Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia

The leading edge (∼1 μm) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 μm of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181–6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.

[1]  P. Janmey,et al.  Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets , 1995, Cell.

[2]  J. Cooper,et al.  Visualization and Molecular Analysis of Actin Assembly in Living Cells , 1998, The Journal of cell biology.

[3]  T. Pollard,et al.  Characterization of actin filament severing by actophorin from Acanthamoeba castellanii , 1991, The Journal of cell biology.

[4]  A. DePace,et al.  The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly , 1997, The Journal of cell biology.

[5]  T. Svitkina,et al.  Correlative light and electron microscopy of the cytoskeleton of cultured cells. , 1998, Methods in enzymology.

[6]  T. Mitchison,et al.  Actin dynamics in vivo. , 1997, Current opinion in cell biology.

[7]  T. Svitkina,et al.  Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles , 1995, The Journal of cell biology.

[8]  D. Bray,et al.  Distribution and cellular localization of actin depolymerizing factor , 1987, The Journal of cell biology.

[9]  Carl Frieden,et al.  Actin polymerization. The mechanism of action of cytochalasin D. , 1986, The Journal of biological chemistry.

[10]  J. Condeelis,et al.  Life at the leading edge: the formation of cell protrusions. , 1993, Annual review of cell biology.

[11]  A. Kapp,et al.  Actin polymerization. , 2000, Methods in molecular biology.

[12]  J H Hartwig,et al.  Mechanisms of actin rearrangements mediating platelet activation , 1992, The Journal of cell biology.

[13]  A. Weeds,et al.  Actophorin preferentially binds monomeric ADP‐Actin over ATP‐bound actin: consequences for cell locomotion , 1994, FEBS letters.

[14]  P. Matsudaira,et al.  Actin crosslinking proteins at the leading edge. , 1994, Seminars in cell biology.

[15]  Walter F. Stafford,et al.  Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba , 1997, The Journal of cell biology.

[16]  M. Way,et al.  Actin and cell pathogenesis. , 1997, Current opinion in cell biology.

[17]  S. Zigmond,et al.  Mechanism of Cdc42-induced Actin Polymerization in Neutrophil Extracts , 1998, The Journal of cell biology.

[18]  T D Pollard,et al.  The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Small,et al.  Actin filament organization in the fish keratocyte lamellipodium , 1995, The Journal of cell biology.

[20]  L. Machesky,et al.  Cell motility: Complex dynamics at the leading edge , 1997, Current Biology.

[21]  T D Pollard,et al.  Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments , 1986, The Journal of cell biology.

[22]  T. Pollard,et al.  Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. , 1991, Biochemistry.

[23]  J. Bamburg,et al.  Actin depolymerizing factor and cofilin phosphorylation dynamics: response to signals that regulate neurite extension. , 1998, Cell motility and the cytoskeleton.

[24]  J. Segall,et al.  EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells. , 1998, Journal of cell science.

[25]  T. Svitkina,et al.  Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton , 1996, The Journal of cell biology.

[26]  D. Drubin,et al.  The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics. , 1995, Molecular biology of the cell.

[27]  M. Carlier,et al.  Control of Actin Dynamics in Cell Motility , 2022 .

[28]  T. Svitkina,et al.  Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells. , 1995, Journal of structural biology.

[29]  Timothy J. Mitchison,et al.  Actin polymerization is induced by Arp 2/3 protein complex at the surface of Listeria monocytogenes , 1997, Nature.

[30]  J. Hartwig,et al.  Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein , 1996, The Journal of cell biology.

[31]  S. Zigmond,et al.  Signal transduction and actin filament organization. , 1996, Current opinion in cell biology.

[32]  J H Hartwig,et al.  Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring , 1990, The Journal of cell biology.

[33]  Marie-France Carlier,et al.  Role of Proteins of the Ena/VASP Family in Actin-based Motility of Listeria monocytogenes , 1999, The Journal of cell biology.

[34]  T D Pollard,et al.  Sequences, structural models, and cellular localization of the actin- related proteins Arp2 and Arp3 from Acanthamoeba , 1995, The Journal of cell biology.

[35]  G. Oster,et al.  Cell motility driven by actin polymerization. , 1996, Biophysical journal.

[36]  D. Lauffenburger,et al.  Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution , 1991, The Journal of cell biology.

[37]  S. Maciver.  How ADF/cofilin depolymerizes actin filaments. , 1998, Current opinion in cell biology.

[38]  T. Steck,et al.  Length distribution of F-actin in Dictyostelium discoideum. , 1990, The Journal of biological chemistry.

[39]  T. Mitchison,et al.  Xenopus Actin Depolymerizing Factor/Cofilin (XAC) Is Responsible for the Turnover of Actin Filaments in Listeria monocytogenes Tails , 1997, The Journal of cell biology.

[40]  T. Mitchison,et al.  Comparison of actin and cell surface dynamics in motile fibroblasts , 1992, The Journal of cell biology.

[41]  B. Imhof,et al.  Actin dynamics in living mammalian cells. , 1998, Journal of cell science.

[42]  M. Kirschner,et al.  The Interaction between N-WASP and the Arp2/3 Complex Links Cdc42-Dependent Signals to Actin Assembly , 1999, Cell.

[43]  J. Small.  Lamellipodia architecture: actin filament turnover and the lateral flow of actin filaments during motility. , 1994, Seminars in cell biology.

[44]  T. Pollard,et al.  Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose , 1994, The Journal of cell biology.

[45]  J. Condeelis,et al.  Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium , 1997, The Journal of cell biology.

[46]  M. Carlier,et al.  Control of actin dynamics in cell motility , 1999 .

[47]  J. Theriot.  Accelerating on a Treadmill: ADF/Cofilin Promotes Rapid Actin Filament Turnover in the Dynamic Cytoskeleton , 1997, The Journal of cell biology.

[48]  T. Pollard,et al.  Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[49]  J. Bamburg,et al.  Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation- regulated protein essential for development , 1996, The Journal of cell biology.

[50]  T. Mitchison,et al.  Control of actin polymerization in live and permeabilized fibroblasts , 1991, The Journal of cell biology.

[51]  A. Weeds,et al.  The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover: pH sensitivity of F-actin binding by human ADF, but not of Acanthamoeba actophorin. , 1998, European journal of biochemistry.

[52]  J. Small.  Getting the actin filaments straight: nucleation-release or treadmilling? , 1995, Trends in cell biology.

[53]  S. Zigmond.  Actin cytoskeleton: The Arp2/3 complex gets to the point , 1998, Current Biology.

[54]  Julie A. Theriot,et al.  Actin microfilament dynamics in locomoting cells , 1991, Nature.

[55]  M. Carlier.  Control of actin dynamics. , 1998, Current opinion in cell biology.

[56]  S. Brown,et al.  Mechanism of action of cytochalasin: evidence that it binds to actin filament ends , 1981, The Journal of cell biology.

[57]  Y. Wang,et al.  Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767] , 1993, The Journal of cell biology.

[58]  K L Gould,et al.  The Arp2/3 complex: a multifunctional actin organizer. , 1999, Current opinion in cell biology.

[59]  A. Wegner,et al.  Head to tail polymerization of actin. , 1976, Journal of molecular biology.

[60]  J. Small,et al.  Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body , 1996, The Journal of cell biology.

[61]  Gary G. Borisy,et al.  Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation , 1997, The Journal of cell biology.

[62]  J. Faber,et al.  Normal table of Xenopus laevis (Daudin). A systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , 1956 .

[63]  A. Lerner,et al.  Action of light on frog pigment cells in culture. , 1990, Pigment cell research.

[64]  K. Ayscough.  Use of latrunculin-A, an actin monomer-binding drug. , 1998, Methods in enzymology.

[65]  J. Bereiter-Hahn,et al.  Ionic control of locomotion and shape of epithelial cells: II. Role of monovalent cations. , 1988, Cell motility and the cytoskeleton.

[66]  E. Nishida,et al.  Distribution among tissues and intracellular localization of cofilin, a 21kDa actin-binding protein. , 1987, Cell structure and function.

[67]  A. Burlingame,et al.  Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins. , 1997, The Biochemical journal.

[68]  T. Mitchison,et al.  Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. , 1998, Science.

[69]  Marie-France Carlier,et al.  Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility , 1997, The Journal of cell biology.

[70]  Jonathan A. Cooper,et al.  Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides , 1996, The Journal of cell biology.