A structural model of the profilin-formin pacemaker system for actin filament elongation.

The formins constitute a large class of multi-domain polymerases that catalyze the localization and growth of unbranched actin filaments in cells from yeast to mammals. The conserved FH2 domains form dimers that bind actin at the barbed end of growing filaments and remain attached as new subunits are added. Profilin-actin is recruited and delivered to the barbed end by formin FH1 domains via the binding of profilin to interspersed tracts of poly-L-proline. We present a structural model showing that profilin-actin can bind the FH2 dimer at the barbed end stabilizing a state where profilin prevents its associated actin subunit from directly joining the barbed end. It is only with the dissociation of profilin from the polymerase that an actin subunit rotates and docks into its helical position, consistent with observations that under physiological conditions optimal elongation rates depend on the dissociation rate of profilin, independently of cellular concentrations of actin subunits.

Tropomyosin assembly intermediates in the control of microfilament system turnover.

Tropomyosin is a coiled-coil alpha-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MW(app) 180,000-250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.

The microfilament system and malignancy.

Increased motile activity, increased rate of cell proliferation and removal of growth inhibiting cell-cell contacts are hallmarks of tumorigenesis. Activation of cell motility and migration is caused by activation of receptors, turning on the growth cycle. Increased expression of metalloproteinases, breaking cell:cell contacts and organ confines, allows the spread of malignant cancer cells to other sites in the organism. It has become increasingly clear that most transmembrane proteins (growth factor receptors, adhesion proteins and ion channels) are either permanently or transiently associated with the sub-membraneous system of actin microfilaments (MF), whose force generating capacity they control. Although there has been great progress in our understanding of the physiological importance of the MF-system, as will be exemplified in this issue of SCB, many aspects of actin microfilament formation and its regulation are still unclear. Redox control of the actin (MF)-system in cell motility and migration and its perturbations in pathophysiology, including cancer, is an emerging field of research.

Tropomyosins regulate the impact of actin binding proteins on actin filaments.

The state of actin depends intimately on its interaction partners in eukaryotic cells. Classically, the cooperative force-generating acto-myosin couple is turned off and on by the calcium-dependent binding and release of tropomyosin molecules. The situation with nonmuscle cells appears to be much more complicated, with tropomyosin isoforms regulating the kinds of tension-producing and stress-bearing structures formed of actin filaments. The polymerization of even the shortest gelsolin-capped filaments is efficiently promoted by the binding of tropomyosin, for example, a process that might occur all the way out to the leading edges of advancing cells. Recently, multimers of tropomyosin have been discovered that appear to be assembly intermediates, formed from identical tropomyosin molecules, which act as ready pools of tropomyosin during the catalytic formation of lamellipodia and filopodia. Remarkably, these multimers apparently reform during the disassembly of cellular actin-containing structures. The existence of these recyclable, tropomyosin isoform-specific structures suggests how cells prevent nonproductive association of non-identical, but closely similar, tropomyosin isoforms.

Molecular and structural basis for redox regulation of beta-actin.

An essential consequence of growth factor-mediated signal transduction is the generation of intracellular H(2)O(2). It operates as a second messenger in the control of actin microfilament dynamics, causing rapid and dramatic changes in the morphology and motile activity of stimulated cells. Little is understood about the molecular mechanisms causing these changes in the actin system. Here, it is shown that H(2)O(2) acts directly upon several levels of this system, and some of the mechanistic effects are detailed. We describe the impact of oxidation on the polymerizability of non-muscle beta/gamma-actin and compare with that of muscle alpha-actin. Oxidation of beta/gamma-actin can cause a complete loss of polymerizability, crucially, reversible by the thioredoxin system. Further, oxidation of the actin impedes its interaction with profilin and causes depolymerization of filamentous actin. The effects of oxidation are critically dependent on the nucleotide state and the concentration of Ca(2+). We have determined the crystal structure of oxidized beta-actin to a resolution of 2.6 A. The arrangement in the crystal implies an antiparallel homodimer connected by an intermolecular disulfide bond involving cysteine 374. Our data indicate that this dimer forms under non-polymerizing and oxidizing conditions. We identify oxidation of cysteine 272 in the crystallized actin dimer, likely to a cysteine sulfinic acid. In beta/gamma-actin, this is the cysteine residue most reactive towards H(2)O(2) in solution, and we suggest plausible structural determinants for its reactivity. No other oxidative modification was obvious in the structure, highlighting the specificity of the oxidation by H(2)O(2). Possible consequences of the observed effects in a cellular context and their potential relevance are discussed.

Tropomyosins are present in lamellipodia of motile cells.

This paper shows that high-molecular-weight tropomyosins (TMs), as well as shorter isoforms of this protein, are present in significant amounts in lamellipodia and filopodia of spreading normal and transformed cells. The presence of TM in these locales was ascertained by staining of cells with antibodies reacting with endogenous TMs and through the expression of hemaglutinin- and green fluorescent protein-tagged TM isoforms. The observations are contrary to recent reports suggesting the absence of TMs in regions,where polymerization of actin takes place, and indicate that the view of the role of TM in the formation of actin filaments needs to be significantly revised.

Activation in isolation: exposure of the actin-binding site in the C-terminal half of gelsolin does not require actin.

Gelsolin requires activation to carry out its severing and capping activities on F-actin. Here, we present the structure of the isolated C-terminal half of gelsolin (G4-G6) at 2.0 A resolution in the presence of Ca(2+) ions. This structure completes a triptych of the states of activation of G4-G6 that illuminates its role in the function of gelsolin. Activated G4-G6 displays an open conformation, with the actin-binding site on G4 fully exposed and all three type-2 Ca(2+) sites occupied. Neither actin nor the type-l Ca(2+), which normally is sandwiched between actin and G4, is required to achieve this conformation.

Tropomyosin and gelsolin cooperate in controlling the microfilament system.

Tropomyosin has been shown to cause annealing of gelsolin-capped actin filaments. Here we show that tropomyosin is highly efficient in transforming even the smallest gelsolin-actin complexes into long actin filaments. At low concentrations of tropomyosin, the effect of tropomyosin depends on the length of the actin oligomer, and the cooperative nature of the process is a direct indication that tropomyosin induces a conformational change in the gelsolin-actin complexes, altering the structure at the actin (+) end such that capping by gelsolin is abolished. At increased concentrations of tropomyosin, heterodimers, trimers, and tetramers are converted to actin filaments. In addition, evidence is presented demonstrating that gelsolin, once removed from the (+) end of the actin, can reassociate with the newly formed tropomyosin-decorated actin filaments. Interestingly, the binding of gelsolin to the tropomyosin-actin filament complexes saturates at 2 gelsolin molecules per 14 actin and 2 tropomyosins, i.e. two gelsolins per tropomyosin-regulatory unit along the filament. These observations support the view that both tropomyosin and gelsolin are likely to have important functions in addition to those proposed earlier.

The role of MeH73 in actin polymerization and ATP hydrolysis.

In actin from many species H73 is methylated, but the function of this rare post-translational modification is unknown. Although not within bonding distance, it is located close to the gamma-phosphate of the actin-bound ATP. In most crystal structures of actin, the delta1-nitrogen of the methylated H73 forms a hydrogen bond with the carbonyl of G158. This hydrogen bond spans the gap separating subdomains 2 and 4, thereby contributing to the forces that close the interdomain cleft around the ATP polyphosphate tail. A second hydrogen bond stabilizing interdomain closure exists between R183 and Y69. In the closed-to-open transition in beta-actin, both of these hydrogen bonds are broken as the phosphate tail is exposed to solvent. Here we describe the isolation and characterization of a mutant beta-actin (H73A) expressed in the yeast Saccharomyces cerevisiae. The properties of the mutant are compared to those of wild-type beta-actin, also expressed in yeast. Yeast does not have the methyl transferase necessary to methylate recombinant beta-actin. Thus, the polymerization properties of yeast-expressed wild-type beta-actin can be compared with normally methylated beta-actin isolated from calf thymus. Since earlier studies of the actin ATPase almost invariably employed rabbit skeletal alpha-actin, this isoform was included in these comparative studies on the polymerization, ATP hydrolysis, and phosphate release of actin. It was found that H73A-actin exchanged ATP at an increased rate, and was less stable than yeast-expressed wild-type actin, indicating that the mutation affects the spatial relationship between the two domains of actin which embrace the nucleotide. At physiological concentrations of Mg(2+), the kinetics of ATP hydrolysis of the mutant actin were unaffected, but polymer formation was delayed. The comparison of methylated and unmethylated beta-actin revealed that in the absence of a methyl group on H73, ATP hydrolysis and phosphate release occurred prior to, and seemingly independently of, filament formation. The comparison of beta and alpha-actin revealed differences in the timing and relative rates of ATP hydrolysis and P(i)-release.

Novel sulfoxides facilitate GC-rich template amplification.

Certain organic solvents, such as DMSO and betaine, have been reported to enhance PCR amplification, particularly for hard-to-amplify high-GC templates. As a result of extensive structure-activity studies between two groups of compounds--amides and sulfones--we have recently discovered several other potent PCR enhancers. Here we describe the effects of a series of different sulfoxides on GC-rich template amplification and report several of these to be exceptionally effective, often outperforming DMSO. We introduce them as novel PCR enhancers. We identify tetramethylene sulfoxide as the most potent sulfur-oxygen compound in the enhancement of PCR amplification and as one of the most potent PCR enhancers currently known.

A cross-linked profilin-actin heterodimer interferes with elongation at the fast-growing end of F-actin.

Profilin and beta/gamma-actin from calf thymus were covalently linked using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in combination with N-hydroxysuccinimide, yielding a single product with an apparent molecular mass of 60 kDa. Sequence analysis and x-ray crystallographic investigations showed that the cross-linked residues were glutamic acid 82 of profilin and lysine 113 of actin. The cross-linked complex was shown to bind with high affinity to deoxyribonuclease I and poly(l-proline). It also bound and exchanged ATP with kinetics close to that of unmodified profilin-actin and inhibited the intrinsic ATPase activity of actin. This inhibition occurred even in conditions where actin normally forms filaments. By these criteria the cross-linked profilin-actin complex retains the characteristics of unmodified profilin-actin. However, the cross-linked complex did not form filaments nor copolymerized with unmodified actin, but did interfere with elongation of actin filaments in a concentration-dependent manner. These results support a polymerization mechanism where the profilin-actin heterodimer binds to the (+)-end of actin filaments, followed by dissociation of profilin, and ATP hydrolysis and P(i) release from the actin subunit as it assumes its stable conformation in the helical filament.