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      Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

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          Abstract

          Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

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          Most cited references59

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          Use of T7 RNA polymerase to direct expression of cloned genes.

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            Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins.

            Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.
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              Actin and actin-binding proteins. A critical evaluation of mechanisms and functions.

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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                24 March 1997
                : 136
                : 6
                : 1307-1322
                Affiliations
                [* ]Dynamique du Cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France; []Laboratory of Plant Cell Biology, Institute of Molecular Agrobiology, National University of Singapore, Singapore 118240; and [§ ]Laboratory of Plant Molecular Biology, Rockefeller University, New York 10021
                Article
                10.1083/jcb.136.6.1307
                2132522
                9087445
                61b41353-3cb0-41a8-8b63-5189a3a8d1ff
                Copyright @ 1997
                History
                : 8 November 1996
                : 23 January 1997
                Categories
                Article

                Cell biology
                Cell biology

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