The structure of FMNL2–Cdc42 yields insights into the mechanism of lamellipodia and filopodia formation (original) (raw)
Related papers
FMNL2 Drives Actin-Based Protrusion and Migration Downstream of Cdc42
Current Biology, 2012
Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE . It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex . Here we show that the Diaphanousrelated formin FMNL2, also known as FRL3 or FHOD2 [3], accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.
Journal of Cell Science, 2013
Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.
2004
Otto Hahn Strasse 11 tional GTPase binding domain (GBD) which is involved D-44227 Dortmund in binding the GTP-bound form of Rho proteins (Evange-Germany lista et al., 1997; Kohno et al., 1996; Watanabe et al., ), connected to the FH3 domain of undefined length University of Kent and structure. In Drfs the activity of the protein is regu-Canterbury, Kent CT2 7NJ lated by the C-terminal diaphanous autoregulatory do-United Kingdom main (DAD), which binds to the GBD and inhibits biologi-3 Research Group for Fluorescence Spectroscopy cal activity (Watanabe et al., 1999; Alberts, 2001). While Office for Academy Research Groups Attached mDia is directly mediating Rho-induced actin reorganito Universities and Other Institutions zation (Watanabe et al., 1997, 1999), it has also been 4 Department of Biophysics implicated in microtubule alignment and stabilization Faculty of Medicine (Ishizaki et al., 2001; Palazzo et al., 2001). University of Pé cs It has been shown that the FH2 domain plays an im-H-7601 Pé cs portant role for the function of formin family proteins. Hungary Mutations in the FH2 domain abolish both the bundling 5 Department of Pharmacology of actin filaments and the alignment of microtubules Kyoto University Faculty of Medicine induced by an active mutant of mDia1 lacking the GBD Yoshida, Sakyo-ku, Kyoto 606-8501 (Ishizaki et al., 2001). A close link exists between the Japan control of cytoskeletal organization and the transcription factor serum response factor (SRF), where SRF is negatively regulated by the actin monomer pool in the Summary cells (Posern et al., 2002; Sotiropoulos et al., 1999). For this system it has been shown in cell culture studies Diaphanous-related formins (Drf) are activated by Rho that the lysophosphatidic acid (LPA)-induced activation GTP binding proteins and induce polymerization of of SRF is mediated by mDia and in particular by the unbranched actin filaments. They contain three formin region encompassing the FH2 domains (Copeland and homology domains. Evidence as to the effect of for-Treisman, 2002). mins on actin polymerization were obtained using Fragments of various length covering the FH2 do-FH2/FH1 constructs of various length from different mains of formins exhibited activity to nucleate actin in Drfs. Here we define the core FH2 domain as a proteovitro (Pruyne et al., 2002; Sagot et al., 2002b; Pring et lytically stable domain of approximately 338 residues. al., 2003; Kovar et al., 2003; Li and Higgs, 2003). All the The monomeric FH2 domains from mDia1 and mDia3 FH2 fragments from either S. cerevisiae Bni1p, S. pombe inhibit polymerization of actin and can bind in a 1:1 Cdc12p, or mDia1 used in the previous studies (Figure complex with F-actin at micromolar concentrations. 1A) have in common the property to affect barbed-end The X-ray structure analysis of the domain shows an kinetics of F-actin via a complete or partial capping elongated, crescent-shaped molecule consisting of mechanism. Although the FH1 domain is dispensable three helical subdomains. The most highly conserved for in vitro actin polymerization induced by the FH2 doregions of the domain span a distance of 75 Å and are main, it modulates the activity of the FH2 domain by its both required for barbed-end inhibition. A construct interaction with profilin (Pruyne et al., 2002; Sagot et al., containing an additional 72 residue linker has dramati-2002b; Kovar et al., 2003; Li and Higgs, 2003). It has cally different properties: It oligomerizes and induces been also proposed that these fragments form a dimer actin polymerization at subnanomolar concentration. or higher oligomers, which is likely related to their function (Li and Higgs, 2003; Zigmond et al., 2003; Takeya Introduction and Sumimoto, 2003). The exact domain boundaries of the FH2 domain and The formin proteins play important roles in many differthe minimal functional unit for actin nucleation were ent cellular processes such as cytokinesis, vesicular not clear. To dissect and structurally define the domain trafficking, and the maintenance of cell polarity by reguboundaries of these proteins and to understand the conlating actin and microtubule cytoskeletons (Wallar and tribution of the core FH2 domain (as defined in this Alberts, 2003). They have in common a poly-proline-rich
Journal of Biological Chemistry
The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), that enhances RhoA-mediated activation of the Diaphanous-related formin, DIAPH1 and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro. In contrast, the actin capping protein Flightless-I, in conjuncti...
The Cdc42 effector IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics
The Journal of biological chemistry, 2008
The Cdc42 effector IRSp53 is a strong inducer of filopodia formation and consists of an Src homology domain 3 (SH3), a potential WW-binding motif, a partial-Cdc42/Rac interacting binding region motif, and an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain. We show that IRSp53 interacts directly with neuronal Wiskott-Aldrich syndrome protein (N-WASP) via its SH3 domain and furthermore that N-WASP is required for filopodia formation as IRSp53 failed to induce filopodia formation in N-WASP knock-out (KO) fibroblasts. IRSp53-induced filopodia formation can be reconstituted in N-WASP KO fibroblasts by full-length N-WASP, by N-WASPDeltaWA (a mutant unable to activate the Arp2/3 complex), and by N-WASPH208D (a mutant unable to bind Cdc42). IRSp53 failed to induce filopodia in mammalian enabled (Mena)/VASP KO cells, and N-WASP failed to induce filopodia when IRSp53 was knocked down with RNA interference. The IRSp53 I-BAR domain alone induces dynamic membrane protrusions that lack actin and are ...
Mechanism and Function of Formins in the Control of Actin Assembly
Annual Review of Biochemistry, 2007
Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain proteins associate with a variety of other cellular factors and directly nucleate actin polymerization through a novel mechanism. The signature formin homology 2 (FH2) domain initiates filament assembly and remains persistently associated with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed formins in animals and fungi are autoinhibited through intramolecular interactions and appear to be activated by Rho GTPases and additional factors. Other classes of formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.
Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics
Biology open, 2016
Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling.
Arp2/3 Branched Actin Network Mediates Filopodia-Like Bundles Formation In Vitro
PLoS ONE, 2008
During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.
Current Biology, 2001
Most eukaryotic cells rely on localized actin Results Although the nucleation activity of the Arp2/3 complex polymerization to generate and sustain the protrusion activity necessary for cell movement has been extensively explored [5-7, 9-11, 30, 31], the branching activity of the complex, an essential step in the [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dendritic nucleation model [5], is much less well characterized, particularly in terms of its relevance in vivo. While dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] it seems clear now that the branching occurs during polymerization rather than after [6, 7], branching has been and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. modeled in two ways, either as a separate activity of the Arp2/3 complex [5, 6, 30] or as an integral part of the The complex has thus been proposed to generate the actin network at the leading edge of crawling nucleation mechanism [7]. Furthermore, the complex does indeed localize at Y branches of actin filaments in cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to vivo [3, 4], but multiple other actin cross-linking proteins are present at the leading edge that could also cause protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 branching [12, 13], and there is no direct evidence that the branching activity of the Arp2/3 complex is required complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side for the extension of the lamellipod. binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, Structural studies of the Arp2/3 complex from Acanthamicroinjection of these antibodies into cells moeba using chemical cross-linking have shown that of specifically inhibits lamellipod extension without the seven subunits of the complex, only two, Arp3 and affecting the EGF-stimulated appearance of free p35, seem to bind actin directly [14]. The atomic model barbed ends in situ. These results indicate that of Arp3, based on its sequence homology to actin, predicts while the side binding activity of the Arp2/3 complex that this subunit is likely to be involved in the nucleation is required for nucleation in vitro and for protrusive function of the complex by forming a heterodimer with force in vivo, it is not required for EGF-stimulated Arp2 that could serve as a pointed end nucleus for actin increases in free barbed ends in vivo. This suggests polymerization [14, 15]. The p35 subunit, on the other that the branching activity of the Arp2/3 complex is hand, is a potential candidate for binding to the side of essential for lamellipod extension, while the the actin filament, since it binds actin but is not obviously generation of nucleation sites for actin involved in the nucleation activity [14]. We have generpolymerization is not sufficient. ated a peptide antibody against p34, the human homolog Addresses: * Albert Einstein College of Medicine, Department of of the Acanthamoeba p35 subunit [16, 17]. This antibody Anatomy and Structural Biology,