Kurt J. Amann

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.

Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy

Existing methods for studying actin filament dynamics have allowed analysis only of bulk samples or individual filaments after treatment with the drug phalloidin, which perturbs filament dynamics. Total internal reflection fluorescence microscopy with rhodamine-labeled actin allowed us to observe polymerization in real time, without phalloidin. Direct measurements of filament growth confirmed the rate constants measured by electron microscopy and established that rhodamine actin is a kinetically inactive tracer for imaging. In the presence of activated Arp2/3 complex, growing actin filaments form branches at random sites along their sides, rather than preferentially from their barbed ends.

The structural basis of actin filament branching by the Arp2/3 complex.

The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.

A cluster of basic repeats in the dystrophin rod domain binds F-actin through an electrostatic interaction.

The dystrophin rod domain is composed of 24 spectrin-like repeats and was thought to act mainly as a flexible spacer between the amino-terminal actin binding domain and carboxyl-terminal membrane-associated domains. We previously demonstrated that a fragment of the dystrophin rod domain also binds F-actin. However, the nature and extent of rod domain association with F-actin is presently unclear. To begin addressing these questions, we characterized two recombinant proteins representing adjacent regions of the dystrophin rod. DYS1416 (amino acids 1416–1880) bound F-actin with a K d of 14.2 ± 5.2 μm and a stoichiometry of 1 mol:mol of actin. However, DYS1030 (amino acids 1030–1494) failed to bind F-actin, suggesting that not all rod domain repeats are capable of binding F-actin. Interestingly, DYS1416 corresponds to a unique region of the dystrophin rod rich in basic amino acids, whereas DYS1030 is composed mainly of acidic repeats. This observation suggested that DYS1416 may interact with acidic actin filaments through an electrostatic interaction. Supporting this hypothesis, actin binding by DYS1416 was dramatically inhibited by increasing ionic strength. We suggest that electrostatic interactions between basic spectrin-like repeats and actin filaments may contribute to the actin binding activity of other members of the actin cross-linking protein family.

EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

Utrophin lacks the rod domain actin binding activity of dystrophin.

We previously identified a cluster of basic spectrin-like repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419–28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11–17) or the homologous region of the utrophin rod domain (UTROR11–16). Although UTROR11–16 is 64% similar and 41% identical to DYSR11–17, UTROR11–16 (pI = 4.86) lacks the basic character of the repeats found in DYSR11–17 (pI = 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly α-helical fold and existed as a highly soluble monomer. DYSR11–17 bound F-actin with an apparentK d of 7.3 ± 1.3 μm and a molar stoichiometry of 1:5. Significantly, UTROR11–16 failed to bind F-actin at concentrations as high as 100 μm. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin.

Dystrophin binding to nonmuscle actin

We purified actin from bovine brain by DNase I affinity chromatography in order to compare the binding of dystrophin to muscle actin with its binding to nonmuscle actin. While both beta- and gamma-nonmuscle actins are expressed in brain, Western blot analysis with isoform-specific antibodies indicated that our purified brain actin was exclusively the gamma-isoform. The recombinant amino-terminal, actin-binding domain of dystrophin bound to muscle and brain actin in a saturable manner (approximately 1 mol/mol actin) with similar Kd values of 13.7+/-3.5 and 10.6+/-3.7 microM, respectively. We further demonstrate that intact dystrophin in the dystrophin-glycoprotein complex bound with equal avidity to muscle and brain F-actin. These data argue that a preferential binding of dystrophin to nonmuscle actin is not the basis for its targeting to the muscle cell plasmalemma but do support the hypothesis that dystrophin is capable of interacting with filamentous actin in nonmuscle tissues.

Assembly properties of the Bacillus subtilis actin, MreB.

The bacterial actin MreB has been implicated in a variety of cellular roles including cell shape determination, cell wall synthesis, chromosome condensation and segregation, and the establishment and maintenance of cell polarity. Toward elucidating a clearer understanding of how MreB functions inside the bacterial cell, we investigated biochemically the polymerization of MreB from Bacillus subtilis. Light scattering and sedimentation assays revealed pH-, ionic-, cationic-, and temperature-dependent behavior. B. subtilis MreB polymerizes in the presence of millimolar divalent cations in a protein concentration-dependent manner. Polymerization is favored by decreasing pH and inhibited by monovalent salts and low temperatures. Although B. subtilis MreB binds and hydrolyzes both ATP and GTP, it does not require a bound nucleotide for assembly and polymerizes indistinguishably regardless of the nucleotide species bound, with a critical concentration of approximately 900 nM. A number of the presently reported properties of B. subtilis MreB differ significantly from those of T. maritima MreB1 (Bean and Amann [2008]: Biochemistry 47: 826-835), including the nucleotide requirements and temperature and ionic effects on polymerization state. These observations collectively suggest that additional factors interact with MreB to account for its complex dynamic behavior in cells.

Cellular regulation of actin network assembly

Bray D: Cell Movements, 2nd edn. London: Garland Publishing; 2000. Higgs HN, Pollard TD: Regulation of actin polymerization by Arp2/3 complex and WASp/Scar proteins. J Biol Chem 1999, 274:32531-32534. Kreis T, Vale R: Guidebook to the Cytoskeletal and Motor Proteins, 2nd edn. Oxford: Oxford University Press; 1999. Pollard TD, Blanchoin L, Mullins RD: Molecular mechanisms controlling actin filament dynamics in nonmuscle cells.Ann Rev Biophys Biomolec Struct 2000, 29:545-576.