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      Single-molecule visualization of a formin-capping protein ‘decision complex' at the actin filament barbed end

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          Abstract

          Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells.

          Abstract

          Formins promote the polymerization of actin filaments at barbed ends and capping protein prevents polymerization. Here the authors use single molecule imaging to directly visualize a decision complex consisting of the formin mDia1 and capping protein bound simultaneously to the filament barbed end and the dynamic mechanisms by which it forms and dissociates.

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          Most cited references42

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          Mechanism and function of formins in the control of actin assembly.

          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.
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            Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility.

            Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia.
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              Control of the assembly of ATP- and ADP-actin by formins and profilin.

              Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                13 November 2015
                2015
                : 6
                : 8707
                Affiliations
                [1 ]Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454-9110, USA
                [2 ]Department of Biology, Brandeis University , Waltham, Massachusetts 02454-9110 USA
                [3 ]New England Biolabs , Ipswich, Massachusetts 01938, USA
                Author notes
                Author information
                http://orcid.org/0000-0002-3169-6878
                http://orcid.org/0000-0001-7910-3421
                Article
                ncomms9707
                10.1038/ncomms9707
                4660045
                26566078
                a2fbdc41-741b-4bb0-9dd4-9f9e5b5ee90e
                Copyright © 2015, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 02 June 2015
                : 22 September 2015
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