Joern Linkner

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Vasodilator‐stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration‐dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane‐associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.

Inhibitory signalling to the Arp2/3 complex steers cell migration

Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott–Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac–Arpin–Arp2/3 inhibitory circuit with the Rac–WAVE–Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.

Molecular mechanism of Ena/VASP-mediated actin-filament elongation

Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro, they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G‐actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP‐mediated filament elongation depends on G‐actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP‐mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP‐mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled.

CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high‐density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53‐dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.

The application of the Cre-loxP system for generating multiple knock-out and knock-in targeted loci.

Dictyostelium discoideum is an exceptionally powerful eukaryotic model to study many aspects of growth, development, and fundamental cellular processes. Its small-sized, haploid genome allows highly efficient targeted homologous recombination for gene disruption and knock-in epitope tagging. We previously described a robust system for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of the Cre recombinase. We have now further optimized the system for higher efficiency and, additionally, coupled it to both, knock-out and knock-in gene targeting, allowing the characterization of multiple and cooperative gene functions in a single cell line.

Analysis of Actin Assembly by In Vitro TIRF Microscopy

Since directed movement toward an extracellular chemoattractant requires rapid and continuous reorganization of the actin cytoskeleton to form complex structures such as a protruding lamellipodium, it is of great interest to analyze and understand the individual contribution of proteins specifically involved in this process. Over the last decade, enormous progress has been made toward understanding the versatile molecular mechanisms underlying actin-based cell motility and the regulation of site-specific F-actin assembly and disassembly. In spite of this wealth of knowledge and due to the constant discovery of novel regulatory factors, many questions remain to be answered. In this chapter, we describe a powerful method that allows to study the effects of actin-binding proteins on the assembly of single filaments by in vitro total internal reflection fluorescence (TIRF) microscopy using purified proteins and fluorescently labeled actin.

The inverse BAR domain protein IBARa drives membrane remodeling to control osmoregulation, phagocytosis and cytokinesis

ABSTRACT Here, we analyzed the single inverse Bin/Amphiphysin/Rvs (I-BAR) family member IBARa from Dictyostelium discoideum. The X-ray structure of the N-terminal I-BAR domain solved at 2.2 Å resolution revealed an all-&agr;-helical structure that self-associates into a 165-Å zeppelin-shaped antiparallel dimer. The structural data are consistent with its shape in solution obtained by small-angle X-ray scattering. Cosedimentation, fluorescence anisotropy, and fluorescence and electron microscopy revealed that the I-BAR domain bound preferentially to phosphoinositide-containing vesicles and drove the formation of negatively curved tubules. Immunofluorescence labeling further showed accumulation of endogenous IBARa at the tips of filopodia, the rim of constricting phagocytic cups, in foci connecting dividing cells during the final stage of cytokinesis and most prominently at the osmoregulatory contractile vacuole (CV). Consistently, IBARa-null mutants displayed defects in CV formation and discharge, growth, phagocytosis and mitotic cell division, whereas filopodia formation was not compromised. Of note, IBARa-null mutants were also strongly impaired in cell spreading. Taken together, these data suggest that IBARa constitutes an important regulator of numerous cellular processes intimately linked with the dynamic rearrangement of cellular membranes.

Highly effective removal of floxed Blasticidin S resistance cassettes from Dictyostelium discoideum mutants by extrachromosomal expression of Cre.

The inactivation of proteins in cells is inevitable to study their physiological role in various cellular processes. In contrast to strategies to alter the amount of active proteins in cells, only a gene knockout guarantees complete removal of the protein of interest. For Dictyostelium discoideum cells, the gene replacement construct typically consists of a Blasticidin S resistance (Bsr) cassette flanked by fragments of the target gene to allow insertion by homologous recombination. More advanced knockout constructs additionally carry loxP sites on both sides of the Bsr cassettes for subsequent removal of the selection marker by transient expression of Cre recombinase, thus allowing generation of multiple knockouts using just a single selection marker. However, due to its design, the available neomycin selection-based Cre expression plasmid occasionally tends to integrate into the genome and also yield only a moderate number of transfectants in liquid media. In some cases, for instance in SCAR-null cells, it was not possible to remove the Bsr cassette without stable integration of the Cre expression vector into the genome. To circumvent these difficulties we designed the extrachromosomal Cre-recombinase expression vector pTX-NLS-Cre. We verified the greatly improved efficacy of this novel Cre-loxP approach by removal of the Bsr cassette in five different cell lines including the SCAR-null mutant. As a consequence, this vector will be a highly valuable means for the rapid generation of single or multiple mutants remaining sensitive to the most reliable selection markers Blasticidin S and neomycin.

High-Resolution X-Ray Structure of the Trimeric Scar/ WAVE-Complex Precursor Brk1

The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration. We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes. Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography. Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle. Notably, this structure is highly similar to the heterotrimeric α-helical bundle of HSPC300/WAVE1/Abi2 within the human Scar/WAVE-complex. This finding, together with the fact that Brk1 is collectively sandwiched by the remaining subunits and also constitutes the main subunit connecting the triple-coil domain of the HSPC300/WAVE1/Abi2/ heterotrimer to Sra1(Pir1), implies a critical function of this subunit in the assembly process of the entire Scar/WAVE-complex.

Differential functions of WAVE regulatory complex subunits in the regulation of actin-driven processes

The WAVE regulatory complex (WRC) links upstream Rho-family GTPase signaling to the activation of the ARP2/3 complex in different organisms. WRC-induced and ARP2/3 complex-mediated actin nucleation beneath the plasma membrane is critical for actin assembly in the leading edge to drive efficient cell migration. The WRC is a stable heteropentamer composed of SCAR/WAVE, Abi, Nap, Pir and the small polypeptide Brk1/Hspc300. Functional interference with individual subunits of the complex frequently results in diminished amounts of the remaining polypeptides of the WRC complex, implying the complex to act as molecular entity. However, Abi was also found to associate with mammalian N-WASP, formins, Eps8/SOS1 or VASP, indicating additional functions of individual WRC subunits in eukaryotic cells. To address this issue systematically, we inactivated all WRC subunits, either alone or in combination with VASP in Dictyostelium cells and quantified the protein content of the remaining subunits in respective WRC knockouts. The individual mutants displayed highly differential phenotypes concerning various parameters, including cell morphology, motility, cytokinesis or multicellular development, corroborating the view of additional roles for individual subunits, beyond their established function in WRC-mediated Arp2/3 complex activation. Finally, our data uncover the interaction of the actin polymerase VASP with WRC-embedded Abi to mediate VASP accumulation in cell protrusions, driving efficient cell migration.