Four polypeptides of 47, 44, 40, and 35 kD have been identified that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurify as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that vary in their proportions to the other polypeptides. Partial protein sequences show that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin (Machesky, 1994).
The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14. The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. Structural and topological models for the Arp2/3 complex are proposed and it is suggested that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba (Mullins, 1997).
Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. An essential gene has been identified and characterized from Schizosaccharomyces pombe, sop2+, a mutation that rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and act1-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the beta-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species, and a human cDNA highly related to sop2+, SOP2 Hs, has been isolated, that functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba beta-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex that also contains the actin-related protein, Arp3p. Immunofluorescence studies reveal the presence of Sop2p in (1) punctate structures distributed throughout the cell, (2) cables that extend the length of the cell, and (3) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin (Balasubramanian, 1996).
Migration of cells through the reorganization of the actin cytoskeleton is essential for morphogenesis of multicellular animals. In a cell culture system, the actin-related protein (Arp) 2/3 complex functions as a nucleation core for actin polymerization when activated by the members of the WASP (Wiskott-Aldrich syndrome protein) family. However, the regulation of cell motility in vivo remains poorly understood. Homologs of the mammalian Arp2/3 complex and N-WASP in Caenorhabditis elegans play an important role in hypodermal cell migration during morphogenesis, a process known as ventral enclosure. In the absence of one of any of the C. elegans Arp2/3 complex subunits (ARX-1, ARX-2, ARX-4, ARX-5, ARX-6 or ARX-7) or of N-WASP (WSP-1), hypodermal cell migration led by actin-rich filopodia formation is inhibited during ventral enclosure owing to the reduction of filamentous actin formation. However, there is no effect on differentiation of hypodermal cells and dorsal intercalation. Disruption of the function of ARX-1 and WSP-1 in hypodermal cells also results in hypodermal cell arrest during ventral enclosure, suggesting that their function is cell autonomous. WSP-1 protein activates Arp2/3-mediated actin polymerization in vitro. Consistent with these results, the Arp2/3 complex and WSP-1 colocalize at the leading edge of migrating hypodermal cells. The stable localization of WSP-1 is dependent on the presence of Arp2/3 complex, suggesting an interaction between the Arp2/3 complex and WSP-1 in vivo (Sawa, 2003).
The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits that include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc. The predicted amino acid sequence of all seven subunits has been determined. Each has homologs in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. Attempts were made to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion (Welch, 1997).
Human neutrophils contain a complex of proteins similar to the actin-related protein 2/3 (Arp2/3) complex of Acanthamoeba. Peptide sequence information has been obtained for each member of the putative seven-protein complex that has been described for Acanthamoeba and human platelets. From the peptide sequences cDNA species encoding three novel proteins in this complex were identified. In addition to Arp2 and Arp3, this complex contains a relative of the human (Suppressor of Profilin) SOP2Hs protein and four previously unknown proteins. These proteins localize in the cytoplasm of fibroblasts that lack lamellipodia, but are enriched in lamellipodia on stimulation with serum or platelet-derived growth factor. A conserved and dynamic role for this complex in the organization of the actin cytoskeleton is proposed (Machesky, 1997).
Structural modeling and biochemical experiments in vitro have implicated a multi-protein complex containing two actin-related proteins, Arp2 and Arp3, as a potential actin-filament nucleation factor. This 'Arp2/3 complex' has been identified in Acanthamoeba and human cells and has been shown to localize to regions involved in actin-based motility, such as the leading edge of moving cells and the 'tail' of actin that forms behind the intracellular pathogen Listeria. The function of this complex in vivo has not been characterized, however, and the sequences of the non-actin-related subunits remain to be determined. Arp3 homolog from the budding yeast Saccharomyces cerevisiae was found to localize to cortical actin patches, highly motile structures that concentrate at sites of polarized growth during the yeast cell cycle. A conditional arp3 mutant allele inhibits cortical actin motility at the restrictive temperature and eventually disrupts actin patches. Most Arp3 protein is found in a multi-protein complex; this complex was purified and the sequences of each of the protein subunits were determined using a high-accuracy mass peptide-mapping technique. The proteins found in the complex are similar to those in the Acanthamoeba and human Arp2/3 complexes. It is concluded that the Arp2/3 protein complex is conserved from yeast to man, and in yeast the complex is required in vivo for the motility and integrity of cortical actin patches. It is hypothesized that these patches may move by a Listeria-like mechanism driven by actin polymerization (Winter, 1997).
The yeast Arp2/3 complex localizes to cortical actin patches and is required for patch motility and integrity in vivo. This complex contains proteins homologous to each subunit of the Acanthamoeba and human Arp2/3 complex except for a 40-kDa subunit (p40), which was missing from the purified yeast complex. Immunoprecipitation and gel-filtration analysis has been used to demonstrate that Arc40p, the homolog of p40 identified from the yeast genome database, associates with the yeast Arp2/3 complex. Gene disruptions of each subunit of the yeast Arp2/3 complex have been carried out to study each subunit's role in the function of the complex. Surprisingly, only ARC40 is found to be fully essential for cell viability. Strains lacking each of the other subunits exhibit varying degrees of defects in cell growth and viability and in assembly and polarization of cortical actin patches. Each subunit's role in maintaining the structural integrity of the Arp2/3 complex was examined. Arp2p, Arp3p, and Arc40p fall into the monomer pool in Deltaarc19 and Deltaarc35 cells, suggesting that Arc19p and Arc35p are the central scaffolding components of the complex. Arp2p and Arp3p do not have major roles in maintaining complex integrity, and Arc15p is required for association of Arp2p and Arc40p, but not other subunits, with the complex. These results provide evidence that each subunit contributes differently to the assembly and function of the Arp2/3 complex (Winter, 1999).
The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, high-copy suppressors of the S. pombe arp3-c1 mutant were sought; one was identified that was termed asp1+. The asp1+ open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1+ nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1+ leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant is synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain shows a negative genetic interaction with a las17Delta strain. It is concluded that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton (Feoktistova, 1999).
ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains are shown to bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. It is proposed that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and requires simultaneous binding of Arp2 and Arp3 (Zalevsky, 2000).
The Arp2/3 complex is a seven-protein assembly that is critical for actin nucleation and branching in cells. Active human Arp2/3 complex has been reconstituted after expression of all seven subunits in insect cells. Expression of partial complexes reveals that a heterodimer of the p34 and p20 subunits constitutes a critical structural core of the complex, whereas the remaining subunits are peripherally located. Arp3 is crucial for nucleation, consistent with it being a structural component of the nucleation site. p41, p21, and p16 contribute differently to nucleation and stimulation by ActA and WASP, whereas p34/p20 bind actin filaments and likely function in actin branching. This study reveals that the nucleating and organizing functions of Arp2/3 complex subunits are separable, indicating that these activities may be differentially regulated in cells (Gournier, 2001).
Because the recombinant complex (rArp2/3) is indistinguishable from the the native complex (nArp2/3 ) in its subunit composition and biochemical activity, it was possible to use the baculovirus system to evaluate the role of individual subunits in the integrity of the complex. To determine the structural role of each subunit, all possible combinations of six subunits (seven combinations in total) were expressed by infecting Sf9 cells with a subset of viruses. The capacity of the six subunits to form a subcomplex was first analyzed by immunoprecipitation using an anti-p41 antibody. This antibody was chosen because it is the only antibody available that recognizes the native Arp2/3 complex. As a control, cells were infected with viruses encoding all seven subunits. All seven could be coimmunoprecipitated, although the exact 1:1:1:1:1:1:1 stoichiometry was not maintained, suggesting that both the intact complex and partial subcomplexes were present (Gournier, 2001).
When either Arp2 or p21 was omitted during expression, a subcomplex missing only the respective subunit was isolated. In contrast, when Arp3 was omitted, a subcomplex missing both Arp3 and p21 was immunoprecipitated, indicating that Arp3 is important for the association of p21 with the complex. As expected, no subcomplex could be immunoprecipitated with the anti-p41 antibody when p41 was omitted. When p34 was left out, all of the other subunits were isolated, but in reproducibly smaller quantities. No subcomplexes were isolated when p20 or p16 were omitted. Thus, p20 and p16, and to a lesser extent p34, are important either for the association of p41 with the complex, or the complex's overall integrity (Gournier, 2001).
To distinguish between these possibilities, gel filtration chromatography was carried out on lysates prepared from cells expressing all subunits except for p41, p34, p20, or p16. Individual subunits were detected in each column fraction by immunoblotting. When p41 or p16 are omitted, a peak of the other polypeptides is observed in fractions 12-15, corresponding to a slightly smaller size than the intact complex, which peaks in fractions 11-14. This suggests that the loss of these subunits does not affect the complex's general integrity. Moreover, only traces of p41 are present in fractions 12-15 from the extract missing p16, indicating that p16 is important for the association of p41 with the complex or for the expression or stability of p41 in insect cells. When p34 is omitted, a minor peak of the other subunits is observed in fractions 12-15, whereas the major peak is observed in fractions 16-20 and higher. This suggests that a complex missing only p34 can form, but that loss of p34 causes destabilization, resulting in the accumulation of smaller subcomplexes. When p20 is omitted, no clear peak of the other subunits is observed, suggesting that an intact complex is not formed. Therefore, the loss p20 disrupts the general structural integrity of the entire Arp2/3 complex (Gournier, 2001).
Arp2/3 subcomplex missing p16 and p41 (rArp2/3 -p16-p41) but containing the remaining five subunits at the correct stoichiometry were purified. rArp2/3 -p16-p41 possesses some activity when stimulated with ActA or WASP-PWCA , although it is less active than the complex missing p21. This suggests that p16 and p41 are important but not essential for nucleation. To quantify and compare the activities of the intact rArp2/3 complex and two of the subcomplexes, rArp2/3 -p21 and rArp2/3 -p16-p41, the calculated concentration of barbed ends generated by each was plotted versus the concentration of the complex. In the range of concentrations tested, there was a linear relationship between the variables. The slope of this line gave a measure of the number of ends created per complex, or the nucleation efficiency. For the intact rArp2/3 complex, the value was 0.85 ends/complex, or 85% efficiency. For rArp2/3 -p21, it was 0.07 or 7% efficiency, approximately 12-fold lower than the intact complex. For rArp2/3 -p16-p41, the slope was 0.01 or 1% efficiency, approximately 85-fold less than the intact complex. Thus, p21 and p16/p41 contribute differently to the nucleation activity of the Arp2/3 complex (Gournier, 2001).
In addition to nucleating actin polymerization, the Arp2/3 complex can crosslink actin filaments into Y-branched arrays. Because the subcomplexes missing p21 and p16/p41 exhibit defects in nucleation, attempts were made to determine whether they were also deficient in branching activity. Branching was visualized using fluorescence microscopy after staining filaments in polymerization reactions with rhodamine-phalloidin. In this assay, the ActA-stimulated intact rArp2/3 complex and nArp2/3 complex branched actin to the same extent (50% branching at 10 nM Arp2/3, 80% at 20 nM Arp2/3), indicating that the recombinant complex is fully active. Because nucleation and branching are tightly coupled, the branching activity of the two subcomplexes, Arp2/3 -p21 and Arp2/3 -p16-p41, was tested at concentrations approximately 10-fold and 100-fold above the intact complex to normalize for differences in nucleation efficiency. At these concentrations, the ActA-stimulated Arp2/3 -p16-p41 subcomplex branched to approximately the same extent as the intact complex (45% branching), whereas Arp2/3 -p21 exhibited slightly reduced branching frequency (12% branching). Branches formed by both subcomplexes were indistinguishable in appearance from those formed by the intact complex. Thus, p41/p16 and p21, while important for nucleation, are dispensable for branch formation by the Arp2/3 complex (Gournier, 2001).
A crystal structure of bovine Arp2/3 complex, an assembly of seven proteins that initiates actin polymerization in eukaryotic cells, has been determined at 2.0 angstrom resolution. Actin-related protein 2 (Arp2) and Arp3 are folded like actin, with distinctive surface features. Subunits ARPC2 p34 and ARPC4 p20 in the core of the complex associate through long carboxyl-terminal alpha helices and have similarly folded amino-terminal alpha/beta domains. ARPC1 p40 is a seven-blade beta propeller with an insertion that may associate with the side of an actin filament. ARPC3 p21 and ARPC5 p16 are globular alpha-helical subunits. It is predicted that WASp/Scar proteins activate Arp2/3 complex by bringing Arp2 into proximity with Arp3 for nucleation of a branch on the side of a preexisting actin filament (Robinson, 2001).
Dynamic actin assembly is required for diverse cellular processes and often involves activation of Arp2/3 complex. Cortactin and N-WASp activate Arp2/3 complex, alone or in concert. Both cortactin and N-WASp contain an acidic (A) domain that is required for Arp2/3 complex binding. How cortactin and the constitutively active VCA domain of N-WASp interact with Arp2/3 complex was investigated. Structural studies show that cortactin is a thin, elongated monomer. Chemical crosslinking studies demonstrate selective interaction of the Arp2/3 binding NTA domain of cortactin (cortactin NTA) with the Arp3 subunit and VCA with Arp3, Arp2, and ARPC1/p40. Cortactin NTA and VCA crosslinking to the Arp3 subunit are mutually exclusive; however, cortactin NTA does not inhibit VCA crosslinking to Arp2 or ARPC1/p40, nor does it inhibit activation of Arp2/3 complex by VCA. An experiment was conducted in which a saturating concentration of cortactin NTA modestly lowered the binding affinity of VCA for Arp2/3; the results of this experiment provided further evidence for ternary complex formation. Consistent with a common binding site on Arp3, a saturating concentration of VCA abolishes binding of cortactin to Arp2/3 complex. It is concluded that under certain circumstances, cortactin and N-WASp can bind simultaneously to Arp2/3 complex, accounting for their synergy in activation of actin assembly. The interaction of cortactin NTA with Arp2/3 complex does not inhibit Arp2/3 activation by N-WASp, despite competition for a common binding site located on the Arp3 subunit. These results suggest a model in which cortactin may bridge Arp2/3 complex to actin filaments via Arp3 and N-WASp activates Arp2/3 complex by binding Arp2 and/or ARPC1/p40 (Weaver, 2002).
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