Maud Hertzog

Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex.

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53–Eps8 complex, supporting the existence of a Cdc42–IRSp53–Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53–Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes

Neural Wiskott–Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.

Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement

Abstract.Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.

Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s−1, respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

Eps8 regulates axonal filopodia in hippocampal neurons in response to brain-derived neurotrophic factor (BDNF)

A novel signaling cascade controlling actin polymerization in response to extracellular signals regulates filopodia formation and likely also neuronal synapse formation.

Molecular Basis for the Dual Function of Eps8 on Actin Dynamics:Bundling and Capping

The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo.

Structure, Function, and Evolution of the β‐Thymosin/WH2 (WASP‐Homology2) Actin‐Binding Module

Abstract:  β‐thymosins are acknowledged G‐actin sequesterers. However, in the recent years, the conserved β‐thymosins/WH2 actin‐binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using thymosin β4 (Tβ4) and Ciboulot, a 3 β‐thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tβ4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot‐actin shows that the major interaction with G‐actin lies in the N‐terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tβ4 can be changed into a profilin activity. (1H, 15N)‐NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C‐terminal regions of the WH2 domain with subdomains 1 and 2 of G‐actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tβ4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tβ4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.

Functional Characterization of Proteins Regulating Actin Assembly

A very large, ever-increasing repertoire of actin-binding proteins regulates the assembly dynamics and the spatial organization of actin filaments, thus orchestrating the motile behavior of the cell. The authors describe a series of biochemical functional assays that allow one to characterize the function of a putative actin-binding protein in actin filament dynamics. These tests allow the characterization of three types of actin-binding proteins: G-actin-sequestering proteins, profilin-like proteins, and barbed-end capping proteins. Biochemical tests include the use of sedimentation of actin filaments, polymerization assays at the barbed or pointed end of actin filaments derived from fluorescently labeled actin, thermodynamic measurements of actin assembly at steady state and during turnover of actin filaments, measurements of nucleotide exchange on G-actin, and the use of the intrinsic or extrinsic fluorescence of actin to measure direct binding of different protein ligands to G-actin.

Correction to: Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)(PLoS Biol, (2015), 13,6)

The authors have realized that the composition of the gel image in S5D Fig is not clearly explained in the figure legend, nor is it marked adequately in the figure itself. We provide therefore an amended legend and figure, which clarify the compilation of the S5D Fig and the presentation of the data. In addition, and at the editors’ request, the original data related to Fig. 6D in this paper were made available. Following verification of these original data, the editors were satisfied with the data provided. A supplementary S6 Fig, which comprises the original film of the immunoblot (upper panel of Fig. 6D) and the original picture of the entire nitrocellulose membrane (lower panel of Fig. 6D) used to reveal GST fusion proteins employed in the pull down experiment described in Fig. 6D, has been included as supplementary material.