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.

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.

The integrin family of extracellular matrix receptors regulates many aspects of cell life, in particular cell adhesion and migration. These two processes depend on organization of the actin cytoskeleton into adhesive and protrusive organelles in response to extracellular signals. Integrins are important switch points for the spatio-temporal control of actin-based motility in higher eukaryotes. Ligands of integrin cytoplasmic tails are central elements of signalling pathways involving small GTPases as well as protein and lipid kinases in the regulation of F-actin crosslinking, actin treadmilling and de novo nucleation of actin filaments. We present an overview of common pathways and discuss recent evidence for their differential use by individual integrin receptors.

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.

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.

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.

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 complex1,2,3. The physical mechanism of force generation by actin polymerization has been described theoretically using various approaches4,5,6, but lacks support from experimental data. By the use of reconstituted motility medium7, we find that the Wiskott–Aldrich syndrome protein8,9 (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’ Listeria10. 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.

gamma-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs. A minor fraction of the protein shows subsarcolemmal localization. Although gamma-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20. Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon. Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs. The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of gamma-filamin. The two Ig-like domains of the recently described alpha-actinin-binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of gamma-filamin may be indirectly anchored to alpha-actinin in the Z-disc via myotilin. Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease.

Conference Abstract| October 01 2000 The interaction of filamin-C with myotilin: a novel link in the limb girdle muscular dystrophy pathway D. O. Fuerst; D. O. Fuerst 1University of Potsdam, Dept. Cell Biology, Lennestr. 7a, D-14471 Potsdam, Germany Search for other works by this author on: This Site PubMed Google Scholar S. Wiesner; S. Wiesner 1University of Potsdam, Dept. Cell Biology, Lennestr. 7a, D-14471 Potsdam, Germany Search for other works by this author on: This Site PubMed Google Scholar D. Auerbach; D. Auerbach *Inst. Cell Biology, Swiss Fed. Inst. Technol., ETH Hoenggerberg, Zuerich, Switzerland Search for other works by this author on: This Site PubMed Google Scholar M. Himmel; M. Himmel 1University of Potsdam, Dept. Cell Biology, Lennestr. 7a, D-14471 Potsdam, Germany Search for other works by this author on: This Site PubMed Google Scholar S. Kempa; S. Kempa 1University of Potsdam, Dept. Cell Biology, Lennestr. 7a, D-14471 Potsdam, Germany Search for other works by this author on: This Site PubMed Google Scholar K. Hayeß; K. Hayeß 1University of Potsdam, Dept. Cell Biology, Lennestr. 7a, D-14471 Potsdam, Germany Search for other works by this author on: This Site PubMed Google Scholar D. Pacholsky; D. Pacholsky 1University of Potsdam, Dept. Cell Biology, Lennestr. 7a, D-14471 Potsdam, Germany Search for other works by this author on: This Site PubMed Google Scholar R. Schroeder; R. Schroeder **Dept. Neurology, University Hospital Bonn, Germany Search for other works by this author on: This Site PubMed Google Scholar P. F. M. van der Ven P. F. M. van der Ven 1University of Potsdam, Dept. Cell Biology, Lennestr. 7a, D-14471 Potsdam, Germany Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2000) 28 (5): A300. https://doi.org/10.1042/bst028a300b Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation D. O. Fuerst, S. Wiesner, D. Auerbach, M. Himmel, S. Kempa, K. Hayeß, D. Pacholsky, R. Schroeder, P. F. M. van der Ven; The interaction of filamin-C with myotilin: a novel link in the limb girdle muscular dystrophy pathway. Biochem Soc Trans 1 October 2000; 28 (5): A300. doi: https://doi.org/10.1042/bst028a300b Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2000 Biochemical Society2000 Article PDF first page preview Close Modal You do not currently have access to this content.