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At least 19 recordsLinked to original sources
PubMed1986

Stimulated cytokineplasts from human polymorphonuclear leukocytes mobilize calcium and polymerize actin. Cytoplasts made in cytochalasin B retain a defect in actin polymerization.

Biologically active fragments from polymorphonuclear leukocytes (PMN) are simplified systems that can be used to elucidate specific pathways by which cell function is altered. In the current study we have found that cytokineplasts, which are motile fragments derived from the leading front (protopod, lamellipodium) of human PMN, rapidly increase their intracellular free calcium concentration when stimulated by chemotactic formyl peptide or by leukotriene B4, as measured by Quin-2 acetoxymethyl ester fluorescence. As in the parent cell, extracellular EGTA blunts this response only partially. Hence, cytokineplasts retain a mobilizable internal calcium pool, despite a general lack of intracellular organelles. In addition, formyl peptide more than doubles the amount of cytoskeleton-associated (polymerized) actin. In contrast, cytoplasts made by high-speed, discontinuous gradient centrifugation of cytochalasin B-treated leukocytes also increase their intracellular free calcium on stimulation, but cytoskeleton-associated actin increases by only approximately 14%. Thus, defective motile function in the latter cytoplast is associated with compromised effector function (actin polymerization).

Actins↗
PubMed1991

Actin polymerization in cellular oxidant injury.

Microfilaments undergo an ATP-dependent disruption into shortened bundles following cellular exposure to oxidants. This phenomenon does not require a net change in the amount of polymerized actin. However, increased amounts of polymerized actin have been detected in oxidant-injured cells and it was the purpose of this study to determine the conditions under which the actin polymerization may occur. Utilizing the formation of oxidized glutathione (GSSG) as an indicator of cellular sulfhydryl oxidation, conditions were chosen to accentuate sulfhydryl oxidation within the target P388D1 cell line following exposure to the oxidants, H2O2 and diamide. Using the DNase I and flow cytometric assays of actin polymerization, significant polymerization of actin was detected only under conditions in which sulfhydryl oxidation occurred after exposure to the two oxidizing agents. Greater sulfhydryl oxidation early in the course of injury was associated with a greater rate and extent of actin polymerization in the injured cells. Experiments with cells depleted of glutathione (GSH) demonstrated that neither loss of GSH nor absolute levels of GSSG formed during oxidant exposure were responsible for the polymerization of actin. The data presented are consistent with the hypothesis that oxidizing conditions which induce significant sulfhydryl oxidation in target cells are correlated with assembly of polymerized actin and that this represents a process which is distinct and separate from the ATP-dependent gross disruption of microfilaments.

Actins↗
PubMed1990

Actin polymerization in neutrophils is triggered without a requirement for a rise in cytoplasmic Ca2+.

Stimulation of rat neutrophils with the peptide fMetLeuPhe caused (i) the appearance of a 40 kDa protein in the Triton-X-100-insoluble cytoskeleton, (ii) the disappearance of DNAase inhibition from the cytosol and (iii) the appearance of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin) binding sites. All three observations were consistent with a rapid and transient assembly of polymerized actin, peaking at approximately 5 s and returning to near resting levels within 40 s. By experimentally depleting the cells of Ca2+ and increasing the cytoplasmic Ca2+ buffering capacity, the peptide-induced Ca2+ transient was reduced from a peak of 900 nM to 250 nM, without inhibiting actin polymerization, and this peak was sustained for at least 2 min. A further dissociation between the triggering of actin polymerization and peptide-induced Ca2+ elevation and oxidase activation was demonstrated at low concentrations of peptide (1-100 pM), actin polymerization being triggered without an elevation in Ca2+ or activation of the oxidase. Two other agents which induced actin polymerization, phorbol 12-myristate 13-acetate and latex beads, failed to elevate cytoplasmic Ca2+. It was therefore concluded that neither Ca2+ nor those intracellular messengers which act with Ca2+ to trigger the neutrophil oxidase are responsible for triggering actin polymerization in neutrophils.

Actins↗
PubMed1990

Gelsolin-actin interaction and actin polymerization in human neutrophils.

The fraction of polymerized actin in human blood neutrophils increases after exposure to formyl-methionyl-leucyl-phenylalanine (fmlp), is maximal 10 s after peptide addition, and decreases after 300 s. Most of the gelsolin (85 +/- 11%) in resting ficoll-hypaque (FH)-purified neutrophils is in an EGTA resistant, 1:1 gelsolin-actin complex, and, within 5 s after 10(-7) M fmlp activation, the amount of gelsolin complexed with actin decreases to 42 +/- 12%. Reversal of gelsolin binding to actin occurs concurrently with an increase in F-actin content, and the appearance of barbed-end nucleating activity. The rate of dissociation of EGTA resistant, 1:1 gelsolin-actin complexes is more rapid in cells exposed to 10(-7) M fmlp than in cells exposed to 10(-9) M fmlp, and the extent of dissociation 10 s after activation depends upon the fmlp concentration. Furthermore, 300 s after fmlp activation when F-actin content is decreasing, gelsolin reassociates with actin as evidenced by an increase in the amount of EGTA resistant, 1:1 gelsolin-actin complex. Since fmlp induces barbed end actin polymerization in neutrophils and since in vitro the gelsolin-actin complex caps the barbed ends of actin filaments and blocks their growth, the data suggests that in FH neutrophils fmlp-induced actin polymerization could be initiated by the reversal of gelsolin binding to actin and the uncapping of actin filaments or nuclei. The data shows that formation and dissociation of gelsolin-actin complexes, together with the effects of other actin regulatory proteins, are important steps in the regulation of actin polymerization in neutrophils. Finally, finding increased amounts of gelsolin-actin complex in basal FH cells and dissociation of the complex in fmlp-activated cells suggests a mechanism by which fmlp can cause actin polymerization without an acute increase in cytosolic Ca++.

Actins↗
PubMed1991

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.

Actins↗
PubMed1992

Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus.

Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoeba proteus cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin.

Actins↗
PubMed1988

Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments.

Tropomyosin inhibition of the rate of spontaneous polymerization of actin is associated with binding of tropomyosin to actin filaments. Rate constants determined by using a direct electron microscopic assay of elongation showed that alpha alpha- and alpha beta-tropomyosin have a small or no effect on the rate of elongation at either end of the filaments. The most likely explanation for the inhibition of the rate of polymerization of actin in bulk samples is that tropomyosin reduces the number of filament ends by mechanical stabilization of the filaments.

Actin Cytoskeleton↗
PubMed1992

Thrombin promotes actin polymerization in U937 human monocyte-macrophage cells. Analysis of the signalling mechanisms mediating actin polymerization.

The U937 human monocyte-macrophage cell line was used to examine the effect of thrombin, an ill-defined chemoattractant, on the polymerization of actin, a process essential for cell motility. In differentiated macrophage-like U937 cells, thrombin (0.5-50 units/ml) caused a rapid dose-dependent increase in the formation of filamentous (F-) actin, detected by the staining of F-actin with the fluorescent toxin, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In contrast with other chemoattractants such as N-formylmethionyl-leucylphenylalanine or C5a, actin polymerization in response to thrombin occurred via a pertussis-toxin-insensitive G1-(inhibitory G-protein) independent signalling pathway. Further, this response was not affected by the Ca2+ chelator EGTA or by the specific protein kinase C (PKC) inhibitor RO-31-8220. The response to thrombin was not mimicked by the Ca2+ ionophore ionomycin or by the direct PKC activator phorbol 12-myristate 13-acetate. The thrombin response was, however, inhibited by the non-specific protein kinase inhibitor staurosporine. The present results suggest that in U937 cells thrombin stimulates the formation of F-actin via a signalling pathway independent of (i) the activation of PKC, (ii) the mobilization of intracellular Ca2+ and (iii) the activation of Ca(2+)-dependent protein kinases, but dependent on the activation of an undefined staurosporine-sensitive protein kinase.

Actins↗
PubMedDate not supplied

Intracellular movements of Rickettsia conorii and R. typhi based on actin polymerization.

Human vascular endothelial, Vero and human embryonic lung cells infected with rickettsiae for 24 h or 48 h were labelled for polymerized actin with NBD-phallacidin. Between 20 and 68% of the intracellular Rickettsia conorii had an actin tail of between 0.33 and 15 microns, with the longest tails being observed in Vero cells. In the case of R. typhi less than 1% of the organisms had actin tails and these were considerably shorter than those of R. conorii. These findings provide new information concerning the different cytopathic effects observed with the two rickettsial species.

Actins↗
PubMed1978

Polyamine-induced actin polymerization.

Muscle actin has been found to polymerize reversibly upon addition of low concentrations of polyamines. This polymerization, studied by centrifugation, has shown a linear relationship between the actin polymerization yield and the chain length of the polyamine. Among the biological polyamines tested, spermidine and spermine are the most efficient. The polymerization of actin can also be induced by the corresponding mono or diguanidine derivatives of these polyamines but monoamines or amino acids are inactive at the same concentration. The transformation of actin from a globular to a fibrous from upon addition of spermidine is also demonstrated by the changes in the near-ultraviolet circular dichoroic spectrum of this protein. Moreover, the polyamine-induced F -actin exhibits the same properties as the salt-induced F -actin: it strongly activates the Mg2+ -ATPase of myosin, its specific viscosity is enhanced to the same extent and electron micrographs show homogeneous thin filaments.

Actins↗
PubMed1985

Purification and characterization of a protein from chicken gizzard, which inhibits actin polymerization.

An actin-polymerization-inhibiting protein, that occurs in crude preparations of vinculin from chicken gizzard, has been purified by DEAE-cellulose and carboxymethyl ion-exchange chromatography. According to sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis and to gel filtration the polymerization-inhibiting protein is heterogeneous and the molecular mass ranges from 20 kDa to 80 kDa. After treatment with acid the polymerization-inhibiting activity was found to migrate on a SDS/polyacrylamide gel as a single band of molecular mass about 32 kDa. The mechanism of the action of the polymerization-inhibiting protein on actin assembly was investigated by the effect on the kinetics of actin polymerization. The polymerization-inhibiting protein blocks elongation of actin filaments at substoichiometric ratios but does not nucleate actin filaments. The equilibrium constant for binding of the polymerization-inhibiting protein to the barbed end of an actin filament was estimated to be 2 X 10(6) M-1 in 100 mM KCl and 2 mM MgCl2, and 35 X 10(6) M-1 in 2 mM MgCl2.

Actin Depolymerizing Factors↗
PubMed1987

Aging and lymphocyte cytoskeleton: age-related decline in the state of actin polymerization in T lymphocytes from Fischer F344 rats.

T cell functions are known to decline with age, but the underlying cause of the decline is unclear. Because of the importance of cytoskeletal elements in cellular functions, we examined the content and the state of polymerization of actin in lymphocytes from Fischer F344 rats of four different ages (6, 14, 23, and 31 mo). The cellular actin content was determined by a DNAase I inhibition assay. Our results indicate that the total actin content of spleen lymphocytes did not change significantly with age; however, polymeric actin content, particularly in T cells, decreased with age, which might be a result of the shift from the polymeric actin pool to the monomeric pool. Similar changes also occurred in B cells but to a lesser extent. We conclude that the state of polymerization of lymphocytes changed drastically with age, and that this might be an important factor in the age-related decline in the cellular functions of lymphocytes.

Actins↗
PubMed1988

Low concentration of reserpine accelerates actin polymerization via interaction with G-actin.

The effect of reserpine on actin polymerization was examined by measurement of the changes in high shear viscosity and by electron microscopic observation of the actin solution. In the presence of low concentrations of reserpine, the time course of actin polymerization was accelerated dose dependently (up to approximately 0.5 nM), without affecting the final level of viscosity. The effect of reserpine rather decreased with dosages over this concentration. The binding of reserpine to actin was tested by developing the mixture of G- or F-actin and [3H]reserpine through a Sephadex G-50 column. A portion of the reserpine coeluted with G-actin, but little reserpine did with F-actin. This means that reserpine bound to G-actin but scarcely to F-actin. The binding of reserpine to G-actin was also confirmed using the method of photoaffinity labeling. After the irradiation of the mixed G-actin and [3H]reserpine by ultraviolet light, they were subjected to SDS-PAGE followed by fluorography. It was demonstrated that reserpine was bound to G-actin covalently by the ultraviolet light irradiation. This indicated the close interaction of reserpine with G-actin. Thus, the effect of reserpine on actin polymerization seemed to be exerted via interaction with G-actin.

Actins↗
PubMed1988

Calcium mobilization, actin polymerization and right-angle light scatter responses to leukotriene B4, 12(R)- and 12(S)-hydroxyeicosatetraenoic acid in human neutrophils.

The presence of microgram quantities of 12(R)-hydroxyeicosatetraenoic acid (12(R)HETE) in psoriatic scales has been reported. 12(R)HETE has been found to be 5-10 times more potent than its isomer 12(S)HETE in inducing neutrophil locomotion and aggregation. In this study, the ability of these two eicosanoids to elicit a mobilization of calcium and a polymerization of actin was examined and compared to that of leukotriene B4. These two cell-biochemical assays were chosen in view of the likelihood that they are relevant to the motile functions of the neutrophils. 12(R)HETE was found to induce an increase in the cytoplasmic level of free calcium and in the amount of polymerized actin. 12(S)HETE also raised the level of free calcium, though to a lesser extent than 12(R)HETE, but did not induce a detectable polymerization of actin. Leukotriene B4 was more active, on the basis of concentration and maximal response, than either 12(R)HETE or 12(S)HETE. The activity of 12(R)HETE detected and reported in this communication provides support for the suggestion that this eicosanoid may play a significant role in the pathogenesis of the inflammatory reactions in psoriasis.

Actins↗
PubMed1978

Differences between nucleus and cytoplasm in the degree of actin polymerization.

For purposes of studying the degree of polymerization of actin in nuclei, nuclei from 35S-labeled amoebas (Amoeba proteus) were transplanted into unlabeled cells, which were immediately lysed and extracted under conditions considered to stabilize preexisting fibrous actin. The enucleated 35S-donor cells were similarly treated for analysis of cytoplasmic actin. The extraction conditions permitted separation of soluble (unpolymerized or G) actin from pelletable (polymerized or F) actin, and the radioactivity of each was determined after the actin was separated from other proteins by polyacrylamide gel electrophoresis. We found that about 2/3 of the actin within the nucleus is pelletable, whereas only about 1/3 of the cytoplasmic actin is pelletable. We speculate that polymerized actin in the nucleus is involved in the condensation of chromatin.

Actins↗
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↗