ARF1-mediated actin polymerization produces movement of artificial vesicles

Abstract

Vesicular trafficking and actin dynamics on Golgi membranes are both regulated by ADP-ribosylation factor 1 (ARF1) through the recruitment of various effectors, including vesicular coats. Actin assembly on Golgi membranes contributes to the architecture of the Golgi complex, vesicle formation, and trafficking and is mediated by ARF1 through a cascade that leads to Arp2/3 complex activation. Here we addressed the role of Golgi actin downstream of ARF1 by using a biomimetic assay consisting of liposomes of defined lipid composition, carrying an activated form of ARF1 incubated in cytosolic cell extracts. We observed actin polymerization around the liposomes resulting in thick actin shells and actin comet tails that pushed the ARF1 liposomes forward. The assay was used to characterize the ARF1-dependent pathway, leading to actin polymerization, and confirmed a dependency on CDC42 and its downstream effector N-WASP. Overall, this study demonstrates that actin polymerization driven by the complex multicomponent signaling cascade of the Golgi apparatus can be reproduced with a biomimetic system. Moreover, our results are consistent with the view that actin-based force generation at the site of vesicle formation contributes to the mechanism of fission. In addition to its well established function in coat recruitment, the ARF1 machinery also might produce movement- and fission-promoting forces through actin polymerization.

Intracellular traffic is mediated by transport vesicles that bud from a donor membrane through the assembly of specific coat proteins and are then transported and fused with the acceptor organelle (1, 2). One important, yet not fully understood, step of vesicle formation is the fission and separation of the transport carrier from the donor compartment. Extensive studies mostly focusing on the formation of clathrin-coated vesicles during endocytosis from the plasma membrane revealed the facilitating role of actin polymerization for the pinching off of vesicles and their movement away from the donor membrane (3). In the secretory pathway, it is not yet clear whether actin fulfills the same role. However, fundamental similarities between the regulation of actin assembly at the plasma membrane and on Golgi subcompartments have been uncovered (4, 5). Besides its implication in the maintenance of Golgi architecture, the actin cytoskeleton might play a role in the biogenesis of Golgi-derived transport vesicles (6–8), just as actin dynamics is coupled to the formation of clathrin-coated vesicles and cargo exit from the trans-Golgi network (TGN) (9, 10) and subsequent trafficking in the early secretory pathway (11–13).

Roles for Golgi coat proteins, namely coat protein I (COPI) and clathrin coats, in the control of actin cytoskeleton dynamics have been documented, connecting the mechanism of vesicle formation to the actin cytoskeleton (9, 11, 12). Recruitment of COPI and clathrin coat–protein complexes on Golgi subcompartments is regulated by the small GTP-binding protein ADP-ribosylation factor 1 (ARF1), and ARF1 has been shown to regulate actin dynamics on Golgi membranes (14). ARF1-mediated recruitment of COPI on cis-medial Golgi compartments triggers Arp2/3 complex-dependent actin polymerization in a cascade that involves the Rho GTPase CDC42 and its downstream effector, N-WASP (11, 12, 15, 16). Although not completely understood, the mechanism of CDC42 association to Golgi membranes may involve the binding of CDC42 to the COPI γ-subunit (15). In addition, it was recently found that GTP-ARF1 on the Golgi complex recruits a CDC42 GTPase-activating protein (GAP), ARHGAP21, which further regulates CDC42 activity and Arp2/3 complex dynamics on Golgi membranes (17, 18). Whether and how ARF1-mediated assembly of clathrin-coated vesicles is coupled to actin dynamics on the TGN is less clear. It was recently reported that activated ARF1 triggers the recruitment of a cortactin/dynamin-2 complex to Golgi membranes with consequences for post-Golgi transport (10). This pathway downstream of ARF1 would couple Arp2/3 complex activation by cortactin to the stimulation of vesicle fission by dynamin-2 (10). In addition, ARF1 is known to promote the production of phosphoinositides, PtdIns 4-phosphate (PI4P), and PtdIns 4,5-biphosphate (PI4,5P2) at the Golgi complex and, hence, to affect both actin dynamics and membrane traffic (19). These observations suggest that ARF1-regulated actin dynamics contribute to the trafficking of Golgi-derived vesicles by facilitating the formation and possibly the dissociation of transport carriers from Golgi subcompartments.

Here we designed an in vitro reconstituted system analyzing the role of ARF1-dependent actin dynamics on liposomes. This system is composed of lipid vesicles of defined composition, to which ARF1 is bound. GDP-ARF1 is soluble, but it binds to membrane in its GTP-bound form by its myristoylated amphipathic helix, allowing liposomes to be readily functionalized by GTP-ARF1 in vitro. In practice, GTP-γ-S, a nonhydrolyzable analog of GTP, was used to stabilize the association of ARF1 to liposomes. These liposomes were incubated in HeLa cell extracts, as opposed to purified protein mixes, because the required components of ARF1-dependent actin dynamics are not yet fully identified. Under these conditions, liposomes promoted actin assembly at their surface in an ARF1-dependent manner and moved by the polymerization of an actin comet tail. This assay allowed for the characterization of the biochemical pathway downstream of ARF1, leading to actin polymerization and movement.

Results

ARF1 Liposomes.

Liposomes were prepared from a mixture of synthetic lipids for a simple and well controlled system. Unless otherwise indicated, they consisted of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) with 1% 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol 4,5-biphosphate (PIP2) because PIP2 is known to regulate the architecture of the Golgi complex downstream of ARF1 (20, 21). Liposomes of different sizes were obtained by using two different techniques. Giant unilamellar vesicles (GUVs), 1 to 20 μm in diameter, formed by the gentle swelling method (22) were used for microscopy observations and quantification of actin polymerization by epifluorescence. Large unilamellar vesicles (LUVs), 0.8 μm in diameter, formed by the extrusion method (23) were generally used for biochemical characterization. To bind ARF1 to liposomes, we made use of its natural property to insert into membranes upon GTP loading (24). For this purpose, recombinant-purified, myristoylated GDP-ARF1 was loaded with the nonhydrolyzable analog of GTP, GTP-γ-S, at a low-Mg²⁺ concentration in the presence of liposomes, and insertion was verified by sedimentation analysis [see supporting information (SI) Fig. 4].

GTP-ARF1 Induces Actin Shell and Comet Formation.

GUVs loaded with GTP-γ-S-ARF1 (ARF1-GUVs) were incubated in HeLa cell extract supplemented with G-actin (including 5% fluorescently labeled actin) and ATP. An actin shell, visible as a dark ring by phase-contrast microscopy or a fluorescent ring by epifluorescence microscopy, was detected around the liposomes as early as 5 min after adding liposomes to the extract. The thickness of the actin gel increased progressively around the GUVs as a function of time (Fig. 1 A–C). In some cases, after 2 to 3 h, the actin gel growth led to the disappearance of the vesicle, producing a homogeneous ball of actin gel (Fig. 1D). Because ≤10 μM residual-free GTP-γ-S was present in the liposome preparation, a control was performed by using GUVs that were preincubated in GTP-γ-S without ARF1. These GUVs never developed an actin gel (Fig. 1 E and F) (the exposure of the fluorescent images is enhanced 10 times compared with Fig. 1 A–D), although faint patches of fluorescent actin could be seen at the periphery of some of these GUVs (Fig. 1F). To quantify the extent of actin polymerization around ARF1-GUVs, the mean actin fluorescence intensity in the fluorescent ring around randomly selected GUVs was measured 30–45 min after mixing with the extract, as described in Material and Methods (Fig. 2). Then >95% of ARF1-GUVs displayed a pronounced fluorescent signal (Fig. 2A1), contrasting with control GUVs preincubated with GTP-γ-S without ARF1 (Fig. 2A2). We conclude that the residual GTP-γ-S left in the assay is not sufficient to trigger actin polymerization in the absence of ARF1.

Fig. 1.

Actin polymerizing around ARF1 liposomes produces actin shells and comets. (A–F) For all image pairs, the left image is a phase-contrast image, and the right is fluorescence microscopy showing labeled actin. The exposition time for all fluorescence images is the same, except for E and F, where exposition is 10 times longer. (Scale bar: 5 μm.) (A and B) GUV loaded with GTP-γ-S-bound ARF1 incubated for 30 min in cytosolic extract. A shell of actin is visible around the GUVs. (C and D) ARF1-GUV incubated for 2 h in extract. A wide actin shell is observable around the GUV in C, whereas a ball of actin with no enclosed volume is shown in D. (E and F) GUV without ARF1 incubated for 30 min in supplemented extracts. Small faint patches of actin are visible in F. (G and H) The elapsed time between each image is 30 sec. (Scale bars: 2 μm.) (G) ARF1-GUV propelled by an actin comet. All images are phase-contrast microscopy, except for the first image, which is fluorescent microscopy of actin. (H) ARF1-LUV propelled by an actin comet. All images are phase-contrast microscopy, except for the last image, which is fluorescent microscopy of actin (red) and lipids (green).

Fig. 2.

Quantification of actin polymerization around ARF1-GUV. Quantification of actin polymerization around GUV after 30–45 min of incubation in supplemented extracts. The histogram shows the percentage of GUVs as a function of circumferential mean of fluorescence arranged by exponential bin size (i.e., the fluorescence value in a column is twice the value of the column on its left and half the value of the column on its right). (A Top) ARF1-GUV (1% PIP2, 99% DOPC). (A Middle) GUV (1% PIP2, 99% DOPC) with no ARF1. (A Bottom) ARF1-GUV (4% PI, 96% DOPC); statistical comparison with A Top is provided. (B and C) ARF1-GUV (1% PIP2, 99% DOPC) incubated in supplemented extracts with increasing concentrations of drugs or proteins. Statistical comparison with the preceding concentration is provided. (B) Incubation with increasing concentrations of cytochalasin D. Concentration of drug vehicle (DMSO) is kept constant (1‰). (C) Incubation with increasing concentrations of the ARFBD fragment of the ARHGAP21 protein.

Actin comets visible by phase-contrast microscopy were observed on 18% of ARF1-GUVs (66 comets for 376 GUVs) and correlated with movement of these GUVs (Fig. 1G). Of note, comets were never observed for GUVs of >2 μm in diameter, an observation that is consistent with experiments on beads (25). The average speed of rocketing liposomes was 0.52 ± 0.30 μm/min (n = 20), comparable to Arp2/3-mediated movement in other in vitro systems (26). Actin comet-based movement was similarly observed with LUVs, on which ARF1 was bound (Fig. 1H).

To confirm that the formation of actin shells and comets was the result of de novo barbed-end actin polymerization, and not because of recruitment of preexisting actin filaments from the extract, the assay was carried out in the presence of 0.1 to 2 μM cytochalasin D, which is an inhibitor of actin polymerization at barbed ends in this concentration range (27). The amount of actin present around ARF1-GUVs was significantly reduced in the presence of the drug at ≥0.5 μM (P < 0.0001) (Fig. 2B). This inhibition, together with the observed propulsion of small liposomes by actin tails, showed that barbed-end actin polymerization, and not recruitment of preexisting filaments, was occurring around ARF1 liposomes, and that actin dynamics were able to generate a propulsive force.

To address whether the presence of PIP2 was important for the production of actin at the membrane, we used ARF1-GUVs composed of 96% DOPC and 4% phosphatidylinositol (PI). Thus, these vesicles lacked PIP2, but bore the same electrostatic charge as the standard 1% PIP2-containing ARF1-GUVs. When DOPC-PI liposomes were mixed with HeLa cell extracts, we observed a shell of actin, although it was markedly reduced compared with standard 1% PIP2-containing ARF1-GUVs (compare Fig. 2 A3 and A1), demonstrating that PIP2 is required for optimal ARF1-dependent polymerization of actin around liposomes. When standard (1% PIP2) ARF1-GUVs were incubated in buffer containing G-actin and ATP without extract, no actin polymerization was observed around the liposomes (data not shown).

Thus far, these results indicate that the presence of activated ARF1 on synthetic liposome membranes triggered actin polymerization and movement. To provide additional evidence for the dependency of ARF1 in our system, we made use of our recent finding that ARHGAP21, a CDC42 GAP regulating actin dynamics on Golgi, associates with Golgi membranes through the interaction of its central ARF-binding domain (ARFBD) with GTP-bound ARF1 (17). Addition of 0.1 or 1 μM purified recombinant GST-tagged ARFBD to the assay resulted in a dose-dependent inhibition of actin polymerization on ARF1-GUVs, compared with 1 μM GST alone (Fig. 2C). Our interpretation of these data are that ARHGAP21-ARFBD, which binds GTP-bound ARF1 with a high affinity (K_d ≈ 50 nM) (18), prevents further activation of effector pathways involved in actin polymerization downstream of ARF1.

Overall, our data demonstrate the absolute requirement of GTP-ARF1 for actin polymerization around liposomes and the necessity of cytosolic factors, most probably proteins, to support actin polymerization downstream of ARF1.

ARF1-Dependent Actin Polymerization Requires CDC42 and N-WASP.

Although the machinery regulating actin dynamics on Golgi membranes is far from understood, it is now clear that ARF1 plays a critical role in Golgi actin assembly through the control of a cascade leading to CDC42/N-WASP/Arp2/3 complex activation (11, 12, 28). We checked that this activation was at work in our assay by immunoblotting for CDC42 on liposomes sedimented by centrifugation. This experiment revealed that, compared with naked LUVs, ARF1-LUVs recruited significantly higher amounts of CDC42 from the cytosolic extract, and this finding correlated with increased association of F-actin with LUVs (SI Fig. 5).

Because CDC42 has been shown to play a role in actin polymerization downstream of ARF1 on isolated Golgi membranes (11, 12), we characterized the role of CDC42 in our ARF1-liposome assay as follows. Specific inhibitors of the CDC42 cascade were added to the polymerization assay, and the actin shell around ARF1-GUVs was quantified as before. Secramine, a recently described specific inhibitor of CDC42 shown to block traffic of secreted proteins out of the Golgi complex (29), was first added to the assay at concentrations ranging from 1 to 20 μM. The addition of 1 μM secramine was sufficient to significantly reduce ARF1-dependent actin polymerization (P < 0.006) (Fig. 3A). In addition, the CDC42/Rac interactive binding (CRIB) region of PAK-1, which binds GTP-bound CDC42 (and Rac1) (30), was purified as a GST-fusion protein and added to the assay at concentrations ranging from 0.25 to 2 μM. Significant inhibition of actin polymerization around ARF1-GUVs was already observed at the lowest concentration of PAK-CRIB domain used (0.25 μM) (Fig. 3B). These results revealed that GTP-bound ARF1 on liposomes was able to trigger a cascade leading to CDC42 activation and, hence, to actin polymerization.

Fig. 3.

Inhibition of proteins of the ARF1 actin polymerization cascade. Quantification of actin polymerization around GUV after 30- to 45-min of incubation in supplemented extracts. Histograms of the radial mean of fluorescence for different GUVs arranged by bins of exponential size. ARF1-GUV (1% PIP2, 99% DOPC) incubated in supplemented extracts with different concentrations of drugs or proteins. Statistical comparison with the preceding concentration is provided. (A) Incubation with increasing concentrations of the secramine drug targeting CDC42. Concentration of drug vehicle (DMSO) was kept constant (1‰). (B) Incubation with increasing concentrations of the PAK-CRIB domain, which binds to and competes for activated CDC42. The 2 μM concentration of GST was kept constant. (C) Incubation with increasing concentrations of Wiskostatin that targets N-WASP. Concentration of drug vehicle (DMSO) was kept constant (1‰).

In its GTP-bound state, CDC42 binds directly to N-WASP, relieving N-WASP autoinhibition and allowing N-WASP to activate the actin polymerization nucleating capacity of the Arp2/3 complex (31). Wiskostatin was recently characterized as a chemical inhibitor of N-WASP, which stabilizes N-WASP in its autoinhibited conformation insensitive to activation by CDC42 and PIP2 (32). Addition of 10 μM Wiskostatin significantly inhibited actin polymerization at the surface of ARF1-GUVs (P < 0.0001) (Fig. 3C), indicating that ARF1 acts upstream of a well established CDC42/N-WASP/Arp2/3 cascade (33), leading to actin polymerization.

These findings demonstrate that membrane-bound ARF1 is competent for triggering CDC42 recruitment and activity, leading to N-WASP/Arp2/3 complex-dependent actin polymerization.

Role of Actin Dynamics in Membrane Remodeling in Vitro.

Because our ARF1-reconstitution assay reproduced the signaling cascade dissected in cellular systems, it provided a good tool for studying membrane deformations induced by the presence of ARF1. Two types of membrane reorganization were observed: liposomes carrying a comet tail or balls of actin with no enclosed volume (see Fig. 1 D–F). Liposomes with comets were observed 30 to 45 min after the start of actin polymerization and underwent motion by actin comet growth during the 30 min. Balls of actin appeared upon longer incubation times (≥2 h) and were preceded by actin shell growth around the liposomes. Note that balls of actin were in the range of several micrometers in diameter and were thus bigger than the liposomes supporting comet growth (<2 μm in diameter).

Liposome separation (i.e., one part of the vesicle moving away from the other part by the growth of an actin comet) was observed on ARF1-GUVs on rare occasions (see SI Movie 1). These events occurred on vesicles with an unusual oblate shape and an asymmetric actin shell.

Discussion

Our data demonstrate that the ARF1 cascade leads to actin polymerization by CDC42 activation and further triggering of N-WASP and likely the Arp2/3 complex at the cytosol/membrane interface. In addition, GTP-ARF1 appears to be indispensable for actin polymerization on DOPC/PIP2 GUVs, somewhat contrasting with previous findings that liposomes containing phosphoinositides could produce actin comets in cell extracts (34). However, it should be noted that these experiments used liposomes with different lipid composition (equal amounts of PI and PC and 4–33% PIP2) incubated in different extracts (Xenopus eggs instead of HeLa cells) (34). Another important finding from our study is that the effect of several CDC42 inhibitors, including the Golgi-associated, CDC42-specific GAP, ARHGAP21 (17), clearly demonstrates that activation of CDC42 (GTP-loading) downstream of ARF1 is indispensable for actin polymerization, and that this step can be reconstituted at the surface of liposomes incubated in cytosolic extracts in vitro. This finding is interesting given that the mechanisms leading to CDC42 activation on Golgi subcompartments downstream of ARF1 are not understood. The COPI could provide a link between ARF1 and CDC42 because it is recruited on the Golgi by binding to ARF1 and it is known to interact with GTP-bound CDC42 (11, 15). However, COPI alone is probably not directly responsible for CDC42 activation because replacement of GDP for GTP on Rho proteins requires specific guanine nucleotide exchange factors (GEFs). The larger family of Rho GEFs is the Dbl-related GEFs that comprise 69 distinct members in humans (35). Among these, only a few have been localized to the Golgi complex so far, including possibly Fgd1 and Dbs (36, 37). Whether these or other Rho GEFs are involved in CDC42 activation and regulation of actin dynamics on Golgi subcompartments is presently unknown. Moreover, the inhibition of ARF1-dependent actin polymerization by secramine (Fig. 3A) is a further indication that this in vitro system is able to reconstitute the full activation cascade of Cdc42. Indeed, in vitro secramine was shown to inhibit binding of Cdc42 to membranes in a RhoGDI-dependent manner (29). Therefore, this drug is thought to inhibit RhoGDI-dependent shuttling of GDP-bound Cdc42 between the cytosol and the target membrane, where GEF-assisted GDP/GTP exchange takes place. Finally, the inhibition of actin polymerization on GUVs we observed in the presence of the ARF1-binding domain of ARHGAP21 (ARFBD) is likely due to binding to GTP-ARF1 and competitive displacement of cytosolic ARF1 effectors required for actin assembly. We previously reported that expression of ARFBD in cells displaces ARF1 effectors such as AP-1 and COPI from the Golgi complex (17). This assumption also is supported by the nanomolar-range binding affinity of ARFBD for GTP-bound ARF1 (K_d ≈ 50 nM) (18). In addition, we observed a further significant inhibition of actin polymerization in the presence of ARFBD fused with the RhoGAP domain of ARHGAP21 (see SI Fig. 6). This effect might be due to RhoGAP-stimulated GTP hydrolysis on CDC42 associated with ARF1-GUVs and hence inhibition of CDC42-dependent actin polymerization.

Our findings that an unidentified CDC42-GEF is involved downstream of ARF1 in activating CDC42 on Golgi membranes, together with our previous observations that ARF1 recruits the CDC42-GAP ARHGAP21 protein on the Golgi complex (17), indicate that ARF1 is able to control the full GDP/GTP cycle of CDC42, and hence regulates actin dynamics on Golgi membranes. The availability of an in vitro system supporting a complex multicomponent signaling cascade leading to CDC42 activation downstream of ARF1 should help to clarify the implication of upstream regulators in the activation of CDC42 on Golgi membranes.

It is important to note that the positive influence of COPI on the recruitment of CDC42 could only account for actin polymerization on cis-medial Golgi cisternae, and not on the trans-side of the Golgi complex, the TGN, where ARF1 is known to control vesicle budding through clathrin and adaptor proteins, APs and GGAs (14). However, roles for CDC42 for exit of cargos out of the TGN also have been described (38), and additional mechanisms must exist to ensure CDC42-dependent actin polymerization on the TGN. In the endocytic pathway, CDC42 activity is regulated by the intersectin families of CDC42 GEFs, which also bind to N-WASP and are part of a core complex composed of dynamin, cortactin, ABP1, and syndapin involved in clathrin-dependent endocytosis (3, 39, 40). A homologue complex consisting of syndapin II and dynamin II promotes vesicle formation at the TGN (41). Whether this complex is linked to the ARF1/cortactin/dynamin pathway regulating post-Golgi transport (5, 10) and to CDC42 activity is presently unknown.

Analyzing the stresses and forces developed during actin polymerization allows us to understand the formation of either actin comet tails or balls of actin in our assay (see SI Discussion for a more complete analysis). We know from previous studies on polystyrene beads that the growth of an actin gel through Arp2/3 nucleation generates stresses inside the gel that lead to gel rupture and bead rocketing (42). On liposomes, which are water-permeable, forces developed during actin polymerization create an osmotic pressure difference that can lead to membrane rupture and solute leakage. Two scenarios can be imagined: Either the stress in the actin gel produces a comet-propelled liposome before pressure is too high to create solute leakage or the liposome leaks and shrinks slowly while actin continues polymerizing, thus producing balls of actin with no detectable liposome. The osmotic pressure at which the vesicle leaks depends inversely on the radius (see SI Discussion). Therefore, larger liposomes are more likely to leak, whereas smaller liposomes are more stable and allow comet formation to occur, which is what we see in our experiments.

Although balls of actin are probably not relevant in the Golgi, smaller liposomes propulsed by comets are reminiscent of what is observed when vesicles bud from the Golgi. We show in our assay that movement of liposomes carrying ARF1 can be triggered by actin polymerization. The force generated during this process has been well characterized in Arp2/3-based assays (43–46), and this force can lead to membrane separation, as observed in other Arp2/3-based liposome reconstitution assays where actin-associated vesicles were observed budding from larger membrane masses (43, 44). Actin polymerization might not be the sole factor responsible for vesicle separation. However, we propose that actin polymerization mediated by ARF1 might help vesicles to transiently pinch off and move away from donor compartments, reminiscent of models proposed for endocytosis (47, 48). The reconstitution of ARF1-dependent actin polymerization in a model system paves the way for a better understanding of the mechanism of vesicle dynamics on Golgi membranes.

Materials and Methods

Materials.

Rabbit muscle actin was from Cytoskeleton (Denver, CO), cytochalasin D and GTP-γ-S were purchased from Sigma–Aldrich (St. Louis, MO), and Wiskostatin was from Calbiochem (San Diego, CA). Lipids were obtained from Avanti Polar Lipids (Alabaster, AL), Alexa Fluor 594 rabbit muscle actin was from Molecular Probes (Eugene, OR), and anti-PIP2 antibody fluorescein conjugate was from Echelon (Salt Lake City, UT). Secramine was a gift from Eric Macia (Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France).

HeLa cell extracts were prepared as described in SI Materials and Methods. Total protein concentration of the extract, as measured by Bradford assay with a BSA standard, was 14 mg/ml.

Myristoylated ARF1 was prepared from Escherichia coli-coexpressing bovine ARF1 and yeast N-myristoyltransferase by a procedure described previously (24). Purification of GST-fusion protein of the PAK-CRIB and indicated domains of ARHGAP21 has been described previously (17).

Liposome Preparation.

One- to 20-μm-diameter GUVs were prepared by the gentle hydration method (22, 49). Three hundred micrograms of lipids solubilized in organic solvent was deposited on a Teflon disk and dried under vacuum for 2 h to ensure complete evaporation of the solvent. The Teflon disk was placed in a beaker, and 10 ml of a 0.28 M sucrose solution was carefully added. The liposomes grew spontaneously during an overnight incubation. The <0.80-μm-diameter LUVs were prepared by extrusion (23). When noted, 2% NBD-PC was added to the lipids as a fluorescent probe.

The difference in optical index between the sucrose solution inside the liposomes and the outside buffer solutions permitted the observation of the liposomes with phase-contrast microscopy. The osmolarity of these solutions was checked to prevent the bursting of the liposomes. Because PIP2 is notoriously difficult to mix with other lipids, we verified its presence by incubating the GUV with fluorescein-labeled anti-PIP2 antibody.

ARF1 Binding on Membranes.

To bind myr-ARF1 on GUVs, we used a modification of the procedure described in ref. 24. Thirty microliters of the liposome stock solution were mixed with 30 μl of Glu buffer [50 mM Hepes (pH 7.5), 2 mM MgCl₂, 180 mM d-glucose] supplemented with 1.5 mM final DTT, 350 μM final GTP-γ-S, 1.6 μM final of purified myr-ARF1, and 2.6 mM EDTA. GDP/GTP exchange and myr-ARF1 insertion on GUVs were allowed for 45–60 min at room temperature, and then 2.6 mM MgCl₂ was added. We washed the GUV with Glu buffer by 200 × g centrifugation for 20 min to reduce the concentration of free GTP-γ-S that could interact with other G proteins (residual GTP-γ-S concentration ≤10 μM). When theARHGAP21 effect was tested, a second centrifugation step was added (residual GTP-γ-S final concentration ≤1 μM). We prepared ARF1-LUVs in the same way, except that the concentration of liposomes was 0.25 mg/ml and centrifugation was omitted (residual GTP-γ-S concentration ≤10 μM).

Motility and Polymerization Assay.

For motility and polymerization assays, 1-μl extracts were mixed with 1 μl of G-actin (partly labeled) in G buffer, 1 μl of a solution containing ATP, creatin-phosphate and 1,4-diazabicyclo[2.2.2]octane (DABCO), and 4 μl of ARF1-GUVs washed in Glu buffer. When drugs or peptide were added to the assay, it was diluted in 1 μl of Glu buffer, and 1 μl of ARF1-GUVs was omitted instead. Final concentrations were 2 mg/ml extracts, 3.8 μM rabbit muscle G-actin, 290 nM Alexa Fluor 594 G-actin, 1 mM ATP, 20 mM creatin-phosphate, and 135 μM 1,4-diazabicyclo[2.2.2]octane (DABCO). Final salinity was equivalent to 80 mM KCl. A higher salinity (150 mM KCl) did not significantly affect the assay (see SI Fig. 7). GUVs were observed with an IX70 Olympus inverted microscope and an Olympus ×100/N.A. 1.35 phase-contrast objective (Olympus, Tokyo, Japan). Fluorescent-labeled molecules were excited by a 200-W mercury lamp (OSRAM, Munich, Germany). Images were recorded with a CCD camera (Roper Scientific, Trenton, NJ) driven by MetaMorph software (Universal Imaging Corporation, Downingtown, PA).

Quantification of Actin Polymerization Around GUVs.

Fluorescent images were analyzed by using ImageJ software (http://rsb.info.nih.gov/ij) to quantify the amount of fluorescently labeled actin present around the membrane of the GUVs. Using the plug-in “Oval Profile Plot,” the maximum intensity value along a radius starting from the center of the GUV was taken. This measure was repeated 360 times by rotating the radius, producing a circumferential profile. The mean value of this profile was taken as a measure of the quantity of actin around a GUV. For each experimental condition, 40–70 GUVs were analyzed, and results were gathered in a histogram. Because the distribution of actin quantity was similar to a log-normal law, we used a logarithmic scale for the fluorescence intensity (i.e., we distributed the data into bins of exponentially increasing size). Different experimental conditions were compared by using a Wilcoxon rank-sum test. Because of some variability in the assay, statistical analysis was performed only on data sets obtained on the same day with the same batch of ARF1-GUVs and extracts.

Abbreviations

GUV

giant unilamellar vesicle

LUV

large unilamellar vesicle

TGN

trans-Golgi network.