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PubMed2003

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes.

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.

Actins↗

PubMed1984

Action of a 19K protein from porcine brain on actin polymerization: a new functional class of actin-binding proteins.

A 19K protein isolated from porcine brain not only inhibits actin polymerization but depolymerizes actin filaments quickly. The protein reacts stoichiometrically with actin in a 1:1 molar ratio. When actin is induced to polymerize with salts in the presence of the brain 19K protein, the lag phase is prolonged, and the extent of polymerization is decreased, but the half-polymerization time is not increased. This can be explained by assuming that the 19K protein severs growing actin filaments and thus causes an increase in the number of filament ends during the polymerization process, thereby accelerating the overall polymerization. Moreover, the low-shear viscosity of actin filaments is reduced much more than the high-shear viscosity by the 19K protein, suggesting that actin filaments become shorter in the presence of the 19K protein than in its absence. Actin filament depolymerization by the 19K protein is much faster than that by brain profilin or than spontaneous depolymerization. This indicates that the 19K protein depolymerizes actin filaments not only by sequestering actin monomers but also by directly attacking the filaments. The number of actin filaments, measured by assaying the nucleating ability, is increased by substoichiometric concentrations of the 19K protein, irrespective of whether the protein is added to actin monomers before polymerization or added to performed actin filaments. These results suggest that the brain 19K protein not only stabilizes actin monomers but also cuts actin filaments, thereby decreasing the extent of actin polymerization and also changing the filament length. The action on actin of the actin-depolymerizing protein from starfish oocytes resembles that of the brain 19K protein, although the molecular weight of the starfish protein is slightly smaller. The brain 19K protein and starfish protein should be classified into a new functional group of actin-binding proteins.

Actin Depolymerizing Factors↗

PubMed1983

Kinetic analysis of actin polymerization.

The kinetics of actin polymerization were analyzed by measuring the changes in absorbance which accompany the G-F transformation of actin. In these studies, gel-filtered actin was polymerized in the absence of shearing stress under physiological ionic conditions. Self-polymerization was found to be characterized as a process having a lag phase followed by a pseudo-first-order decay process, suggesting that actin polymerization consists of distinct nucleation and elongation phases. The size of the nucleus was estimated to be two to four monomer units by analyzing the actin concentration-dependences of the rate constant for the pseudo-first-order process, the maximal rate of polymerization and the half-polymerization time. The elongation reaction was induced by mixing actin filament seeds with actin monomers under conditions where spontaneous nucleation is slow. This elongation was also found to be a pseudo-first-order process, indicating that spontaneous nucleation was negligible. Our data suggested that the initial rate of elongation was proportional to both the number concentration of actin filaments and the actin monomer concentration above the critical concentration, and that the depolymerization rate was proportional to the number concentration of actin filaments but independent of monomer concentration. The results of direct analysis of the depolymerization reaction were consistent with this suggestion. These studies strongly support the condensation polymerization mechanism as a model for actin polymerization.

Actins↗

PubMed1982

The state of actin polymerization in tetracaine-treated platelets.

Although there is considerable evidence that platelet activation is associated with polymerization of actin, it is not known whether there is some pre-existing F-actin even before activation. We have examined the state of actin polymerization in nonactivated platelets by deoxyribonuclease assay of G-actin and total actin. To fully suppress activation, platelets were prepared in the presence of tetracaine. The G-actin/total actin ratio in tetracaine-treated platelets prepared by three different methods is significantly less than one (average of all results = 0.61). We conclude that about 39% of total actin in nonactivated tetracaine-treated platelets may be present as F-actin.

Actins↗

PubMed2001

The regulation of actin polymerization and cross-linking in Dictyostelium.

It is clear that the polymerization and organization of actin filament networks plays a critical role in numerous cellular processes. Inhibition of actin polymerization by pharmacological agents will completely prevent chemotactic motility, macropinocytosis, endocytosis, and phagocytosis. Recently there has been great progress in understanding the mechanisms that control the assembly and structure of the actin cytoskeleton. Members of the Rho family of GTPases have been identified as major players in the signal transduction pathway leading from a cell surface signal to actin polymerization. The Arp2/3 complex has been added to the list of means by which new actin filaments can be nucleated. However, it is clear that actin polymerization by Arp2/3 complex is not the whole story. In principle, the final structures formed by actin filaments will depend on factors such as: the length of actin filaments, the degree of branching, how they are cross-linked and the tensions imparted on them. In addition, the means by which actin polymerization generates protrusion of membranes is still controversial. A phagosome, filopodium and a lamellipodium all require polymerization of new actin filaments, but each has a characteristic morphology and cytoskeletal structure. In the following chapter, we will discuss actin polymerization and filament organization, especially as it relates to the machinery of phagocytosis in Dictyostelium.

Actin Cytoskeleton↗

PubMed1982

The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin.

Profilin inhibits the rate of nucleation of actin polymerization and the rate of filament elongation and also reduces the concentration of F-actin at steady state. Addition of profilin to solutions of F-actin causes depolymerization. The same steady state concentrations of polymerized and nonpolymerized actin are reached whether profilin is added before initiation of polymerization or after polymerization is complete. The KD for formation of the 1:1 complex between Acanthamoeba profilin and Acanthamoeba actin is in the range of 4 to 11 microM; the KD for the reaction between Acanthamoeba profilin and rabbit skeletal muscle actin is about 60 to 80 microM, irrespective of the concentrations of KCl or MgCl2. The critical concentration of actin for polymerization and the KD for the actin-profilin interaction are independent of each other; therefore, a change in the critical concentration of actin alters the amount of actin bound to profilin at steady state. As a consequence, the presence of profilin greatly amplifies the effects of small changes in the actin critical concentration on the concentration of F-actin. Profilin also inhibits the ATPase activity of monomeric actin, the profilin-actin complex being entirely inactive.

Actins↗

PubMed2005

The adapter protein CrkII regulates neuronal Wiskott-Aldrich syndrome protein, actin polymerization, and tension development during contractile stimulation of smooth muscle.

Actin polymerization has been shown to occur in tracheal smooth muscle tissues and cells in response to contractile stimulation, and there is evidence that the polymerization of actin is required for contraction. In tracheal smooth muscle, agonist-induced actin polymerization is mediated by activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp) and the Arp (actin-related protein) 2/3 complex, and activation of the small GTPase Cdc42 regulates the activation of N-WASp. In the present study, the role of the adapter protein CrkII in the regulation of N-WASp and Cdc42 activation, actin polymerization, and tension development in smooth muscle tissues was evaluated. Stimulation of tracheal smooth muscle tissues with acetylcholine increased the association of CrkII with N-WASp. Plasmids encoding wild type CrkII or a CrkII mutant lacking the SH3 effector-binding ability, CrkII SH3N, were introduced into tracheal smooth muscle tissues, and the tissues were incubated for 2 days to allow for protein expression. Expression of the CrkII SH3N mutant in smooth muscle tissues inhibited the association of CrkII with N-WASp and the activation of Cdc42. The CrkII SH3N mutant also inhibited the increase in the association of N-WASp with Arp2, a major component of the Arp2/3 complex, in response to contractile stimulation, indicating inhibition of N-WASp activation. Expression of the CrkII SH3N mutant also inhibited tension generation and actin polymerization in response to contractile stimulation; however, it did not inhibit myosin light chain phosphorylation. These results suggest that CrkII plays a critical role in the regulation of N-WASp activation, perhaps by regulating the activation of Cdc42, and that it thereby regulates actin polymerization and active tension generation in tracheal smooth muscle. These studies suggest a novel signaling pathway for the regulation of N-WASp activation and active contraction in smooth muscle tissues.

Acetylcholine↗

PubMed1986

Inositol 1,4,5-trisphosphate and calcium stimulate actin polymerization in Dictyostelium discoideum.

The effect of chemoattractants such as cyclic AMP and folate on amoebae of the cellular slime mould Dictyostelium discoideum is to cause a series of rapid intracellular responses. One of the most rapid of these responses is the polymerization of actin associated with the cytoskeleton, an event correlated with pseudopodium formation, which occurs within 3-5 s of chemotactic receptor stimulation. We report that this response can be mimicked by addition of 5 microM-inositol 1,4,5-triphosphate (IP3) or by addition of 100 microM-Ca2+ to saponin-permeabilized amoebae. The data suggest that cytoskeletal actin polymerization occurs in normal cells as a result of IP3 formation in response to cell surface receptor stimulation and the consequent release of Ca2+ from internal stores.

Actins↗

PubMed2006

Integrin-driven actin polymerization consolidates long-term potentiation.

Long-term potentiation (LTP), like memory, becomes progressively more resistant to disruption with time after its formation. Here we show that threshold conditions for inducing LTP cause a rapid, long-lasting increase in polymerized filamentous actin in dendritic spines of adult hippocampus. Two independent manipulations that reverse LTP disrupted this effect when applied shortly after induction but not 30 min later. Function-blocking antibodies to beta1 family integrins selectively eliminated both actin polymerization and stabilization of LTP. We propose that the initial stages of consolidation involve integrin-driven events common to cells engaged in activities that require rapid morphological changes.

Actins↗

PubMed1994

[Ca2+]i-transients and actin polymerization in human neutrophils under stimulation with GRO alpha and complement fragment C5a.

The neutrophil chemotaxins, complement fragment C5a (C5a) and GRO alpha, induced the mobilization of Ca2+ from intracellular stores and the polymerization of actin in human neutrophils as assayed by flow cytometric measurements. [Ca2+]i-transients developed as an "all-or-none" response. Individual neutrophils required different threshold concentrations of added ligand to induce [Ca2+]i-transients which were then always maximal. In contrast, chemotaxin-induced formation of actin filaments in single neutrophils occurred in a dose-dependent manner. Pertussis toxin blocked chemotaxin-induced actin polymerization and [Ca2+]i-transients indicating that both cell responses shared initial activation steps such as ligand binding and activation of guanine nucleotide-binding proteins (G-proteins).

Actins↗

PubMed1994

Actin polymerization, calcium-transients, and phospholipid metabolism in human neutrophils after stimulation with interleukin-8 and N-formyl peptide.

Signal transduction of interleukin-8 (IL-8) was analyzed in neutrophils, and compared with the well known neutrophil activator N-formyl peptide. Stimulation of human neutrophils with IL-8 induced a rapid polymerization of actin as detected by 7-nitrobenz-2-oxa-1,3-diazol-(NBD)-phallacidin staining of f-actin and reduction of monitored right-angle light scatter. Actin polymerization peaked within 10 seconds after the addition of IL-8 and was short-lived as compared to N-formyl peptide-induced stimulation. Analysis of phospholipids by thin-layer chromatography and analysis of deacylation products of lipid extracts by high-pressure liquid chromatography (HPLC) showed that IL-8 triggered a rapid rise of [32P]phosphatidyl-inositol(3,4,5)trisphosphate (PtdInsP3) followed by a slower increase of [32P]phosphatidylinositol(3,4)bisphosphate (PtdIns-3,4-P2) along with a rapid decrease of [32P]phosphatidylinositol(4,5)bisphosphate (PtdIns-4,5-P2). Changes in polyphosphoinositide metabolism were more moderate and transient than those obtained by N-formyl peptide. Moreover, [32P]phosphatidic acid (PA) production stimulated by IL-8 was minimal and transient as compared to the response activated by N-formyl peptide. Both IL-8 and N-formyl peptide induced Ca++ mobilization from intracellular stores, but IL-8 in contrast to N-formyl peptide failed to trigger the secondary influx of Ca++ from the extracellular medium. In summary, IL-8 and N-formyl peptide stimulated similar and distinct patterns of intracellular activation steps. This study indicates that IL-8 is a potent activator of intracellular events presumably required for chemotaxis, but a relatively weak activator for events associated with superoxide anion generation and proinflammatory activity.

Actins↗

PubMed2004

Ca2+-calmodulin regulates fesselin-induced actin polymerization.

Fesselin is a proline-rich actin-binding protein that was isolated from avian smooth muscle. Fesselin bundles actin and accelerates actin polymerization by facilitating nucleation. We now show that this polymerization of actin can be regulated by Ca(2+)-calmodulin. Fesselin was shown to bind to immobilized calmodulin in the presence of Ca(2+). The fesselin-calmodulin interaction was confirmed by a Ca(2+)-dependent increase in 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) fluorescence upon addition of fesselin to MIANS-labeled wheat germ calmodulin. The affinity was estimated to be approximately 10(9) M(-1). The affinity of Ca(2+)-calmodulin to the fesselin F-actin complex was approximately 10(8) M(-1). Calmodulin binding to fesselin appeared to be functionally significant. In the presence of fesselin and calmodulin, the polymerization of actin was Ca(2+)-dependent. Ca(2+)-free calmodulin either had no effect or enhanced the ability of fesselin to accelerate actin polymerization. Ca(2+)-calmodulin not only reversed the stimulatory effect of fesselin but reduced the rate of polymerization below that observed in the absence of fesselin. While Ca(2+)-calmodulin had a large effect on the interaction of fesselin with G-actin, the effect on F-actin was small. Neither the binding of fesselin to F-actin nor the subsequent bundling of F-actin was greatly affected by Ca(2+)-calmodulin. Fesselin may function as an actin-polymerizing factor that is regulated by Ca(2+) levels.

Actins↗

PubMed1982

Purification and characterization of a new mammalian serum protein with the ability to inhibit actin polymerization and promote depolymerization of actin filaments.

A protein with capacity to bind G-actin and the ability to inhibit polymerization and promote depolymerization of actin filaments has been isolated from the serum of rabbit. The protein, SAIP (for serum actin inhibitory protein), has been purified by affinity chromatography of serum over actin-Sepharose followed by protein fractionation with ammonium sulfate and chromatography over DEAE-cellulose. Five milligrams of purified SAIP is obtained from 100 mL of serum. Rabbit SAIP is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two closely related polypeptides of 60000 and 56000 daltons, respectively (ratio 5.1:1). Each of these polypeptides consists of two isoelectric variants. SAIP binds to monomeric actin with a stoichiometry of 1:1 and a Kd of 0.12 microM. The SAIP-actin complex binds to DNase I. Actin polymerization is completely inhibited by incubation of actin with an equal concentration of SAIP. At equimolar concentrations to F-actin, SAIP induces complete depolymerization of the actin filaments. SAIP is also present in calf serum.

Actin Depolymerizing Factors↗

PubMed1986

Fibronectin potentiates actin polymerization in thrombin-activated platelets.

The effect of fibronectin on the polymerization state of actin was studied. Triton X-100-insoluble cytoskeleton was prepared from thrombin-activated platelets, and the conversion of G-actin into F-actin was monitored by an assay involving DNase I inhibition by G-actin. It was found that fibronectin bound to membrane receptors decreased the level of platelet G-actin. This observation suggests that in the presence of fibronectin a larger amount of F-actin becomes incorporated into the Triton X-100-insoluble cytoskeleton. At the same molar concentration, fibrinogen only slightly increased actin polymerization, whereas bovine serum albumin at a much higher concentration caused a small inhibition of actin immobilization. Our data show that fibronectin, through interaction with the platelet actomyosin fibrillar system, facilitates actin polymerization into the cytoskeleton.

Actins↗

PubMed1998

Effect of replacement of the tightly bound Ca2+ by Ba2+ on actin polymerization.

G-actin has a single tight-binding (high-affinity) site for divalent cations per mole of protein, whose occupancy is important for the stability of the molecule. Different tightly bound divalent cations differently influence the polymerization properties of actin. The tightly bound metal ion easily exchanges for free exogenous cations. Moreover, biochemical and structural evidence demonstrates that actin, in both the G- and F-forms, assumes different conformations depending on the metal ion bound with high affinity in the cleft between two main domains of the molecule. In this work, we used proteolytic susceptibility to detect possible local conformational alterations of the actin molecule following a brief incubation of Ca-G-actin with barium chloride and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We found that substitution of Ba2+ for the tightly bound Ca2+ affects the regions around Arg-62 and Lys-68 in subdomain 2 of G-actin, as judged from inhibition of tryptic cleavage at these residues. Using the fluorescent chelator Quin-2, we observed that about 0.95 mol of Ba2+ is released per 1 mol of actin. We also examined the effect of replacement of the tightly bound Ca2+ by Ba2+ on actin polymerization. With respect to Ca-actin, Ba-actin shows an increased polymerization rate, mainly due to its enhanced nucleation and a higher critical concentration.

Actins↗

PubMed1990

Kinetic analysis of chemotactic peptide-induced actin polymerization in neutrophils.

Definition of the kinetics of ligand-activated actin polymerization in the neutrophil is important for ultimately understanding the mechanisms utilized for regulation of actin polymerization in this non-muscle cell. To better define the kinetics of formyl peptide (fMLP)-induced actin polymerization in neutrophils we determined F-actin content at 5 second intervals after activation of human neutrophils with a range (10(-11)-10(-9) M) of fMLP concentrations. The state of actin polymerization was monitored by quantifying F-actin content with NBD phallacidin binding in both flow cytometric and extraction assays. Results demonstrate three successive kinetic periods of fMLP-induced actin polymerization in neutrophils, a lag period, a 5 second period when rate of polymerization is maximal, and a period of declining rate of actin polymerization as F-actin content approaches a maximum. The duration of the lag period, the maximum rate of polymerization, and the maximum extent of polymerization all depend upon the fMLP concentration. The lag period varies from 0 to 12 seconds and is followed in 5-10 seconds by a 5 second burst of actin polymerization when the rate is as great as 9% increase in F-actin content per second. After the 5 second burst of polymerization, the rate of polymerization rapidly declines. The study defines three distinct kinetic periods of fMLP-induced actin polymerization during which important rate-limiting biochemical events occur. The mechanistic and motile implications of kinetic periods are discussed.

Actins↗

PubMed2004

Electron microscopic study of actin polymerization in airway smooth muscle.

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.

Actin Cytoskeleton↗

PubMed1988

Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape.

Platelets gradually lose their disc shape during storage. The authors studied simultaneous changes in platelet cytosolic Ca2+ (Cai) and the polymerization state of actin as related to the shape. Platelet concentrates were stored under blood bank conditions for up to 10 days. Aliquots were removed and analyzed as follows: platelet Cai and increments in Cai induced by adenosine diphosphate (ADP) were determined by fluorescence of fura-2-loaded cells; loss of disc shape was determined by differences in light scattering intensity induced by stirring; and the ratio of globular and total actin (G/T) of platelets in plasma was determined by a modification of the DNase inhibition assay. Globular actin was found to be 86 +/- 3% of total actin in freshly drawn platelets suspended in plasma. The following changes occurred during storage: G/T in platelet concentrates increased from 63 +/- 5 (day 0) to 74 +/- 2% in the first 24 hours then fell to 33 +/- 6% by day 10. The percent discoid platelets also increased from day 0 to day 1 then fell in the ensuing days. There was an initial drop in Cai from day 0 to day 1, after which Cai increased on days 3 and 6. Globular actin polymerization during storage closely correlated with the change in percent discs (r = 0.95). During 6 days of storage Cai was highly correlated with shape change (r = 0.97) and to a lesser extent (r = 0.87) with the ratio of globular actin. The authors conclude that actin polymerization, shape, and Ca2+ change in a related fashion during storage.

Actins↗