion of Coronin 1B leads to a striking narrowing of the zone of barbed ends near the cell edge compared to control cells (Fig. 2C, D). In addition to altering the spatial distribution of barbed ends, Coronin 1B depletion increased the density of barbed ends relative to total F-actin (Fig. 2E). Thus, Coronin 1B inhibits the generation of barbed ends at the leading edge and alters their spatial distribution.
To examine the underlying actin filament architecture at the leading edge of Coronin 1B depleted cells, we used platinum replica electron microscopy. Rat2 cells have a robust and uniform dendritic network of actin filaments at the leading edge that is approximately 2µm wide (Fig. 2F). Cells depleted of Coronin 1B have an abnormal actin network characterized by densely branched filaments at the cell margin and a relative paucity of actin filaments at the rear of the lamellipodium (Fig. 2F). These changes in the organization of actin filaments are not observed in cells expressing a control shRNA or in Coronin 1B-depleted cells rescued with human Coronin 1B-GFP (Fig. S3). Thus, Coronin 1B appears to plays a role in coordinating assembly of actin filaments at the cell edge and disassembly of actin filaments at the rear of the lamellipodium.
Yeast Coronin inhibits actin filament nucleation by Arp2/3 complex in vitro (Humphries et al., 2002). To determine if human Coronin 1B inhibits Arp2/3 complex nucleation activity, we added recombinant Coronin 1B to pyrenyl actin polymerization reactions. Coronin 1B had no effect on the rates of spontaneous actin assembly or of assembly nucleated from Spectrin-F-actin seeds (Fig. S6). However, in reactions containing Coronin 1B and GST-VCA-activated Arp2/3 complex, the rate of actin polymerization was reduced (Fig. 3A, B). To determine if phosphorylation at Ser2 regulates Coronin 1B’s inhibition of Arp2/3 complex, we compared wild-type Coronin 1B (WT), phosphorylated Coronin 1B (p-WT) and a phosphomimetic S2D mutant of Coronin 1B. In contrast to WT Coronin 1B, phosphorylated Coronin 1B (p-WT) and the S2D mutant Coronin 1B weakly inhibit Arp2/3 complex nucleation activity at all doses tested (Fig. 3B, Fig. S7). Furthermore, purified Arp2/3 complex bound directly to wild-type Coronin 1B, but did not bind to the phosphomimetic S2D Coronin 1B mutant (Fig. 3C), which corroborates previous immunoprecipitation experiments (Cai et al., 2005). Thus, Coronin 1B inhibits Arp2/3 complex nucleation in vitro and phosphorylation of Coronin 1B on Ser2 regulates this activity.
The phosphatase that dephosphorylates and activates Coronin 1B is unknown. To identify this 1B phosphatase, we developed an assay in which cells were first treated with PMA to stimulate maximal phosphorylation, followed by PMA washout in the presence of a pan-PKC inhibitor (Ro32-0432) to block further phosphorylation. Using this regime, dephosphorylation of Coronin 1B was detected within one minute and phospho-Coronin 1B returned to basal levels by 10 minutes (Fig. 4A). We examined the sensitivity of this phosphatase to the Ser/Thr phosphatase inhibitor okadaic acid, which potently inhibits PP1 and PP2A (Cohen et al., 1990). Okadaic acid at concentrations from 100nM (Fig. 4B) to 1µM (data not shown) had no effect on the rate of Coronin 1B dephosphorylation, making it unlikely that phospho-Coronin 1B is a substrate of either PP1 or PP2A.
We considered whether the Coronin 1B phosphatase might be Slingshot, which acts on Cofilin. Slingshots are among a small number of Ser/Thr phosphatases resistant to okadaic acid (Niwa et al., 2002). To test this hypothesis, we performed in vitro and in vivo dephosphorylation assays using Slingshot-1L (SSH1L). Recombinant Coronin 1B phosphorylated in vitro with purified PKCα was efficiently dephosphorylated by immunoprecipitated SSH1L-myc (Fig 4C). Since phosphatases often exhibit promiscuous activity on purified proteins, we tested whether SSH1L dephosphorylates Coronin 1B in cell lysates. Coronin 1B was phosphorylated by stimulating cells with PMA prior to lysis and increasing amounts of SSH1L-myc were then added; the phosphorylation status of Coronin 1B and other substrates was monitored by immunoblotting. Coronin 1B was dephosphorylated by SSH1L in a dose-dependent manner (Fig. 4D). Cofilin was also efficiently dephosphorylated and may be a preferred substrate. In contrast, no detectable dephosphorylation of phospho-Erk1/2 or phospho-Paxillin was detected, suggesting that the SSH1L phosphatase activity is highly specific under these conditions.
To determine if Coronin 1B is a substrate of SSH1L in vivo, we performed dephosphorylation assays in two different cell types. First, HEK293 cells were transiently transfected with dominant negative mutant form of SSH1L (SSH1L-CS), which harbors a mutation (C393S) in the phosphatase domain that renders it catalytically inactive (Ohta et al., 2003). In the presence of SSH1L-CS, phosphor-Coronin 1B was elevated after PMA stimulation and the return to basal levels after washing out PMA was delayed (Fig. 4E). Since ectopic SSH1L expression is relatively high in HEK293 cells, we confirmed these observations using Rat2 cell stably expressing lower levels of either wild-type (WT) SSH1L-GFP or dominant negative SSH1L-CS-GFP (Fig. 4F). As expected, GFP-tagged SSH1L localized to stress fibers, focal adhesions and the leading edge (Ohta et al., 2003) (Fig. S1A) in Rat2 cells and the level of phosopho-Cofilin decreased in lysates from Rat2 cells expressing WT SSH1L-GFP and increased in lysates from Rat2 cells expressing CS SSH1L-GFP. We conclude that, like Cofilin, Coronin 1B is a substrate of the SSH1L phosphatase in vitro and in vivo.
Coronin 1B and Arp2/3 complex interact in vivo (Cai et al., 2005). To determine if Slingshot 1L is part of this complex, we immunoprecipitated SSH1L and probed for Coronin 1B and Arp2/3 complex. SSH1L-myc interacted with endogenous Coronin 1B using reciprocal co-immunoprecipitations (Fig. 4H). Arp2/3 complex (as reported by the p34 subunit) was detected in both the Coronin 1B and SSH1L immunoprecipitates. Notably, actin was not detected, despite the fact that Coronin 1B, SSH1L and Arp2/3 complex all bind F-actin, indicating that the interaction among these components is not bridged by residual F-actin.
While these results are consistent with the existence of a ternary complex containing Coronin 1B, SSH1L and Arp2/3 complex, they do not exclude the possibility of two bivalent complexes (a Coronin 1B-Arp2/3 complex and a SSH1L-Arp2/3 complex). To identify a ternary complex, we used a two-step immunoprecipitation protocol in which Coronin 1B was first immuno-depleted from the lysates derived from Rat2:SSH1L-GFP cells followed by a second immunoprecipitation of SSH1L-GFP from the depleted lysate (Fig 4I). In the first step, anti-Coronin 1B (but not control IgG) immunoprecipitated both SSH1L-GFP and Arp2/3 complex. When SSH1L-GFP was immunoprecipitated from the Coronin 1B-depleted lysate, no additional Arp2/3 complex was co-immunoprecipitated. In contrast, SSH1L-GFP co-immunoprecipitated both Coronin 1B and Arp2/3 complex from a mock-depleted lysate.
To confirm this result, we tested for an interaction between SSH1L and Arp2/3 complex in Coronin 1B-depleted cells. No SSH1L was detected when Arp2/3 complex was immunoprecipitated from lysates derived from Coronin 1B-depleted, SSH1L-GFP expressing Rat2 (Fig. 4J). We conclude that SSH1L, Coronin 1B and Arp2/3 exist in a ternary complex that is bridged by Coronin 1B.
Expression of SSH1L-GFP in Rat2 cells induces constitutive hyper-ruffling at the cell periphery (Fig. 5A). Lamellipodia in SSH1L-expressing cells protrude at approximately twice the rate and protrusions are short-lived compared to control cells, with no difference in protrusion distance. Remarkably, depletion of Coronin 1B completely suppressed SSH1L-induced hyper-ruffling. One possible explanation is that Coronin 1B generally influences lamellipodial dynamics by a mechanism unrelated to SSH1L activity. To address this possibility, we investigated lamellipodia in Coronin 1B-depleted cells expressing FP4-CAAX, which induces hyper-ruffling by targeting Ena/VASP proteins to the plasma membrane (Bear et al., 2000; Bear et al., 2002). As expected, expression of FP4-CAAX increased protrusion rate and shortened protrusion persistence, similar to the effects of SSH1L expression, but depletion of Coronin 1B had no effect on the ruffling induced by FP4-CAAX. Thus, the suppression of the SSH1L-induced ruffling in Coronin 1B–depleted cells is a specific, rather than a general, effect on lamellipodial dynamics.
Suppression of SSH1L-induced ruffling upon Coronin 1B depletion may occur via dephosphorylation of Coronin 1B by SSH1L. Thus, a phosphomimetic mutant form of Coronin 1B would be predicted to suppress SSH1L-induced hyper-ruffling by competing with endogenous substrates. To test this hypothesis, lamellipodial dynamics were examined in cells expressing Coronin 1B S2D and SSH1L. To our surprise, the Coronin 1B S2D mutant is ineffective in suppressing SSH1L-induced hyper-ruffling (Fig. 5B). In contrast, expression of the phosphomimetic Cofilin S3D mutant suppresses SSH1L-induced hyper-ruffling (Fig. 5B), confirming that SSH1L-induced ruffling is due to the phosphatase activity of SSH1L. While these results do not preclude a contribution of SSH1L dephosphorylation of Coronin 1B in regulating lamellipodial behavior, they do suggest that regulation of Cofilin by SSH1L influences lamellipodial behavior. Alternately, Cofilin S3D may be a more effective competitor of SSH1L phosphatase than Coronin 1B S2D. Nonetheless, we sought another explanation for the potent suppression of the ectopic SSH1L-induced ruffling upon Coronin 1B depletion.
Coronin 1B may act upstream of SSH1L and thereby influence SSH1L-induced hyper-ruffling. For example, Coronin 1B may allow SSH1L to reach its substrate (Cofilin) and thereby influence lamellipodial dynamics. To test this idea, we compared the localization of SSH1L-GFP in control and in Coronin 1B-depleted cells. shRNAs were co-expressed with SSH1L-GFP using a single lentirviral vector (Fig. 6A), so that every cell expressing SSH1L-GFP also expressed either Coronin 1B-specific or control shRNA. SSH1L-GFP was not as enriched at the leading edge of Coronin 1B-depleted cells as in the control cells (Fig. 6B) when compared to the distribution of Cortactin (see Fig. S1). In control shRNA expressing cells, the maximal peaks of Cortactin and SSH1L-GFP were separated by an average of 0.35 µm. In Coronin 1B-depleted cells, Cortactin was distributed identically to that in control cells, but the peak of SSH1L-GFP signal was approximately twice as far rearward from the peak of Cortactin than in control cells (Fig. 6C). These data suggest that Coronin 1B is required to target SSH1L to a distinct region within lamellipodia and that proper targeting of SSH1L is required to exert an effect on lamellipodial dynamics.
Previous studies suggested that binding of SSH1L to F-actin targeted it to the leading edge (Nagata-Ohashi et al., 2004). To examine the role of F-actin in localizing SSH1L within lamellipodia, we stained cells expressing SSH1L-GFP (+/− Coronin 1B shRNA) with phalloidin (Fig. 6D). In control cells, SSH1L-GFP and F-actin co-localized within lamellipodia, however in Coronin 1B-depleted cells, the distribution of F-actin was indistinguishable from that in control cells at the level of light microscopy, but SSH1L-GFP was excluded from the most distal, F-actin rich region near the leading edge. This result indicates that F-actin is insufficient to target SSH1L to the leading edge, but does not exclude a contribution of F-actin-SSH1L interactions for stimulating phosphatase activity (Nagata-Ohashi et al., 2004; Soosairajah et al., 2005).
To test whether Coronin 1B depletion also inhibited endogenous Cofilin phosphatase activity, we compared the phospho-Cofilin levels in cells expressing Coronin 1B-shRNA and control shRNA. Cells depleted of Coronin 1B had higher levels of phospho-Cofilin relative to controls (Fig. 7A). This effect is not due activation of Cofilin kinases because Coronin 1B-depletion does not alter the level of active LIMK 1/2. To confirm the effect of Coronin 1B depletion on phospho-Cofilin levels, we determined the ratio of active-to-total Cofilin in Coronin 1B-depleted cells using ratiometric immunofluorescent imaging (Fig. 7B). In control cells, the ratio of active-to-total Cofilin was similar to that in surrounding uninfected cells; in Coronin 1B-depleted cells, the ratio of active-to-total Cofilin is lower than in uninfected cells, particularly in lamellipodial regions (Fig. 7B). The ratio of active-to-total Cofilin across the whole cell is approximately two fold lower in Coronin 1B–depleted cells (Fig. 7C). Taken together these results indicate that Coronin 1B enhances dephosphorylation and activation of Cofilin. Moreover, these findings are consistent with the biochemical and cellular effects of ectopic expression of SSH1L-GFP and suggest Coronin 1B influences Cofilin activity via a Slingshot-dependent mechanism.
Since Coronin 1B-depletion increases the amount of phospho-Cofilin, we posit that the effects on lamellipodial dynamics might be due, in part, to a failure to activate Cofilin at the leading edge. To test this idea, we co-expressed an activated Cofillin mutant (S3A) along with Coronin 1B or control shRNA. Lamellipodial protrusions extended faster and were shorter-lived in cells expressing Cofilin (S3A)-GFP and a control shRNA than those in either control cells expressing no Cofilin (S3A)-GFP or non-transfected cells (Fig. 7D), similar to the effects of expressing SSH1L-GFP. Co-expression of Cofilin (S3A)-GFP and Coronin 1B shRNA partially suppressed the effects on lamellipodial dynamics of Coronin 1B depletion. Lamellipodial protrusion rate decreased and protrusion persistence was modestly increased, but did not reach control levels. It is important to note that, although Cofilin S3A rescued the lamellipodial dynamics associated with depletion of Coronin 1B, it did not rescue the effects on whole cell motility (data not shown). Together, these data suggest that Coronin 1B promotes the activation of Cofilin at the leading edge.
Our data indicate that Coronin 1B is a key regulator of whole cell motility and lamellipodial dynamics. Although Coronin 1B is not absolutely required for cells to move or lamellipodia to protrude, it enhances these processes and is a likely target of signal transduction cascades that regulate motility. Coronin 1B influences motility through control of actin filament dynamics and architecture at the leading edge. In the absence of Coronin 1B, retrograde actin flow is reduced and the dendritic actin meshwork of the lamellipodia is abnormal. These two observations are likely to be aspects of the same underlying phenomenon, as the architecture of the actin network reflects the balance of actin assembly at the front and disassembly at the back of the lamellipodia. Future studies will dissect the relative contributions of actin assembly/disassembly and myosin-based contraction to Coronin 1B’s contribution to retrograde flow.
Most striking is the different distribution and density of actin barbed ends in the lamellipodia of cells lacking Coronin 1B. Cells depleted of Coronin 1B have higher barbed end density than controls and the barbed ends are concentrated in a narrower zone at the leading edge. Increased barbed ends could arise by three mechanisms: failure to cap or uncapping of existing filaments, severing of filaments or de novo nucleation of new filaments. Since Coronin 1B has no detectable capping activity (Fig. S6), failure to cap filaments can be eliminated from consideration. The increased barbed ends observed with Coronin 1B depletion are also unlikely to result from increased filament severing since Cofilin is less active in the lamellipodia of knockdown cells. We cannot exclude an effect on Cofilin-independent severing (eg. Gelsolin), but since Cofilin is required for protrusion, it is probably the predominant severing factor in lamellipodia. We suggest that increased nucleation of filaments most likely results in increased barbed ends in lamellipodia of Coronin 1B-depleted cells.
Consistent with the increased density of barbed ends in lamellipodia of Coronin 1B-depleted cells, recombinant Coronin 1B inhibits the nucleation activity of Arp2/3 in vitro. This inhibitory effect on Arp2/3 activity is a conserved property of Coronins from yeast to humans (Humphries et al., 2002). Other proteins such as Tropomyosin, Caldesmon and EPLIN inhibit Arp2/3 complex nucleation activity, but these proteins are thought to bind tightly to the sides of actin filaments and compete with binding of Arp2/3 complex to the mother actin filament (Blanchoin et al., 2001; Maul et al., 2003; Yamakita et al., 2003) to indirectly inhibit filament-dependent Arp2/3 nucleation activity. In contrast, inhibition by Coronin 1B appears to arise from direct binding of Coronin to Arp2/3 complex and stabilization of its “open”, inactive form (Rodal et al., 2005). Our findings support this notion since Coronin 1B S2D binds poorly to Arp2/3 and has weaker inhibitory activity. Further studies will be required to elucidate the molecular mechanism of Coronin 1B’s inhibition of Arp2/3 complex.
Our results suggest that limiting filament nucleation activity via Coronin 1B is important for efficient lamellipodial protrusion. This conclusion is consistent with other studies in which nucleation of actin filaments was rampant and lamellipodial protrusions formed in response to EGF were blocked in cells microinjected with the Arp2/3 complex activating VCA (WA) domain of SCAR or WASP (Shao et al., 2006). Coronin 1B apparently serves to temper filament nucleation by Arp2/3 complex in lamellipodia, especially close to the membrane where several WASP/SCAR co-activating factors, such as Rac, Cdc42 and PIP2, are enriched. In the absence of Coronin 1B, nucleation by Arp2/3 complex may be overactive, leading to the increased free barbed ends and the faster rate of protrusion observed in Coronin 1B depleted cells. Without a means to limit Arp2/3 complex activity, explosive generation of new filaments could result in short-lived protrusions that stall as G-actin becomes limiting, consistent with the decreased protrusion persistence and distance observed in the Coronin 1B-depleted cells. Previous models for the control of filament nucleation in lamellipodia focused on the localized activation of WASP/SCAR/Formin proteins by inositol lipids and Rho-type GTPases. Our studies suggest that nucleation control is more complex than current models predict and involves a balance between nucleation promoting factors and inhibitors.
Coronin 1B is required for SSH-induced lamellipodial activity and for targeting SSH1L within lamellipodia. Through these processes, Coronin 1B could significantly influence the activity of Cofilin at the leading edge. This mechanism for regulating Cofilin activity is analogous to the mechanisms regulating many kinases and phosphatases via targeting subunits and scaffolds (Cohen, 2002; Sim and Scott, 1999). It is important to note that targeting of SSH1L does not override other mechanisms of Slingshot regulation such as phosphorylation or F-actin binding (Nagata-Ohashi et al., 2004). Rather, it is likely that appropriate targeting of SSH1L works in conjunction with these other regulatory events to control Slingshot’s activity spatially and temporally.
Our studies in fibroblasts link Coronins and Cofilin activities via SSH, however there is precedence for such a connection in other systems (Goode et al., 1999). Mutations in the single yeast Coronin gene are benign except when combined with mutations in the Cofilin gene. F-actin accumulates in abnormal aggregates in double Coronin/Cofilin mutants, but not with each single mutant, suggesting that these proteins function together to promote actin turnover in this organism. Recent studies also identified Coronin 1A as a factor leading to actin depolymerization in Listeria actin tails (Brieher et al., 2006). This study suggested that Coronin 1A-bound F-actin has increased affinity for Cofilin, perhaps due to altered helical twist. One prediction of this idea is that Cofilin may not be recruited to the leading edge in the absence of Coronin. We observed no change in Cofilin distribution at the leading edge in Coronin 1B-depleted cells (Fig. S8), but future studies are needed to explicitly test whether Coronin 1B has similar activity to Coronin 1A in this regard. It is possible that Coronins direct Cofilin recruitment and activation via SSH targeting to synergistically control Cofilin activity. Regardless of the precise mechanistic details, a conserved functional connection between Coronins and Cofilin in regulating filament dynamics exists from yeast to humans.
Why is a factor that coordinates the activities of Arp2/3 complex and Cofilin in lamellipodia necessary? Cell motility is a system-based process that requires precise spatial and temporal control of the individual components. Chemotaxis requires localized and coordinate cytoskeletal remodeling to achieve directional movement towards a chemotactic signal. Our work identifies Coronin 1B as a link between two major components, Cofilin and Arp2/3 complex, that control cytoskeletal remodeling in many cells. Since factors that coordinate cytoskeletal dynamics are likely to be crucial for directional sensing and motility, Coronin 1B is likely regulated by chemotactic signaling pathways. This idea is strongly supported by the recent observations that thymocytes from Coronin 1A knockout mice are severely defective in chemotaxis towards chemokines (Foger et al., 2006). Future studies will focus on the role that Coronin 1B plays in directed cell motility and on elucidating the molecular mechanism of Coronin 1B action.
Antibodies were obtained from Cell Signaling Technologies (pSerPKC, pLIMK1/2, LIMK1), Upstate Biotechnology (p34Arc, Cortactin), Cytoskeleton (Cofilin), Chemicon (actin), Biosource (pPaxillinSer126, pErk1/2), Roche (GFP), Sigma (Myc) and Jackson ImmunoResearch (Cy2, Rhodamine Red-X, Cy5, and HRP conjugated secondary antibodies). AlexaFluor phalloidins were from Molecular Probes. Cells were obtained from ATCC. Inhibitors Ro32-0432 and okadaic acid were from Calbiochem. Antibodies to Coronin 1B (4245.Exp) were affinity purified as described (Cai et al., 2005).
Cell culture, immunoprecipitation and immunoblotting were performed as described (Cai et al., 2005). Transient transfections were performed using FuGENE 6 (Roche) for HEK293 cells, PolyFect (Qiagen) for Rat2 cells and Lipofectamine 2000 (Invitrogen) for NIH3T3 cells. Retroviral packaging, infections, and fluorescence-activated cell sorting were as described (Bear et al., 2000). Lentivirus production and infection were as described (Rubinson et al., 2003).
Descriptions of the construction of plasmids are in Supplemental Experimental Procedures.
Migration of Rat2 fibroblasts was analyzed as described with slight modifications (Cai et al., 2005). Cells were infected with lentivirus for expressing Coronin1B or control shRNA (with and without rescue proteins) together with GFP. GFP-expressing cells were tracked and whole cell speed calculated using Tracking Analysis software (Andor Bioimaging). GFP negative cells were analyzed as an internal control. For kymography, 300 images were captured at 1 second intervals and processed using an ImageJ plugin (http://www.embl-heidelberg.de/eamnet/html/body_kymograph.html). Kymographs were generated from protrusive areas of at least five cells per treatment and lamellipodial parameters were calculated as described (Bear et al., 2002). Data were exported to Prism for statistical analysis.
Rat2 cells were infected with lentivirus to express shRNA against Coronin 1B or control shRNA together with GFP-actin. Images were captured at 1 sec intervals using a Nipkow-type spinning disk confocal microscope (Yokogawa CSU-10 and IX-81, Olympus) equipped with a 100x objective and a Hamamatsu CCD camera (model C4742-80-12AG). Kymographs were generated and analyzed as described above.
Free actin filament barbed ends were detected in permeabilized fibroblasts as described in Supplemental Experimental Procedures using AlexaFluor568-actin (Bryce et al., 2005). AlexaFluor568-actin incorporation was quantified from the intensity profile around the cell periphery as described for Fig. S1. The upper 50% of intensities were used to determine the width of the zone of free barbed ends (Fig. S2). The ratio of the barbed end intensity to phalloidin intensity along the edge was normalized and plotted as a function of distance from the cell margin (Fig S2).
Rat2 cells were infected with lentivirus expressing Coronin 1B or control shRNA for 4 days. Cells were plated onto coverslips coated with 10 µg/ml fibronectin for 4 hours and samples were prepared for platinum replica electron microscopy as described (Svitkina, 2005). Images were collected at 6300X using a Philips CM12 microscope at 60 kV using Kodak SO-163 negative film, scanned using an Imacon FlexTight 848 scanner (Hasselblad AB) at resolution 2500 dpi and adjusted for publication using Photoshop (Adobe).
Recombinant Coronin 1B was expressed and purified either from Drosophila S2 cells (Invitrogen) or from HEK293 cells as described in Supplemental Experimental Procedures. Phosphorylated Coronin 1B was obtained from HEK293 cells treated for 30 min with 50nM PMA prior to harvesting and sodium orthovanadate (4 mM) was included in all buffers. The phosphorylation status of Coronin 1B was verified by pSerPKC blotting, immunoprecipitation and by mass spectrometry (Fig. S5).
Spectrin F-actin seeds (SAS), actin, pyrene labeled actin, GST-VCA and Arp2/3 complex were prepared as described (Bryan and Coluccio, 1985; DiNubile et al., 1995; Egile et al., 1999; Higgs et al., 1999; Spudich and Watt, 1971). Seeded actin polymerization reactions were performed as described (Barzik et al., 2005) with 1 µM actin (5% pyrene labeled) and 0.2 nM SAS. VCA-induced Arp2/3 nucleation reactions were performed as follows: Coronin 1B and Arp2/3 (20 nM) were mixed in MKEI-50 Buffer and incubated at room temperature for 5 min; reactions were initiated by the simultaneous addition of 1.5 µM actin (5% pyrene labeled, primed with 1 mM EGTA and 0.1 mM MgCl2 for 90 sec) and GST-VCA (1 nM or 10 nM). The delay between mixing reactants and recording fluorescence was 20 sec. Fluorescence was converted to the molar concentration of F-actin from the fluorescence of completely polymerized (24 hours post reaction) and unpolymerized actin, assuming a critical concentration of 0.1 µM. Maximal actin assembly rates were determined from linear fits of the rate as 0.5–1 µM actin polymer formed during the reactions.
Rat2 cells were treated with 100 nM PMA for 30 min to stimulate Coronin 1B phosphorylation. Cells were then washed with fresh media and incubated in media containing the pan-PKC inhibitor, 1 µM Ro32-4032, for the indicated time. Okadaic acid (up to 1 µM) was included in the washout media in some experiments. Reactions were stopped by lysing the cells in RIPA Buffer and Coronin 1B was immunoprecipitated and blotted with pSerPKC antibody as described (Cai et al., 2005).
The in vitro Slingshot phosphatase assay was as described (Nagata-Ohashi et al., 2004; Niwa et al., 2002) with slight modifications; a detailed description is in the Supplemental Experimental Procedures.
Immunofluorescent staining and imaging were performed as described (Cai et al., 2005). For co-localization analyses, a custom ImageJ macro was used to extract pixel intensity as a function of distance from the leading edge. A detailed explanation of this method is in the Supplemental Experimental Procedures.
To assess active/total Cofilin ratios, cells were stained with antibodies against Cofilin (MAB22; 1:100) and phospho-Cofilin (4321; 1:170) and processed for immunofluorescence imaging as described in the Supplemental Experimental Procedures. DIC images indicated the outline of the cells. The ImageJ plugin, Ratio Plus (http://rsb.info.nih.gov/ij/plugins/ratio-plus.html) was used to calculate the ratio between phospho-Cofilin and Cofilin staining. The ratio was converted into active/total Cofilin ratio by [(Total Cofilin-phosphoCofilin) / Total Cofilin], normalized and presented using a rainbow look-up table.
We are grateful to J. Bamburg (Colorado State University) for providing antibodies to pCofilin (4321) and Cofilin (MAB22) and the protocol to quantify active Cofilin by ratio imaging prior to publication, to A. Makhov and H. Mekeel for help with electron microscopy, to K. Burridge, M. Schaller, F. Gertler, J. Bamburg and D. Roadcap for critical reading of the manuscript, to E. Izaurralde, M. Wilm, Y. Durocher for reagents and to T. Kotova and F. Tariq for technical assistance. Electron microscopy work was supported by NIH grant CA-16086 to JD Griffith. This work was supported by NIH (GM067222) to DAS and funds from the V Foundation and Melanoma Research Foundation to JEB.
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