Purification of Arp2/3 complex from Saccharomyces cerevisiae.

Much of the cellular control over actin dynamics comes through regulation of actin filament initiation. At the molecular level, this is accomplished through a collection of cellular protein machines, called actin nucleation factors, which position actin monomers to initiate a new actin filament. The Arp2/3 complex is a principal actin nucleation factor used throughout the eukaryotic family tree. The budding yeast Saccharomyces cerevisiae has proven to be not only an excellent genetic platform for the study of the Arp2/3 complex, but also an excellent source for the purification of endogenous Arp2/3 complex. Here we describe a protocol for the preparation of endogenous Arp2/3 complex from wild type Saccharomyces cerevisiae. This protocol produces material suitable for biochemical study and yields milligram quantities of purified Arp2/3 complex.

Purification of native Arp2/3 complex from bovine thymus.

The Arp2/3 complex is an actin filament nucleator involved in cell motility and vesicle trafficking. Owing to the role the complex plays in important and fundamental cell biological processes, the purified complex is used in biochemical assays, reconstituted motility assays, and structural biology. As this is a eukaryotic complex assembled from seven polypeptides, the complex is purified from eukaryotic sources. Described here is a detailed method for purification of the complex from a mammalian tissue, bovine thymus.

Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

Nucleotide- and activator-dependent structural and dynamic changes of arp2/3 complex monitored by hydrogen/deuterium exchange and mass spectrometry.

Arp2/3 complex plays a central role in the de novo nucleation of filamentous actin as branches on existing filaments. The complex must bind ATP, protein activators [e.g., Wiskott-Aldrich syndrome protein (WASp)], and the side of an actin filament to form a new actin filament. Amide hydrogen/deuterium exchange coupled with mass spectrometry was used to examine the structural and dynamic properties of the mammalian Arp2/3 complex in the presence of both ATP and the activating peptide segment from WASp. Changes in the rate of hydrogen exchange indicate that ATP binding causes conformational rearrangements of Arp2 and Arp3 that are transmitted allosterically to the Arp complex (ARPC)1, ARPC2, ARPC4, and ARPC5 subunits. These data are consistent with the closure of nucleotide-binding cleft of Arp3 upon ATP binding, resulting in structural rearrangements that propagate throughout the complex. Binding of the VCA domain of WASp to ATP-Arp2/3 further modulates the rates of hydrogen exchange in these subunits, indicating that a global conformational reorganization is occurring. These effects may include the direct binding of activators to Arp3, Arp2, and ARPC1; alterations in the relative orientations of Arp2 and Arp3; and the long-range transmission of activator-dependent signals to segments proposed to be involved in binding the F-actin mother filament.

Phosphorylation of the Arp2/3 complex is necessary to nucleate actin filaments.

The actin-related protein 2/3 (Arp2/3) complex is the primary nucleator of new actin filaments in most crawling cells. Nucleation-promoting factors (NPFs) of the Wiskott-Aldrich syndrome protein (WASP)/Scar family are the currently recognized activators of the Arp2/3 complex. We now report that the Arp2/3 complex must be phosphorylated on either threonine or tyrosine residues to be activated by NPFs. Phosphorylation of the Arp2/3 complex is not necessary to bind NPFs or the sides of actin filaments but is critical for binding the pointed end of actin filaments and nucleating actin filaments. Mass spectrometry revealed phosphorylated Thr237 and Thr238 in Arp2, which are evolutionarily conserved residues. In cells, phosphorylation of only the Arp2 subunit increases in response to growth factors, and alanine substitutions of Arp2 T237 and T238 or Y202 inhibits membrane protrusion. These findings reveal an additional level of regulation of actin filament assembly independent of WASP proteins, and show that phosphorylation of the Arp2/3 complex provides a logical "or gate" capable integrating diverse upstream signals.

Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex.

Capping protein (CP) is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between CP and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of CP and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility.

X-ray scattering study of activated Arp2/3 complex with bound actin-WCA.

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.

Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium.

We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2/3 complex, as bait. After denaturation and SDS-PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified.

Arp2/3 complex-mediated actin polymerisation occurs on specific pre-existing networks in cells and requires spatial restriction to sustain functional lamellipod extension.

The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre-existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex-mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex-mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network.

In vitro reconstitution of cdc42-mediated actin assembly using purified components.

In the accompanying chapter, we describe an in vitro system that uses Xenopus egg extracts to study actin assembly induced by phosphatidylinositol (4,5)bisphosphate (PIP2) and Cdc42. Biochemical fractionation and candidate screening experiments conducted in the extract system have identified the Arp2/3 complex, the N-WASP-WIP (or N-WASP-CR16) complex, and the Cdc42-binding protein Toca-1 as important mediators of PIP2- and Cdc42-actin signaling. Toward our ultimate goal of reconstituting an in vitro system that recapitulates the signaling properties observed in vivo, we then developed a purified actin assembly assay system consisting of the regulatory components that we discovered from extracts. In these assays, the stereotypical sigmoidal kinetics of actin polymerization are monitored by pyrene-actin fluorescence in the presence of defined recombinant or purified proteins, enabling the detailed study of mechanism and protein function. In this chapter, we describe the preparation of the components used in these purified actin assembly reactions, as well as the assay conditions under which we monitor actin polymerization kinetics in vitro.

Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast.

Yeast actin patches are dynamic structures that form at the sites of cell growth and are thought to play a role in endocytosis. We used biochemical analysis and live cell imaging to investigate actin patch assembly in fission yeast Schizosaccharomyces pombe. Patch assembly proceeds via two parallel pathways: one dependent on WASp Wsp1p and verprolin Vrp1p converges with another dependent on class 1 myosin Myo1p to activate the actin-related protein 2/3 (Arp2/3) complex. Wsp1p activates Arp2/3 complex via a conventional mechanism, resulting in branched filaments. Myo1p is a weaker Arp2/3 complex activator that makes unstable branches and is enhanced by verprolin. During patch assembly in vivo, Wsp1p and Vrp1p arrive first independent of Myo1p. Arp2/3 complex associates with nascent activator patches over 6-9 s while remaining stationary. After reaching a maximum concentration, Arp2/3 complex patches move centripetally as activator proteins dissociate. Genetic dependencies of patch formation suggest that patch formation involves cross talk between Myo1p and Wsp1p/Vrp1p pathways.