Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin Dynamics

ABSTRACT In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.

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Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1

Journal of Cell Science ◽

10.1242/jcs.111.16.2433 ◽

1998 ◽

Vol 111 (16) ◽

pp. 2433-2443 ◽

Cited By ~ 9

Author(s):

S.A. Weed ◽

Y. Du ◽

J.T. Parsons

Keyword(s):

Actin Polymerization ◽

Intracellular Localization ◽

Adhesion Formation ◽

Small Gtpases ◽

Small Gtpase ◽

Dominant Negative ◽

Actin Binding ◽

Cell Cortex ◽

Cell Periphery ◽

Membrane Ruffling

Small GTPases of the Rho family regulate signaling pathways that control actin cytoskeletal structures. In Swiss 3T3 cells, RhoA activation leads to stress fiber and focal adhesion formation, Rac1 to lamellipoda and membrane ruffles, and Cdc42 to microspikes and filopodia. Several downstream molecules mediating these effects have been recently identified. In this report we provide evidence that the intracellular localization of the actin binding protein cortactin, a Src kinase substrate, is regulated by the activation of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and this translocation is blocked by expression of dominant negative Rac1N17. Expression of constitutively active Rac1L61 evoked the translocation of cortactin from cytoplasmic pools into peripheral membrane ruffles. Expression of mutant forms of the serine/threonine kinase PAK1, a downstream effector of Rac1 and Cdc42 recently demonstrated to trigger cortical actin polymerization and membrane ruffling, also led to the translocation of cortactin to the cell cortex, although this was effectively blocked by coexpression of Rac1N17. Collectively these data provide evidence for cortactin as a putative target of Rac1-induced signal transduction events involved in membrane ruffling and lamellipodia formation.

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Osmotic stress-induced remodeling of the cortical cytoskeleton

AJP Cell Physiology ◽

10.1152/ajpcell.00018.2002 ◽

2002 ◽

Vol 283 (3) ◽

pp. C850-C865 ◽

Cited By ~ 103

Author(s):

Caterina Di Ciano ◽

Zilin Nie ◽

Katalin Szászi ◽

Alison Lewis ◽

Takehito Uruno ◽

...

Keyword(s):

Osmotic Stress ◽

De Novo ◽

Small Gtpases ◽

Dominant Negative ◽

Actin Binding ◽

Cell Cortex ◽

Actin Assembly ◽

Signaling Mechanism ◽

Actin Binding Domain ◽

Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

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βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0726 ◽

2003 ◽

Vol 14 (10) ◽

pp. 4155-4161 ◽

Cited By ~ 11

Author(s):

Kathleen N. Riley ◽

Angel E. Maldonado ◽

Patrice Tellier ◽

Crislyn D'Souza-Schorey ◽

Ira M. Herman

Keyword(s):

Western Blot ◽

Molecular Mechanisms ◽

Active Form ◽

Leading Edge ◽

Dominant Negative ◽

Actin Dynamics ◽

Actin Binding ◽

Capping Protein ◽

Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

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Basic Characterization of Plant Actin Depolymerizing Factors: A Simplified, Streamlined Guide

10.21203/rs.2.16466/v1 ◽

2019 ◽

Author(s):

Sonali Sengupta ◽

Kanniah Rajasekaran ◽

Niranjan Baisakh

Keyword(s):

Ionic Strength ◽

Actin Filaments ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Monomer ◽

Oligomeric State ◽

Binding Partners ◽

Helical Twist

Abstract Actin depolymerizing factors (ADFs) are small monomeric actin-binding proteins that alter the oligomeric state of cellular actin. Members of the ADF family can bind both the G-actin and F-actin in plants, and their functions are regulated by cellular pH, ionic strength and availability of other binding partners. Actin depolymerization activity is reportedly essential for plant viability. By binding to the ADP-bound form of actin, ADFs severe actin filaments and thereby provide more barbed filament ends for polymerization. They also increase the rate of dissociation of F-actin monomer by changing the helical twist of the actin filament. These two activities together make ADF the major regulator of actin dynamics in plant cell. Therefore, it is essential to measure the binding and depolymerization activity of the plant ADFs. Here, we present a simplified, streamlined step-by-step protocol to quickly measure these important functions of the ADF proteins in vitro.

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αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

Biochemical Journal ◽

10.1042/bj20041502 ◽

2005 ◽

Vol 388 (2) ◽

pp. 631-638 ◽

Cited By ~ 35

Author(s):

Björn ROTTER ◽

Odile BOURNIER ◽

Gael NICOLAS ◽

Didier DHERMY ◽

Marie-Christine LECOMTE

Keyword(s):

Binding Protein ◽

Focal Adhesions ◽

Membrane Skeleton ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Binding Protein ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

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The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200106089 ◽

2001 ◽

Vol 154 (6) ◽

pp. 1209-1224 ◽

Cited By ~ 178

Author(s):

Åsa E.Y. Engqvist-Goldstein ◽

Robin A. Warren ◽

Michael M. Kessels ◽

James H. Keen ◽

John Heuser ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Live Cells ◽

Cell Cortex ◽

Coated Pits ◽

Actin Binding Protein ◽

Real Time Analysis

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

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h3/Acidic Calponin: An Actin-binding Protein That Controls Extracellular Signal-regulated Kinase 1/2 Activity in Nonmuscle Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e09-06-0451 ◽

2010 ◽

Vol 21 (8) ◽

pp. 1409-1422 ◽

Cited By ~ 21

Author(s):

Sarah Appel ◽

Philip G. Allen ◽

Susanne Vetterkind ◽

Jian-Ping Jin ◽

Kathleen G. Morgan

Keyword(s):

Wound Healing ◽

Binding Protein ◽

Actin Binding ◽

Cell Cortex ◽

Extracellular Signal Regulated Kinase ◽

Actin Binding Protein ◽

Fibroblast Migration ◽

Extracellular Signal ◽

Nonmuscle Cells

Migration of fibroblasts is important in wound healing. Here, we demonstrate a role and a mechanism for h3/acidic calponin (aCaP, CNN3) in REF52.2 cell motility, a fibroblast line rich in actin filaments. We show that the actin-binding protein h3/acidic calponin associates with stress fibers in the absence of stimulation but is targeted to the cell cortex and podosome-like structures after stimulation with a phorbol ester, phorbol-12,13-dibutyrate (PDBu). By coimmunoprecipitation and colocalization, we show that extracellular signal-regulated kinase (ERK)1/2 and protein kinase C (PKC)α constitutively associate with h3/acidic calponin and are cotargeted with h3/acidic calponin in the presence of PDBu. This targeting can be blocked by a PKC inhibitor but does not require phosphorylation of h3/acidic calponin at the PKC sites S175 or T184. Knockdown of h3/acidic calponin results in a loss of PDBu-mediated ERK1/2 targeting, whereas PKCα targeting is unaffected. Caldesmon is an actin-binding protein that regulates actomyosin interactions and is a known substrate of ERK1/2. Both ERK1/2 activity and nonmuscle l-caldesmon phosphorylation are blocked by h3/acidic calponin knockdown. Furthermore, h3/acidic calponin knockdown inhibits REF52.2 migration in an in vitro wound healing assay. Our findings are consistent with a model whereby h3/acidic calponin controls fibroblast migration by regulation of ERK1/2-mediated l-caldesmon phosphorylation.

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Basic Characterization of Plant Actin Depolymerizing Factors: A Simplified, Streamlined Guide

10.21203/rs.2.16466/v2 ◽

2019 ◽

Author(s):

Sonali Sengupta ◽

Kanniah Rajasekaran ◽

Niranjan Baisakh

Keyword(s):

Ionic Strength ◽

Actin Filaments ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Monomer ◽

Oligomeric State ◽

Binding Partners ◽

Helical Twist

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Actin-Dependent Alterations of Dendritic Spine Morphology in Shankopathies

Neural Plasticity ◽

10.1155/2016/8051861 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 20

Author(s):

Tasnuva Sarowar ◽

Andreas M. Grabrucker

Keyword(s):

Pdz Domain ◽

Autism Spectrum ◽

Small Gtpases ◽

Actin Dynamics ◽

Actin Binding ◽

Sam Domain ◽

Wide Range ◽

Spine Morphogenesis

Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains—the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, andβPIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiplein vitroandin vivomodels. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling.

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Villin Severing Activity Enhances Actin-based Motility In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0423 ◽

2007 ◽

Vol 18 (3) ◽

pp. 827-838 ◽

Cited By ~ 22

Author(s):

Céline Revenu ◽

Matthieu Courtois ◽

Alphée Michelot ◽

Cécile Sykes ◽

Daniel Louvard ◽

...

Keyword(s):

Shigella Flexneri ◽

Actin Dynamics ◽

Actin Binding ◽

Loss Of Function ◽

Mesenchymal Transition ◽

Capping Protein ◽

Function Strategy ◽

In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

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