scholarly journals Cortactin Localization to Sites of Actin Assembly in Lamellipodia Requires Interactions with F-Actin and the Arp2/3 Complex

2000 ◽  
Vol 151 (1) ◽  
pp. 29-40 ◽  
Author(s):  
Scott A. Weed ◽  
Andrei V. Karginov ◽  
Dorothy A. Schafer ◽  
Alissa M. Weaver ◽  
Andrew W. Kinley ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Actin Polymerization ◽  
Spatial Organization ◽  
Pdz Domain ◽  
Actin Binding ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Amino Terminal ◽  
Receptor Complexes

Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain–containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.

scholarly journals CLIC4 is regulated by RhoA-mDia2 signaling through Profilin-1 binding to modulate filopodium length

2018 ◽  
Author(s):  
Elisabetta Argenzio ◽  
Katarzyna M. Kedziora ◽  
Leila Nahidiazar ◽  
Tadamoto Isogai ◽  
Anastassis Perrakis ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Binding Protein ◽  
Feedback Mechanism ◽  
Actin Binding ◽  
Actin Assembly ◽  
Wild Type ◽  
Cortical Actin ◽  
Conserved Residues ◽  
Rhoa Activation ◽  
Necessary And Sufficient

AbstractCLIC4 is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by G12/13-coupled receptor agonists and then partly co-localizes with β1 integrins. Receptor-mediated CLIC4 translocation depends on actin polymerization, but the mechanism and functional significance of CLIC4 trafficking are unknown. Here we show that RhoA activation by either LPA or EGF is necessary and sufficient for CLIC4 translocation, with a regulatory role for the RhoA effector mDia2, an inducer of actin polymerization. We find that CLIC4 directly interacts with the G-actin-binding protein Profilin-1 via conserved residues that are required for CLIC4 trafficking and lie in a concave surface. Consistently, silencing of Profilin-1 impaired CLIC4 trafficking induced by either LPA or EGF. CLIC4 knockdown promoted the formation of long integrin-dependent filopodia, a phenotype rescued by wild-type CLIC4 but not by trafficking-incompetent CLIC4(C35A). Our results establish CLIC4 as a Profilin-1-binding protein and suggest that CLIC4 translocation provides a feedback mechanism to modulate mDia2/Profilin-1-driven cortical actin assembly and membrane protrusion.

scholarly journals Haematopoietic lineage cell-specific protein 1 (HS1) promotes actin-related protein (Arp) 2/3 complex-mediated actin polymerization

2003 ◽  
Vol 371 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Takehito URUNO ◽  
Peijun ZHANG ◽  
Jiali LIU ◽  
Jian-Jiang HAO ◽  
Xi ZHAN
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Primary Component ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Specific Gene ◽  
Actin Assembly ◽  
Related Protein ◽  
Filamentous Actin ◽  
Cortical Actin

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (Kd) of 880nM. Although HS1 is a modest F-actin-binding protein with a Kd of 400nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott–Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Small GTP-binding Protein TC10 Differentially Regulates Two Distinct Populations of Filamentous Actin in 3T3L1 Adipocytes

2002 ◽  
Vol 13 (7) ◽  
pp. 2334-2346 ◽  
Author(s):  
Makoto Kanzaki ◽  
Robert T. Watson ◽  
June Chunqiu Hou ◽  
Mark Stamnes ◽  
Alan R. Saltiel ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Actin Polymerization ◽  
Coat Proteins ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Amino Terminal ◽  
Gtp Binding ◽  
Subcellular Compartmentalization ◽  
Constitutively Active

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (ΔN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/ΔVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

Abstract 20484: Enah/Vasp-Like Protein is a Critical Component in S1P-Mediated Endothelial Lamellipodia Formation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Joseph B Mascarenhas ◽  
Ghassan Mouneimne ◽  
Carol C Gregorio ◽  
Mary E Brown ◽  
Ting Wang ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Human Lung ◽  
Actin Binding ◽  
Lipid Mediator ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Pulmonary Endothelial Cells ◽  
Sphingosine 1 Phosphate ◽  
Barrier Regulation ◽  
Lamellipodia Formation

Ena/VASP like protein, or EVL, is an actin-binding protein that regulates cancer cell lamellipodia protrusive activity and cell motility via an actomyosin contractility-dependent mechanism. The function of EVL in human lung endothelial cell (EC) barrier regulation, especially by the endogenous bioactive lipid mediator sphingosine-1-phosphate (S1P), is largely unknown. In this current study, we demonstrated that EVL is an active component in S1P-mediated EC barrier enhancement and lamellipodia formation. Compared to other focal adhesion (FA) proteins such as paxillin, EVL protein expression is very low in human pulmonary endothelial cells (ECs). S1P (1 μM) challenge stimulates translocation of cytosolic EVL to FAs in ECs, which was attenuated by EVL knockdown (KD) by its selective siRNA. S1P also promoted significant EVL translocation to lamellipodia, further confirmed by tracking translocation of EVL-GFP fusion protein upon S1P stimulation in a time-dependent manner. In addition, S1P-mediated cortical actin filament formation is attenuated by EVL KD, further confirming the function of EVL in S1P-induced lamellipodia formation/cortical actin polymerization. S1P stimulates EVL phosphorylation by tyrosine kinase c-Abl which is attenuated by the c-Abl inhibitor, imatinib. Finally, EVL KD attenuated S1P-mediated EC barrier enhancement and paracellular gap resealing reflected by reduced transendothelial electrical resistance (TER) measurements. These findings confirm a novel role for EVL in human lung vascular barrier enhancement and cytoskeleton rearrangement by S1P.

scholarly journals Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin

2003 ◽  
Vol 160 (4) ◽  
pp. 565-575 ◽  
Author(s):  
Qiang Wang ◽  
Yi Xie ◽  
Quan-Sheng Du ◽  
Xiao-Jun Wu ◽  
Xu Feng ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Cell Periphery ◽  
Cytoskeletal Organization ◽  
Capping Protein ◽  
Tyrosine Kinase 2 ◽  
Multiple Cells ◽  
Actin Rings

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion–activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion–targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2–gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.

scholarly journals The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

2000 ◽  
Vol 150 (6) ◽  
pp. 1321-1334 ◽  
Author(s):  
Zhengshan Dai ◽  
Xiaoyan Luo ◽  
Hongbo Xie ◽  
H. Benjamin Peng
Keyword(s):  
Actin Cytoskeleton ◽  
Acetylcholine Receptor ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Actin Binding ◽  
Heparin Binding ◽  
Actin Assembly ◽  
Achr Cluster ◽  
Green Fluorescent ◽  
Receptor Clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin–binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule–coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead–muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein–coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

scholarly journals The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

scholarly journals Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein.

1990 ◽  
Vol 110 (3) ◽  
pp. 681-692 ◽  
Author(s):  
A Shariff ◽  
E J Luna
Keyword(s):  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Actin Binding ◽  
Heat Denaturation ◽  
Actin Assembly ◽  
Binding Studies ◽  
Actin Nucleation ◽  
Equilibrium Binding ◽  
Nucleation Activity

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1-pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre-activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.

scholarly journals Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

1999 ◽  
Vol 10 (4) ◽  
pp. 1061-1075 ◽  
Author(s):  
Kathryn R. Ayscough ◽  
Jennifer J. Eby ◽  
Thomas Lila ◽  
Hilary Dewar ◽  
Keith G. Kozminski ◽  
...  
Keyword(s):  
Cell Walls ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Actin Binding Protein ◽  
Actin Patch ◽  
Mutant Forms ◽  
Modular Component ◽  
Terminal Repeat Region

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme β-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.  Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified inSchizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 inSaccharomyces cerevisiae.

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