We have previously shown that Exo70 directly interacts with the Arp2/3 complex [7
]. However, the molecular consequence and functional implication of this interaction is unknown. Here, we purified Exo70 and the Arp2/3 complex (Figure S1A, B
) and performed polymerization assays using pyrene-labeled actin. Since the WAVE complexes are usually inactive [10
], we used the recombinant WAVE2 as the nucleation-promoting factor in our assay. Exo70 alone or together with the Arp2/3 complex did not affect F-actin assembly. WAVE2 stimulated the Arp2/3 complex; further addition of Exo70 significantly enhanced actin polymerization (). The concentration of barbed ends in our experiment setting (2μM G-actin, 25 nM WAVE2, and 15 nM Arp2/3) was 0.22 nM in the absence of Exo70, and was 0.41 nM in the presence of Exo70. The stimulatory effect of Exo70 is dose-dependent (). Furthermore, in the presence of saturating concentration of Exo70 (1 μM), significantly less WAVE2 is required (reduced from 25 nM to 6.25 nM) for Arp2/3 activation (Figure S1C
Exo70 stimulates Arp2/3-mediated actin polymerization
We have previously found that Exo70 lacking residues 628-630 had a weaker interaction with the Arp2/3 complex [7
]. Indeed, Exo70(Δ628–630) had a much lower stimulatory effect on actin polymerization than the wild type Exo70 (). Addition of Exo70 reduced the time to reach half maximal polymerization by 2.4-fold, whereas Exo70(Δ628–630) had a much weaker effect (). The maximal rate of actin polymerization was increased by 100% with Exo70, but only 34% with Exo70(Δ628–630) ().
Upon activation by WAVE2, the Arp2/3 complex generates new actin branches on the side of pre-existing actin filaments, which can be monitored by total internal reflection fluorescence microscopy (TIRFM) [11
]. Here we examined the effect of Exo70 on actin branching in real-time using TIRFM. To better visualize branching and elongation of the new filaments, we performed dual-color imaging to better differentiate the pre-existing and newly formed actin filaments. Rhodamine-labeled G-actin was allowed to polymerize for 3 mins and captured onto the surface of a coverslip coated with NEM-myosin. Then Cy5-labeled G-actin together with Arp2/3, WAVE2, wild type or mutant Exo70 was flown into the chamber to replace Rhodamine-labeled G-actin. The newly generated actin filaments were monitored over time. In the presence of Arp2/3 and WAVE2, branches were generated from the sides of actin filaments (, upper panel; see Movie S1
for both audio and visual presentation of the generation of new branches; see Figure S2
for enlarged images). Addition of Exo70 significantly stimulated branch formation (, middle panel; Movie S2 and Figure S2
). The Exo70(Δ628–630) mutant barely had any stimulatory effect (, bottom panel; Movie S3
). Addition of Exo70 in the absence of WAVE2 had no effect on actin (Movie S4
). After 240 seconds, the branching ratio (number of branches/number of total actin filaments) was 6-fold higher in the presence of Exo70 (). The lengths of the newly generated actin filaments were also measured. In reactions with Exo70, the actin filaments were slightly shorter than those without Exo70 (), suggesting that, when the total amount of G-actin is limited (0.8 μM of 8% Cy5-labeled G-actin, in contrast to 2 μM G-actin in the pyrene actin assay), an increase in actin branching results in a reduction of the final length of the filaments.
Exo70 stimulates the branching activity of the Arp2/3 complex in the presence of WAVE2
Since the effect of Exo70 is shown only in the presence of WAVE2, Exo70 itself is unlikely to be an actin nucleator or a nucleation-promoting factor (NPF). Then what is the molecular mechanism by which Exo70 stimulates the Arp2/3 complex? Exo70 binds to the Arp2/3 complex through its ARPC1 subunit (a.k.a. p40 or Arc40), which physically contacts N-WASP/WAVE [12
]. It is thus possible that Exo70 affects the WAVE2-Arp2/3 interaction, which in turn promotes the Arp2/3 complex activation. To test this possibility, we first performed in vitro
binding assay using GST-WAVE2 and purified Arp2/3 complex (). In the presence of Exo70, the amount of Arp2/3 bound to GST-WAVE2 increased by 3.5-fold (). As a negative control, GST-Rabin8, a fusion protein with a similar molecular weight to Exo70, did not bind to the Arp2/3 complex. The affinity of the Arp2/3 complex to WAVE2 is much higher in the presence of Exo70 (kd=210 nM) than without Exo70 (kd=470 nM) (Figure S3A
). To examine whether Exo70 affects the WAVE2-Arp2/3 interaction in cells, immunoprecipitation experiments were carried out using cells expressing FLAG-WAVE2 (). When Exo70 was knocked down by siRNA, only 44% of Arp3 was co-precipitated with WAVE2 comparing with control siRNA-treated cells (). Reciprocally, when ARPC1 was knocked down by siRNA, endogenous Exo70 was no longer co-precipitated with WAVE2 (). Consistent with the finding, the Exo70(Δ628–630) mutant interacts much weakly with WAVE2 in comparison to the wild type Exo70 as tested by co-immunoprecipitation experiments () and pull down experiments (Figure S3B
). Taken together, our data strongly suggested that Exo70 positively regulates the interaction between the Arp2/3 complex and WAVE2, and provide a molecular basis for our observed stimulatory effect of Exo70 on Arp2/3 activation.
Exo70 promotes the interaction between the Arp2/3 complex and WAVE2
To examine the physiological function of Exo70 in the stimulation of the Arp2/3 complex, we generated human MDA-MB-231 stable cell lines expressing GFP-tagged rat wild type Exo70 and a Exo70(Δ628-630) mutant that is defective in stimulating the Arp2/3 complex (see above). The endogenous Exo70 was then knocked down by siRNA in these cells (Figure S4A
). The levels of other exocyst subunits, WAVE2 and Arp3 were not affected in the knockdown cells (Figure S4B
), and Exo70(Δ628-630) binds to Sec8 to the same extent as the wild type (Figure S4C
). As shown in , the Exo70-knockdown cells expressing Exo70(Δ628-630) failed to form lamellipodia as indicated by Arp3 and F-actin staining. The “lamellipodia ratio”, defined as the lamellipodia length divided by the total cell perimeter, was significantly lower in the knockdown cells expressing Exo70(Δ628-630) (). The migratory properties of these cells were also examined. In transwell assays, the number of mutant Exo70 cells migrated to the lower chamber was much smaller than the control cells (). Also, using a wound-healing assay, we found that Exo70 siRNA knockdown cells expressing Exo70(Δ628-630) took more time for wound closure ( and Figure S4D
). By tracking the trajectories of individual cells over time, we found that cells expressing Exo70(Δ628-630) were not only slower in migration, but also less capable of maintaining the direction of their movement (). The directional persistence, quantified as the ratio of net displacement to the total migration distance (“D:T ratio”), was much smaller in cells expressing Exo70(Δ628-630) (). These results suggest that the function of Exo70 in stimulating the Arp2/3 complex plays an important role in membrane protrusion formation and directional cell migration.
The function of Exo70 in Arp2/3 stimulation is required for lamellipodia formation and directional cell migration
Our experiments, for the first time, revealed that Exo70 is able to kinetically stimulate the Arp2/3 complex in the presence of WAVE2 for actin polymerization and branching. Exo70 binds to the Arp2/3 complex through ARPC1, the subunit that physically contacts N-WASP/WAVE for activation [12
]. Our data show that Exo70 stimulates the interaction between the Arp2/3 complex and WAVE2, which may provide a molecular mechanism for the observed stimulatory effect of Exo70 on the Arp2/3 complex activation. During membrane protrusion formation, associated with actin assembly is dynamic membrane reorganization [15
]. Exo70, as a member of the exocyst complex, is involved in post-Golgi exocytosis and membrane remodeling ([17
]; Guo lab unpublished data). The effect of Exo70 on Arp2/3-mediated actin polymerization and branching may couple actin dynamics and membrane reorganization for cell morphogenesis and effective migration.
We have previously shown that the Exo70-Arp2/3 complex interaction is stronger in cells treated with EGF [7
], or expressing Y527F c-Src [18
], which are known to stimulate membrane protrusion and cell migration. These observations suggest that, in cells, the effect of Exo70 on the Arp2/3 complex is up-regulated by signaling events to levels above what we observed here using recombinant proteins. It is possible that the activation of Exo70 and/or the Arp2/3 complex by small GTP-binding proteins or kinases in response to growth factors stimulates their interaction. Future investigation of this regulation will lead to better understanding of many processes such as chemotaxis and tumor cell invasion.