EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/−0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.

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Side-binding proteins modulate actin filament dynamics

10.1101/008128 ◽

2014 ◽

Author(s):

Alvaro H. Crevenna ◽

Marcelino Arciniega ◽

Aurelie Dupont ◽

Kaja Kowalska ◽

Oliver Lange ◽

...

Keyword(s):

Actin Filament ◽

Brownian Dynamics ◽

Actin Polymerization ◽

Actin Dynamics ◽

Central Feature ◽

Filament Dynamics ◽

Filament Structure ◽

The Rich ◽

Dynamics Simulations ◽

Pointed End

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of the filament to polymerize and depolymerize at its ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. Here, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by proteins that bind to the lateral filament surface. We also show that the less dynamic end, called the pointed-end, has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of filament flexibility and Brownian dynamics simulations suggest that the observed kinetic diversity arises from structural alteration. Tuning filament kinetics by exploiting the natural malleability of the actin filament structure may be a ubiquitous mechanism to generate the rich variety of observed cellular actin dynamics.

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Small-Molecule Screen Identifies ROS as Key Regulators of Actin Dynamics in Neutrophils

Blood ◽

10.1182/blood.v112.11.4641.4641 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4641-4641

Author(s):

Hidenori Hattori ◽

Kulandayan K Subramanian ◽

Hongbo R. Luo

Keyword(s):

Small Molecule ◽

Actin Polymerization ◽

Neutrophil Migration ◽

Physiological Role ◽

Leading Edge ◽

Actin Dynamics ◽

Functional Screening ◽

Cellular Processes

Abstract Precise spatial and temporal control of actin polymerization and depolymerization is essential for mediating various cellular processes such as migration, phagocytosis, vesicle trafficking and adhesion. In this study, we used a small-molecule functional screening approach to identify novel regulators of actin dynamics during neutrophil migration. Here we show that NADPH-oxidase dependent Reactive Oxygen Species act as negative regulators of actin polymerization. Neutrophils with pharmacologically inhibited oxidase or isolated from Chronic Granulomatous Disease (CGD) patient and mice displayed enhanced F-actin polymerization, multiple pseudopods formation and impaired chemotaxis. ROS localized to pseudopodia and inhibited actin polymerization by driving actin glutathionylation at the leading edge of migrating cells. Consistent with these in vitro results, adoptively transferred CGD murine neutrophils also showed impaired in vivo recruitment to sites of inflammation. Together, these results present a novel physiological role for ROS in regulation of action polymerization and shed new light on the pathogenesis of CGD.

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

The Journal of Cell Biology ◽

10.1083/jcb.110.3.681 ◽

1990 ◽

Vol 110 (3) ◽

pp. 681-692 ◽

Cited By ~ 23

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.

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Profilin connects actin assembly with microtubule dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e15-11-0799 ◽

2016 ◽

Vol 27 (15) ◽

pp. 2381-2393 ◽

Cited By ~ 25

Author(s):

Michaela Nejedla ◽

Sara Sadi ◽

Vadym Sulimenko ◽

Francisca Nunes de Almeida ◽

Hans Blom ◽

...

Keyword(s):

Actin Polymerization ◽

Actin Dynamics ◽

Control Element ◽

Actin Assembly ◽

Actin Nucleation ◽

Superresolution Microscopy ◽

Drug Treatments ◽

And Migration ◽

Wasp Family Proteins ◽

Microscopy Techniques

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects microtubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

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Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2207 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2207-2213 ◽

Cited By ~ 40

Author(s):

A L Hall ◽

V Warren ◽

S Dharmawardhane ◽

J Condeelis

Keyword(s):

Dictyostelium Discoideum ◽

Actin Polymerization ◽

Developmental Regulation ◽

Reversible Inhibitor ◽

Actin Nucleation ◽

Low Speed ◽

Polymerization Inhibitor ◽

Nucleation Activity ◽

Triton X

Actin polymerization occurs in amebae of Dictyostelium discoideum after chemotactic stimulation (Hall, A. L., A. Schlein, and J. Condeelis. 1988. J. Cell. Biochem. 37:285-299). When cells are lysed with Triton X-100 during stimulation, an actin nucleation activity is detected in lysates by measuring the rate of pyrene-labeled actin polymerization. This stimulated nucleation activity is closely correlated with actin polymerization observed in vivo in its kinetics, developmental regulation, and cytochalasin D sensitivity. Actin polymerization is coordinate with pseudopod extension in synchronized populations of cells and is correlated with the accumulation of F actin in pseudopods. The stimulated actin nucleation activity is present in low-speed pellets from Triton lysates (cytoskeletons) within 3 s of stimulation and is stable compared with the nucleation activity of whole cell lysates. Low-speed supernatants contain a reversible inhibitor of the actin nucleation activity that is itself regulated by chemotactic stimulation. Neither activity requires Ca2+ and both are fully expressed in 10 mM EGTA. Fractions containing the inhibitor do not sever actin filaments but do inhibit actin polymerization that is seeded by fragments of purified F actin. These results indicate that chemotactic stimulation of Dictyostelium discoideum generates both an actin-nucleating activity and an actin-polymerization inhibitor, and suggest that the parallel regulation of these two activities leads to the transient phases of actin polymerization observed in vivo. The different compartmentation of these two activities may account for polarized pseudopod extension in gradients of chemoattractant.

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Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

The Journal of Cell Biology ◽

10.1083/jcb.139.5.1243 ◽

1997 ◽

Vol 139 (5) ◽

pp. 1243-1253 ◽

Cited By ~ 45

Author(s):

R.J. Eddy ◽

J. Han ◽

J.S. Condeelis

Keyword(s):

Actin Cytoskeleton ◽

Actin Polymerization ◽

De Novo ◽

Leading Edge ◽

Actin Assembly ◽

Directed Movement ◽

Capping Protein ◽

End Capping

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

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Regulation of leading edge microtubule and actin dynamics downstream of Rac1

The Journal of Cell Biology ◽

10.1083/jcb.200303082 ◽

2003 ◽

Vol 161 (5) ◽

pp. 845-851 ◽

Cited By ~ 182

Author(s):

Torsten Wittmann ◽

Gary M. Bokoch ◽

Clare M. Waterman-Storer

Keyword(s):

Rho Gtpases ◽

Actin Polymerization ◽

Leading Edge ◽

Time Lapse ◽

Dominant Negative ◽

Actin Dynamics ◽

Retrograde Flow ◽

Rho Proteins ◽

Migrating Cells

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.

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Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor

The Journal of Cell Biology ◽

10.1083/jcb.200705122 ◽

2007 ◽

Vol 179 (2) ◽

pp. 239-245 ◽

Cited By ~ 83

Author(s):

Chun Xiang Sun ◽

Marco A.O. Magalhães ◽

Michael Glogauer

Keyword(s):

Actin Cytoskeleton ◽

De Novo ◽

Leading Edge ◽

Small Gtpases ◽

Actin Dynamics ◽

Membrane Protrusion ◽

Actin Assembly ◽

Unique Combination ◽

High Affinity ◽

Fmlp Receptor

Actin assembly at the leading edge of migrating cells depends on the availability of high-affinity free barbed ends (FBE) that drive actin filament elongation and subsequent membrane protrusion. We investigated the specific mechanisms through which the Rac1 and Rac2 small guanosine triphosphatases (GTPases) generate free barbed ends in neutrophils. Using neutrophils lacking either Rac1 or Rac2 and a neutrophil permeabilization model that maintains receptor signaling to the actin cytoskeleton, we assessed the mechanisms through which these two small GTPases mediate FBE generation downstream of the formyl-methionyl-leucyl-phenylalanine receptor. We demonstrate here that uncapping of existing barbed ends is mediated through Rac1, whereas cofilin- and ARP2/3-mediated FBE generation are regulated through Rac2. This unique combination of experimental tools has allowed us to identify the relative roles of uncapping (15%), cofilin severing (10%), and ARP2/3 de novo nucleation (75%) in FBE generation and the respective roles played by Rac1 and Rac2 in mediating actin dynamics.

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Association of Mouse Actin-binding Protein 1 (mAbp1/SH3P7), an Src Kinase Target, with Dynamic Regions of the Cortical Actin Cytoskeleton in Response to Rac1 Activation

Molecular Biology of the Cell ◽

10.1091/mbc.11.1.393 ◽

2000 ◽

Vol 11 (1) ◽

pp. 393-412 ◽

Cited By ~ 107

Author(s):

Michael M. Kessels ◽

Åsa E. Y. Engqvist-Goldstein ◽

David G. Drubin

Keyword(s):

Actin Cytoskeleton ◽

Tyrosine Kinases ◽

Actin Polymerization ◽

De Novo ◽

Leading Edge ◽

Membrane Dynamics ◽

Cellular Growth ◽

Actin Binding ◽

Cortical Actin ◽

Binding Domains

Yeast Abp1p is a cortical actin cytoskeleton protein implicated in cytoskeletal regulation, endocytosis, and cAMP-signaling. We have identified a gene encoding a mouse homologue of Abp1p, and it is identical to SH3P7, a protein shown recently to be a target of Src tyrosine kinases. Yeast and mouse Abp1p display the same domain structure including an N-terminal actin-depolymerizing factor homology domain and a C-terminal Src homology 3 domain. Using two independent actin-binding domains, mAbp1 binds to actin filaments with a 1:5 saturation stoichiometry. In stationary cells, mAbp1 colocalizes with cortical F-actin in fibroblast protrusions that represent sites of cellular growth. mAbp1 appears at the actin-rich leading edge of migrating cells. Growth factors cause mAbp1 to rapidly accumulate in lamellipodia. This response can be mimicked by expression of dominant-positive Rac1. mAbp1 recruitment appears to be dependent on de novo actin polymerization and occurs specifically at sites enriched for the Arp2/3 complex. mAbp1 is a newly identified cytoskeletal protein in mice and may serve as a signal-responsive link between the dynamic cortical actin cytoskeleton and regions of membrane dynamics.

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Filopodia formation and Disabled degradation downstream of Reelin

Biochemical Journal ◽

10.1042/bj20041588 ◽

2004 ◽

Vol 384 (1) ◽

Author(s):

Steven J. WINDER

Keyword(s):

Actin Polymerization ◽

Leading Edge ◽

Negative Control ◽

Actin Dynamics ◽

Abelson Tyrosine Kinase ◽

Motile Cells ◽

Biochemical Journal ◽

Subtle Effect ◽

Important Regulator ◽

Syndrome Protein

During Drosophila embryogenesis, Abl (Abelson tyrosine kinase) is localized in the axons of the CNS (central nervous system). Mutations in Abl have a subtle effect on the morphology of the embryonic CNS, and the mutant animals survive to the pupal and adult stages. However, genetic screens have identified several genes that, when mutated along with the Abl gene, modified the phenotypes. Two prominent genes that arose from these screens were enabled (Ena) and disabled (Dab). It has been known for some time that Enabled and its mammalian homologues are involved in the regulation of actin dynamics, and promote actin polymerization at the leading edge of motile cells. It was a defect in actin polymerization in migrating neurons in particular that resulted in the identification of Enabled as an important regulator of neuronal migration. Defects in Disabled, in both Drosophila and mammals, also gave rise to neuronal defects which, in mice, were indistinguishable from phenotypes observed in the reeler mouse. These observations suggested that mDab1 (mammalian Disabled homologue 1) acted in a pathway downstream of Reelin, the product of the reelin gene found to be defective in reeler mice. Now, in this issue of the Biochemical Journal, Takenawa and colleagues have demonstrated that Disabled also acts in a pathway to regulate actin dynamics through the direct activation of N-WASP (neuronal Wiskott–Aldrich syndrome protein). Furthermore, they were also able to link several lines of investigation from other groups to show that the ability of mDab1 to regulate actin dynamics during cell motility was under the negative control of tyrosine phosphorylation, leading to ubiquitin-mediated degradation of mDab1.

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