Depletion of Coronin 1B reduces whole cell motility and modulates lamellipodial dynamics
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 () decreased the level of Coronin 1B in mouse or rat cells (, ) (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 (). 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.
Depletion of Coronin 1B slows whole cell migration and alters lamellipodial dynamics
Interactions between Coronin 1B and Slingshot 1L
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 (). 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 (). Thus, Coronin 1B modulates lamellipodial dynamics, but is not absolutely required for protrusions to form.
Depletion of Coronin 1B slows retrograde actin flow, influences barbed end distribution and density, and actin architecture at the leading edge
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 (), supporting the idea that Coronin 1B modulates the dynamics of the actin network at the leading edge.
Depletion of Coronin 1B slows retrograde actin flow, influences barbed end distribution and filament architecture 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 (). In addition to altering the spatial distribution of barbed ends, Coronin 1B depletion increased the density of barbed ends relative to total F-actin (). 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 (). 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 (). 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.
Coronin 1B inhibits Arp2/3 complex activity in a phosphorylation-dependent manner
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 (). 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. S7
). Furthermore, purified Arp2/3 complex bound directly to wild-type Coronin 1B, but did not bind to the phosphomimetic S2D Coronin 1B mutant (), 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.
Coronin 1B inhibits actin nucleation by Arp2/3 complex in vitro
Coronin 1B is rapidly dephosphorylated by an okadiac acid-insensitive phosphatase
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 (). 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 () 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.
Coronin 1B is a substrate of the Slingshot-1L phosphatase
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 (). 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 (). 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 (). 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 (). 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, Slingshot 1L and Arp2/3 form a complex in vivo that is bridged by Coronin 1B
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 (). 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 (). 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 (). We conclude that SSH1L, Coronin 1B and Arp2/3 exist in a ternary complex that is bridged by Coronin 1B.
Depletion of Coronin 1B inhibits SSH1L-induced membrane ruffling
Expression of SSH1L-GFP in Rat2 cells induces constitutive hyper-ruffling at the cell periphery (). 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.
Depletion of Coronin 1B alters lamellipodial dynamics and inhibits SSH1L-induced membrane ruffling
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 (). In contrast, expression of the phosphomimetic Cofilin S3D mutant suppresses SSH1L-induced hyper-ruffling (), 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.
Depletion of Coronin 1B disrupts normal Slingshot 1L targeting to the leading edge
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 (), 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 () 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 (). 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.
Depletion of Coronin 1B disrupts targeting of Slingshot 1L to the leading edge
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 (). 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
Depletion of Coronin 1B inhibits endogenous Cofilin phosphatases
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 (). 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 (). 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 (). The ratio of active-to-total Cofilin across the whole cell is approximately two fold lower in Coronin 1B–depleted cells (). 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.
Depletion of Coronin 1B increases phosphorylation of Cofilin and expression of activated Cofilin (S3A) partially rescues the effects of Coronin 1B depletion on lamellipodia dynamics
Activated Cofilin partially rescues the effects of Coronin 1B depletion on lamellipodial dynamics
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 (), 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.