Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells.

A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.

[1]  C. Cohan,et al.  Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia. , 2001, Cell motility and the cytoskeleton.

[2]  J. Condeelis,et al.  Imaging Sites of N-WASP Activity in Lamellipodia and Invadopodia of Carcinoma Cells , 2004, Current Biology.

[3]  T. Takenawa,et al.  CRMP-2 Is Involved in Kinesin-1-Dependent Transport of the Sra-1/WAVE1 Complex and Axon Formation , 2005, Molecular and Cellular Biology.

[4]  D. Hanein,et al.  Ena/VASP Proteins Enhance Actin Polymerization in the Presence of Barbed End Capping Proteins*[boxs] , 2005, Journal of Biological Chemistry.

[5]  Gary G. Borisy,et al.  Formation of filopodia-like bundles in vitro from a dendritic network , 2003, The Journal of cell biology.

[6]  T. Svitkina,et al.  Correlative Light and Electron Microscopy of the Cytoskeleton , 2006 .

[7]  M. Steketee,et al.  Filopodial initiation and a novel filament-organizing center, the focal ring. , 2001, Molecular biology of the cell.

[8]  Charles Boone,et al.  Role of Formins in Actin Assembly: Nucleation and Barbed-End Association , 2002, Science.

[9]  S. Zigmond.  Formin-induced nucleation of actin filaments. , 2004, Current opinion in cell biology.

[10]  M. Dailey,et al.  The organization of myosin and actin in rapid frozen nerve growth cones , 1989, The Journal of cell biology.

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

[12]  John G. Collard,et al.  Interaction between Tiam1 and the Arp2/3 complex links activation of Rac to actin polymerization. , 2006, The Biochemical journal.

[13]  T. Svitkina,et al.  Cascade pathway of filopodia formation downstream of SCAR , 2004, Journal of Cell Science.

[14]  L. Machesky,et al.  Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex. , 2004, The Biochemical journal.

[15]  Lorene M Lanier,et al.  Arp2/3 Is a Negative Regulator of Growth Cone Translocation , 2004, Neuron.

[16]  Alissa M. Weaver,et al.  Integration of signals to the Arp2/3 complex. , 2003, Current opinion in cell biology.

[17]  Charles Boone,et al.  Formin Leaky Cap Allows Elongation in the Presence of Tight Capping Proteins , 2003, Current Biology.

[18]  Robert E. Lewis,et al.  The formin-homology-domain-containing protein FHOD1 enhances cell migration , 2003, Journal of Cell Science.

[19]  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.

[20]  Stanislav S Zakharenko,et al.  Plasma membrane recycling and flow in growing neurites , 2000, Neuroscience.

[21]  M. Dichter,et al.  Properties of inhibitory and excitatory synapses between hippocampal neurons in very low density cultures , 1994, Synapse.

[22]  Josephine C. Adams,et al.  Roles of fascin in cell adhesion and motility. , 2004, Current opinion in cell biology.

[23]  J. Grosshans,et al.  Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion. , 2007, Genes & development.

[24]  Y. Wang,et al.  Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling , 1985, The Journal of cell biology.

[25]  Yoichi Kato,et al.  Wnt/Frizzled Activation of Rho Regulates Vertebrate Gastrulation and Requires a Novel Formin Homology Protein Daam1 , 2001, Cell.

[26]  B. Baum,et al.  Abi, Sra1, and Kette Control the Stability and Localization of SCAR/WAVE to Regulate the Formation of Actin-Based Protrusions , 2003, Current Biology.

[27]  Giorgio Scita,et al.  Novel Roles of Formin mDia2 in Lamellipodia and Filopodia Formation in Motile Cells , 2007, PLoS biology.

[28]  H. Mellor,et al.  The Rho Family GTPase Rif Induces Filopodia through mDia2 , 2005, Current Biology.

[29]  Gary G. Borisy,et al.  Mechanism of filopodia initiation by reorganization of a dendritic network , 2003, The Journal of cell biology.

[30]  K. Rottner,et al.  The making of filopodia. , 2006, Current opinion in cell biology.

[31]  J. Hartwig,et al.  Arp2/3 complex-deficient mouse fibroblasts are viable and have normal leading-edge actin structure and function. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Dylan T Burnette,et al.  Myosin II functions in actin-bundle turnover in neuronal growth cones , 2006, Nature Cell Biology.

[33]  H. Higgs,et al.  Actin Dynamics: Growth from Dendritic Branches , 2005, Current Biology.

[34]  T. Svitkina,et al.  Actin machinery: pushing the envelope. , 2000, Current opinion in cell biology.

[35]  R. Vale,et al.  Molecular requirements for actin-based lamella formation in Drosophila S2 cells , 2003, The Journal of cell biology.

[36]  Timothy J Mitchison,et al.  Single-Molecule Speckle Analysis of Actin Filament Turnover in Lamellipodia , 2002, Science.

[37]  C. Otey,et al.  B35 neuroblastoma cells: an easily transfected, cultured cell model of central nervous system neurons. , 2003, Methods in cell biology.

[38]  P. J. Smith,et al.  Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones. , 1999, Molecular biology of the cell.

[39]  G. Borisy,et al.  Kinetic-structural analysis of neuronal growth cone veil motility , 2007, Journal of Cell Science.

[40]  T. Mitchison,et al.  Regulated Actin Cytoskeleton Assembly at Filopodium Tips Controls Their Extension and Retraction , 1999, The Journal of cell biology.

[41]  Lorene M Lanier,et al.  Critical Role of Ena/VASP Proteins for Filopodia Formation in Neurons and in Function Downstream of Netrin-1 , 2004, Neuron.

[42]  Gary G. Borisy,et al.  Role of fascin in filopodial protrusion , 2006, The Journal of cell biology.

[43]  Till Bretschneider,et al.  Subsecond reorganization of the actin network in cell motility and chemotaxis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[44]  G. Gallo.  RhoA-kinase coordinates F-actin organization and myosin II activity during semaphorin-3A-induced axon retraction , 2006, Journal of Cell Science.

[45]  Gary G. Borisy,et al.  Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia , 1999, The Journal of cell biology.

[46]  Vladimir Gelfand,et al.  Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein. , 1987, Experimental cell research.

[47]  L. Van Aelst,et al.  The role of the Rho GTPases in neuronal development. , 2005, Genes & development.

[48]  Klemens Rottner,et al.  Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes. , 2006, Molecular biology of the cell.

[49]  T. Bretschneider,et al.  The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia , 2005, Nature Cell Biology.

[50]  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.

[51]  Gary G. Borisy,et al.  Lamellipodial Versus Filopodial Mode of the Actin Nanomachinery Pivotal Role of the Filament Barbed End , 2004, Cell.

[52]  Jun Peng,et al.  Disruption of the Diaphanous-Related Formin Drf1 Gene Encoding mDia1 Reveals a Role for Drf3 as an Effector for Cdc42 , 2003, Current Biology.

[53]  Gary G. Borisy,et al.  Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility , 2002, Cell.

[54]  H. Yee,et al.  RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor. , 2006, Journal of neurobiology.

[55]  J. Small,et al.  Polarity of actin at the leading edge of cultured cells , 1978, Nature.

[56]  T. Svitkina,et al.  Speckle microscopic evaluation of microtubule transport in growing nerve processes , 1999, Nature Cell Biology.