Intrinsic dynamic behavior of fascin in filopodia.

Recent studies showed that the actin cross-linking protein, fascin, undergoes rapid cycling between filopodial filaments. Here, we used an experimental and computational approach to dissect features of fascin exchange and incorporation in filopodia. Using expression of phosphomimetic fascin mutants, we determined that fascin in the phosphorylated state is primarily freely diffusing, whereas actin bundling in filopodia is accomplished by fascin dephosphorylated at serine 39. Fluorescence recovery after photobleaching analysis revealed that fascin rapidly dissociates from filopodial filaments with a kinetic off-rate of 0.12 s(-1) and that it undergoes diffusion at moderate rates with a coefficient of 6 microm(2)s(-1). This kinetic off-rate was recapitulated in vitro, indicating that dynamic behavior is intrinsic to the fascin cross-linker. A computational reaction-diffusion model showed that reversible cross-linking is required for the delivery of fascin to growing filopodial tips at sufficient rates. Analysis of fascin bundling indicated that filopodia are semiordered bundles with one bound fascin per 25-60 actin monomers.

[1]  A. Koleske.  Do Filopodia Enable the Growth Cone to Find Its Way? , 2003, Science's STKE.

[2]  P. Bridgman,et al.  Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity , 1992, The Journal of cell biology.

[3]  T. Mitchison,et al.  Actin-Based Cell Motility and Cell Locomotion , 1996, Cell.

[4]  F. Matsumura,et al.  Cell-matrix adhesions differentially regulate fascin phosphorylation. , 1999, Molecular biology of the cell.

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

[6]  F. Matsumura,et al.  Phosphorylation of Human Fascin Inhibits Its Actin Binding and Bundling Activities (*) , 1996, The Journal of Biological Chemistry.

[7]  T. Pollard,et al.  Dependence of the mechanical properties of actin/α-actinin gels on deformation rate , 1987, Nature.

[8]  R. Pego,et al.  Analysis of binding reactions by fluorescence recovery after photobleaching. , 2004, Biophysical journal.

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

[10]  D. DeRosier,et al.  Growth conditions control the size and order of actin bundles in vitro. , 1991, Biophysical journal.

[11]  A L Pearlman,et al.  Extension of filopodia by motor-dependent actin assembly. , 1992, Cell motility and the cytoskeleton.

[12]  T D Pollard,et al.  Dynamic Cross-linking by α-Actinin Determines the Mechanical Properties of Actin Filament Networks* , 1998, The Journal of Biological Chemistry.

[13]  D. Wirtz,et al.  Strain Hardening of Actin Filament Networks , 2000, The Journal of Biological Chemistry.

[14]  G. Albrecht-Buehler,et al.  Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function? , 1976, The Journal of cell biology.

[15]  S. B. Kater,et al.  A sensory role for neuronal growth cone filopodia , 1993, Nature.

[16]  J. Spudich,et al.  The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. , 1971, The Journal of biological chemistry.

[17]  T. Pollard,et al.  Actin and Myosin Biochemistry in Relation to Cytokinesis a , 1990, Annals of the New York Academy of Sciences.

[18]  A. Mogilner,et al.  The physics of filopodial protrusion. , 2005, Biophysical journal.

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

[20]  Maddy Parsons,et al.  Interaction of fascin and protein kinase Cα: a novel intersection in cell adhesion and motility , 2003, The EMBO journal.

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

[22]  E. Dent,et al.  Cytoskeletal Dynamics and Transport in Growth Cone Motility and Axon Guidance , 2003, Neuron.

[23]  F. Matsumura,et al.  Purification and characterization of an F-actin-bundling 55-kilodalton protein from HeLa cells. , 1985, The Journal of biological chemistry.

[24]  Niels Volkmann,et al.  An Atomic Model of Actin Filaments Cross-Linked by Fimbrin and Its Implications for Bundle Assembly and Function , 2001, The Journal of cell biology.

[25]  L. G. Tilney,et al.  Regulation of Actin Filament Cross-Linking and Bundle Shape in Drosophila Bristles , 2000, The Journal of cell biology.

[26]  J. Bryan,et al.  Separation and interaction of the major components of sea urchin actin gel. , 1978, Journal of molecular biology.

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

[28]  T. Pollard,et al.  Progressing actin: Formin as a processive elongation machine , 2004, Nature Cell Biology.

[29]  Hiroshi Suzuki,et al.  Short‐term retention of actin filament binding proteins on lamellipodial actin bundles , 2006, FEBS letters.

[30]  G. Borisy,et al.  Quantitative determination of the proportion of microtubule polymer present during the mitosis-interphase transition. , 1994, Journal of cell science.

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

[32]  T. Mitchison,et al.  Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex , 2004, The Journal of cell biology.

[33]  P. Janmey,et al.  Viscoelastic properties of vimentin compared with other filamentous biopolymer networks , 1991, The Journal of cell biology.

[34]  A. Mehta,et al.  In vitro assays of processive myosin motors. , 2000, Methods.

[35]  A. Verkman,et al.  Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. , 1997, Biophysical journal.

[36]  J. Gnarra,et al.  Identification of an Actin Binding Region and a Protein Kinase C Phosphorylation Site on Human Fascin* , 1997, The Journal of Biological Chemistry.

[37]  H. Isambert,et al.  Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide, phalloidin, and muscle regulatory proteins , 1995, The Journal of Biological Chemistry.

[38]  D. DeRosier,et al.  Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate. , 1986, Developmental biology.

[39]  D. DeRosier,et al.  Actin Filaments , Stereocilia , and Hair Cells of the Bird Cochlea . V . How the Staircase Pattern of Stere iliary Lengths Is Generated , 2002 .

[40]  Thomas D Pollard,et al.  Counting Cytokinesis Proteins Globally and Locally in Fission Yeast , 2005, Science.