Actin cytoskeleton: formins lead the way.

Formins are eukaryotic proteins that potently influence actin polymerization dynamics. Recent evidence strongly suggests that these proteins move processively with the elongating barbed ends of actin filaments.

Actin nucleation: nucleation-promoting factors are not all equal.

Arp2/3 complex plays a key role in regulated actin polymerization. A recent study has revealed marked differences in the ability of two nucleation-promoting factors - N-WASP and Scar/WAVE1 - to activate the Arp2/3 complex. Further insights have come from determination of the Arp2/3 crystal structure.

Crystal structure of Arp2/3 complex.

We determined a crystal structure of bovine Arp2/3 complex, an assembly of seven proteins that initiates actin polymerization in eukaryotic cells, 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. We predict 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.

The verprolin-like central (vc) region of Wiskott-Aldrich syndrome protein induces Arp2/3 complex-dependent actin nucleation.

Wiskott-Aldrich Syndrome protein (WASp) and related proteins stimulate actin filament nucleation by Arp2/3 complex. The isolated C-terminal VCA domain of WASp (containing Verprolin-like, Central and Acidic regions) is constitutively active but autoinhibited in the full-length protein. This study compared the ability of parts of VCA fused to the C terminus of glutathione S-transferase (GST) to bind actin and Arp2/3 complex in vitro and to activate actin polymerization in vitro and in cells. Fluorescence anisotropy measurements showed that GST-CA and GST-A bound Arp2/3 complex with K(d) values of 0.11 microm and 1.0 microm, respectively, whereas GST-VC displayed almost undetectable binding (K(d) > 1 mm). However, GST-VC activated actin nucleation through Arp2/3 complex in vitro, though requiring 70-fold higher concentration than GST-VCA while neither GST-CA nor GST-A activated Arp2/3 complex in vitro, though both GST-CA and GST-A inhibited Arp2/3 complex activation by WASp VCA. None of these constructs bound WASp from macrophage lysates. Both GST-VC and GST-CA induced actin accumulations when microinjected into primary human macrophages or human endothelial vein cells. However, only microinjection of GST-VC led to a significant increase of cellular polymerized actin. Additionally, endogenous Arp2/3 complex, but not WASp, colocalized with these GST-VC-induced actin accumulations. These data suggest that WASp constructs lacking the A region, previously thought to be indispensable for actin nucleation, are able to bind and activate Arp2/3 complex in vitro and in vivo.

Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins.

Actin filament assembly and turnover drive many forms of cellular motility, particularly extension of the leading edge of locomoting cells and rocketing of pathogenic microorganisms through host cell cytoplasm. De novo nucleation of actin filaments appears to be required for these movements. A complex of seven proteins called Arp2/3 complex is the best characterized cellular initiator of actin filament nucleation. Arp2/3 complex is intrinsically inactive, relying on nucleation promoting factors for activation. WASp/Scar family proteins are prominent cellular nucleation promoting factors. They bring together an actin monomer and Arp2/3 complex in solution or on the side of an existing actin filament to initiate a new filament that grows in the barbed end direction. WASp and N-WASP are intrinsically autoinhibited, and their activity is regulated by Rho-family GTPases such as Cdc42, membrane polyphosphoinositides, WIP/verprolin, and SH3 domain proteins. These interactions provide a final common pathway for many signaling inputs to regulate actin polymerization. Microorganisms either activate Arp2/3 complex directly or usurp N-WASP to initiate actin polymerization.

Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex.

The Wiskott-Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a Kd of 0.6 microM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable DeltaH of -10 kcal mol(-1) drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 microM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott-Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 complex.

Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex.

We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 microM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP(2)) micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP(2) and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42 was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 microM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction.

Membrane lipids have multiple effects on interfacial catalysis by a phosphatidic acid-preferring phospholipase A1 from bovine testis.

We previously purified a cytosolic phospholipase A1 that could catalyze the preferential hydrolysis of phosphatidic acid in mixed-micelle assays. Here we studied the enzyme's interactions with unilamellar lipid membranes and examined effects of the lipids on enzyme binding, stability, and catalysis. A major finding was that membrane lipids could influence the stability, activity, and specificity of the enzyme under conditions where enzyme binding to the membranes was likely to be saturated. Thus, the enzyme was unstable at 37 degrees C in the absence of membranes but bound to membranes that contained anionic phosphoglycerides and could be stabilized by these membranes in the presence of albumin. The overall activity of the bound enzyme toward membrane phosphoglycerides, assayed in the presence of albumin, increased when phosphatidylethanolamine was substituted for phosphatidylcholine. Furthermore, the enzyme's catalytic preference for phosphatidic acid increased when cholesterol and diacylglycerol were included in the membranes, sn-1-stearoyl-2-arachidonoylphosphatidylethanolamine was substituted for sn-1-palmitoyl-2-oleoylphosphatidylethanolamine, and the concentration of phosphatidic acid was increased from 0 to 10 mol % of the total membrane phosphoglycerides. Finally, changes in the relative contents of phosphatidylcholine and phosphatidylserine in the membranes influenced the enzyme's catalytic preference for different molecular species of phosphatidic acid. These results provide the first available information about the enzyme's ability to interact with membranes and identify conditions that yield high enzyme activity toward membrane-associated phosphatidic acid.

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization.

The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent K(d) of 0.4 microM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp WA and <10 nM Arp2/3 complex, there is a 1:1 correspondence between the Arp2/3 complex and the concentration of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determined to be 9.7 microM in human neutrophils. Preformed filaments increase the rate of nucleation by WASp WA and the Arp2/3 complex but not the number of filaments that are generated. We propose that filament side binding by the Arp2/3 complex enhances its activation by WASp WA.

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.

The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.

Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex.

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament-a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

Interactions of Acanthamoeba profilin with actin and nucleotides bound to actin.

Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity of profilin for actin. The affinity for rabbit skeletal muscle actin is about 4-fold lower. The affinity for both actins is 5-8-fold lower with ADP bound to actin rather than ATP. Pyrenyliodoacetamide labeling of cysteine 374 of muscle actin reduces the affinity for profilin 10-fold. The affinity of profilin for nucleotide-free actin is approximately 3-fold higher than for Mg-ATP-actin and approximately 24-fold higher than for Mg-ADP-actin. As a result, profilin binding reduces the affinity of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. Mg-ATP dissociates 8 times faster from actin-profilin than from actin and binds actin-profilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as fast as actin. Thus, profilin promotes the exchange of ADP for ATP. These properties allow profilin to bind a high proportion of unpolymerized ATP-actin in the cell, suppressing spontaneous nucleation but allowing free barbed ends to elongate at more than 500 subunits/second.

Cloning of a phosphatidic acid-preferring phospholipase A1 from bovine testis.

We report the molecular cloning and expression of a phosphatidic acid-preferring phospholipase A1 from bovine testis. The open reading frame encoded an 875-amino acid protein with a calculated molecular mass of 97,576 daltons and a pI of 5.61. The sequence included a region similar to a lipase consensus sequence containing the putative active site serine and also included a potential, coiled-coil-forming region. Expression of the open reading frame in COS1 cells resulted in a 20-44-fold increase in phosphatidic acid phospholipase A1 activity over that of control cells. Mutation of the putative active site serine (amino acid 540) demonstrated that it was essential for this increase in enzyme activity. Northern blot analysis revealed at least five different messages with the highest overall message levels in mature testis, but detectable message in all tissues examined. Two possible alternately spliced regions in the open reading frame also were identified. Finally, a search of the data base identified six related proteins: a potential counterpart of the phospholipase A1 in Caenorhabditis elegans, two putative lipases in yeast, and three proteins separately encoded by the Drosophila retinal degeneration B gene and its mouse and human homologues.

Purification and properties of a phosphatidic acid-preferring phospholipase A1 from bovine testis. Examination of the molecular basis of its activation.

We recently identified a cytosolic phospholipase A1 activity in bovine brain and testis that preferentially hydrolyzes phosphatidic acid substrates. We also showed that the enzyme displays sigmoidal kinetics toward phosphatidic acid substrates in Triton X-100 mixed micelle assay system (Higgs, H.N., and Glomset J.A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9574-9578). In the present work we purified the bovine testis enzyme 14,000-fold and used a combination of size exclusion chromatography, labeling with the phospholipase A inhibitor, methyl arachidonyl fluorophosphonate, and SDS-polyacrylamide gel electrophoresis to provide evidence that it is a homotetramer of 110-kDa subunits. Studies of the molecular basis of the enzyme reaction in Triton micelles revealed that (a) a nonhydrolyzable sn-1-alkyl-2-oleoyl-analogue of phosphatidic acid activated the enzyme 30-fold in a sigmoidal fashion (Hill coefficient 3.2, EC50 4 mol %) without substantially affecting its preference for specific diacyl phosphoglyceride substrates, (b) the activator promoted tight binding of the enzyme to micelles, and (c) the enzyme's activity toward unsaturated phosphatidic acid substrates was affected by the location and nature of the fatty acyl chain double bonds.

Identification of a phosphatidic acid-preferring phospholipase A1 from bovine brain and testis.

Recent experiments in several laboratories have provided evidence that phosphatidic acid functions in cell signaling. However, the mechanisms that regulate cellular phosphatidic acid levels remain obscure. Here we describe a soluble phospholipase A1 from bovine testis that preferentially hydrolyzes phosphatidic acid when assayed in Triton X-100 micelles. Moreover, the enzyme hydrolyzes phosphatidic acid molecular species containing two unsaturated fatty acids in preference to those containing a combination of saturated and unsaturated fatty acyl groups. Under certain conditions, the enzyme also displays lysophospholipase activity toward lysophosphatidic acid. The phospholipase A1 is not likely to be a lysosomal enzyme because its optimum pH is 7.5-8.5. Furthermore, it is probably not a general lipid metabolic enzyme because high levels of activity are found in mature testis and brain but no measurable activity is seen in liver, spleen, or heart. The fact that the activity of the phospholipase A1 in mature bovine testis is > 10-fold higher than that in newborn calf testis raises the possibility that the enzyme may play a regulatory role in spermatogenesis or sperm function.

Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.

Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.