The Ru(IV)=O-catalyzed sulfoxidation: a gated mechanism where O to S linkage isomerization switches between different efficiencies.

Two Ru(IV)=O catalysts with either a pentadentate bispidine ligand L(1) or a bidentate pyrazolate L(2)/terpy L(3) combination of ligands have very different efficiencies as oxygen transfer catalysts for the selective oxidation of sulfides to sulfoxides: the [Ru(II)(L(1))(solvent)](2+)/iodosyl benzene system has an initial TOF of approx. 40 h(-1) and quantitative yield, with [Ru(II)(L(2))(L(3))(solvent)](+) the initial TOF is approx. 12 h(-1) with a maximum yield of approx. 60%. By experiment (cyclovoltametry) it is shown that there is S- to O-linkage isomerization of the Ru(II) sulfoxide product complex, and this may partially switch off the catalytic cycle for the L(2)/L(3)-based catalyst. It emerges that the reasons for the reduced efficiency in the case of the L(2)/L(3), in comparison with the L(1)-based catalyst, are a more efficient linkage isomerization, a more stable S-bonded, in comparison with the O-bonded, Ru(II)-based isomer, and inefficient ligand exchange in the product (hydrolysis produces the free sulfoxide and the Ru(II) precatalyst). These interpretations are qualitatively in good agreement with preliminary DFT-based data.

Iron vs. ruthenium--a comparison of the stereoselectivity in catalytic olefin epoxidation.

The synthesis and the full characterization of a new ruthenium(II) complex with the pentadentate bispidine ligand L(1) is reported and shown to be a very active catalyst for olefin epoxidation. The selectivity in the epoxidation of cis- and trans-beta-methylstyrene with the formation of cis and trans products, each, was determined and compared with that of the iron bispidine complex of L(1). There is a significant difference in selectivity between the two catalysts in the epoxidation of cis-beta-methylstyrene but the epoxidation of trans-beta-methylstyrene is highly stereoselective with both catalysts. Based on these results, electrochemical and labeling studies, a radical pathway for the epoxidation and isomerization is proposed, and this is supported by computational data. DFT indicates that, with both catalysts, the epoxidation is based on a stepwise mechanism, which, in the first step leads to a radical-based intermediate. This exists in two configurations, which interconvert with a relatively low energy barrier. The product ratio depends on the relative energies of the two configurations of the radical intermediate and the height of the energy barriers to the cis- and trans-epoxide products. For the Fe-based system there is, as expected, the additional complication of the availability of various spin levels, and multi-state reactivity is observed. The computed structures and energies are in agreement with the observed data.

Profilin 1 is required for abscission during late cytokinesis of chondrocytes.

Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1-mediated actin filament elongation. Neither an actin nor a poly-proline binding-deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.

Local call: from integrins to actin assembly.

Integrins link the extracellular matrix to the actin cytoskeleton by triggering the assembly of different types of adhesion complex. One of their major components is filamentous actin (F-actin), and they are important signaling hubs for actin cytoskeleton reorganization in response to chemical and mechanical signals. In an exciting publication, Butler et al. have demonstrated for the first time that purified adhesion complexes possess the entire machinery necessary to actively assemble F-actin as a function of integrin activity and clustering.

Visualizing cytoskeleton dynamics in mammalian cells using a humanized variant of monomeric red fluorescent protein.

Fluorescent proteins are versatile tools for live cell imaging studies. In particular, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation and intracellular fluorescence. mRFPmars, a red fluorescent protein designed especially for the use in Dictyostelium, proved to be a brilliant label for different cytoskeletal elements. Here we report on the synthesis of a humanized version of a monomeric RFP, mRFPruby, which differs in sequence from mRFPmars in four amino acids and has a codon usage that is optimized for the application in mammalian cells. In order to demonstrate the usefulness of this new mRFP variant, mRFPruby fused to beta-actin was expressed in different mouse cell lines and used to visualize actin cytoskeleton dynamics by live cell microscopy.

Actin-based motility as a self-organized system: mechanism and reconstitution in vitro.

Site-directed actin polymerisation in response to signalling is responsible for the formation of cell protrusions. These elementary 'actin-based motility processes' are involved in cell locomotion, cell metastasis, organ morphogenesis and microbial pathogenesis. We have reconstituted actin-based propulsive movement of particles of various sizes and geometries (rods, microspheres) in a minimum motility medium containing five pure proteins. The ATP-supported treadmilling of actin filaments, regulated by Actin Depolymerizing Factor (ADF/cofilin), profilin and capping proteins provides the thermodynamic basis for sustained actin-based movement. Local activation of Arp2/3 complex at the surface of the particle promotes autocatalytic barbed end branching of filaments, generating a polarized arborescent array. Barbed end growth of branched filaments against the surface generates a propulsive force and is eventually arrested by capping proteins. Understanding the mechanism of actin-based movement requires elucidation of the biochemical properties and mode of action of Arp2/3 complex in filament branching, in particular the role of ATP binding and hydrolysis in Arp2/3, and a physical analysis of the movement of functionalised particles. Because the functionalisation of the particle by an activator of Arp2/3 complex (N-WASP or the Listeria protein ActA) and the concentrations of effectors in the medium are controlled, the reconstituted motility assay allows an analysis of the mechanism of force production at the mesoscopic and molecular levels.

Actin-based motility: from molecules to movement.

Extensive progress has been made recently in understanding the mechanism by which cells move and extend protrusions using site-directed polymerization of actin in response to signalling. Insights into the molecular mechanism of production of force and movement by actin polymerization have been provided by a crosstalk between several disciplines, including biochemistry, biomimetic approaches and computational studies. This review focuses on the biochemical properties of the proteins involved in actin-based motility and shows how these properties are used to generate models of force production, how the predictions of different theoretical models are tested using a biochemically controlled reconstituted motility assay and how the changes in motility resulting from changes to the concentrations of components of the assay can help understand diverse aspects of the motile behavior of living cells.

A biomimetic motility assay provides insight into the mechanism of actin-based motility.

Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 microm) nor by increasing the viscosity of the medium by 10(5)-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.

The dynamics of actin-based motility depend on surface parameters.

In cells, actin polymerization at the plasma membrane is induced by the recruitment of proteins such as the Arp2/3 complex, and the zyxin/VASP complex. The physical mechanism of force generation by actin polymerization has been described theoretically using various approaches, but lacks support from experimental data. By the use of reconstituted motility medium, we find that the Wiskott Aldrich syndrome protein (WASP) subdomain, known as VCA, is sufficient to induce actin polymerization and movement when grafted on microspheres. Changes in the surface density of VCA protein or in the microsphere diameter markedly affect the velocity regime, shifting from a continuous to a jerky movement resembling that of the mutated 'hopping' Listeria. These results highlight how simple physical parameters such as surface geometry and protein density directly affect spatially controlled actin polymerization, and play a fundamental role in actin-dependent movement.