The diacylglycerol kinase inhibitor R59022 potentiates superoxide production but not secretion induced by fMet-Leu-Phe: effects of leupeptin and the protein kinase C inhibitor H-7

The addition of low concentrations of the chemotactic factor fMet-Leu-Phe to rabbit neutrophils in the absence of cytochalasin B produces very little superoxide. This level of superoxide can be greatly increased in neutrophils pretreated for 30 min with 10 microM of the diacyl-glycerol kinase inhibitor R59022. This potentiation occurs also in the presence of cytochalasin B. In addition, while the small level of superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7), the increase by R59022 is completely abolished by this compound. In addition, this increase can be potentiated further by leupeptin. Unlike superoxide generation, the release of lysozyme or N-acetyl-beta-glucosaminidase produced by fMet-Leu-Phe is not stimulated by R59022. The results presented here suggest that stimulation of the oxidative burst requires the generation and the maintenance of a sufficient amount of diacylglycerol and/or the rearrangement of the cytoskeleton such as the inhibition of actin polymerization. Furthermore, the membrane-associated form of protein kinase C is the one responsible for the activation of the oxidative burst. The relationship between protein kinase C activation and the stimulated oxidative burst and the physiological role of chemotactic factors in the functions of the neutrophils are discussed.     

Association of leukotriene B4 with the cytoskeleton of rabbit neutrophils. Effect of chemotactic factor and phorbol esters

Following its addition to a suspension of rabbit neutrophils, leukotriene B4 is rapidly (less than 1 min) recovered from the cytoskeletal fraction (Triton X-100 insoluble pellet) of these cells. The association of leukotriene B4 with the cytoskeleton can be competed with by leukotriene B4 itself and by 20-OH leukotriene B4 but not by 20-COOH leukotriene B4. In addition, the preincubation of the cells with fMet-Leu-Phe or with phorbol 12-myristate 13-acetate, but not with 4 alpha-phorbol 12,13-didecanoate, results in a greatly decreased association of leukotriene B4 with the cytoskeleton. These results suggest that a specific association between the leukotriene B4 receptors and the cytoskeleton may be involved in signal transduction in the leukotriene B4 stimulated neutrophils.     

Stimulus-induced selective association of actin-associated proteins (alpha-actinin) and protein kinase C isoforms with the cytoskeleton of human neutrophils.

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein alpha-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal alpha-actinin and actin. Increased association of PKCbetaI and betaII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, alpha-actinin, and PKCbetaII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal alpha-actinin and PKCbetaII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 microM) completely blocked PMA-induced increases in cytoskeletal alpha-actinin but reduced cytoskeletal recruitment of PKCbetaII only by 16%. Higher concentrations of latrunculin A (4 microM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCbetaII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils

Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dose-dependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.     

Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: mediation by cyclic AMP.

Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP.     

The hyaluronate receptor is associated with actin filaments

The cell-surface receptor for hyaluronate is an integral membrane glycoprotein of Mr 85,000 (Underhill, C. B., A. L. Thurn, and B. E. Lacy, 1985, J. Biol. Chem., 260:8128-8133) that is thought to mediate many of the effects that hyaluronate has on cell behavior, such as migration, angiogenesis, and phagocytosis. To determine if the receptor is associated with the underlying cytoskeleton, Swiss 3T3 cells were extracted with a solution of Triton X-100, which solubilized most of the cellular components, but which left behind an insoluble residue containing the cytoskeleton. This detergent-insoluble residue was found to contain the bulk of the hyaluronate-binding activity, suggesting that the receptor might indeed be associated with the cytoskeleton. To further define the cytoskeletal element with which the receptor interacts, 3T3 cells were extracted with Triton X-100 under a variety of different ionic conditions. In each case, the amount of hyaluronate-binding activity in the detergent-insoluble residue was related to the amount of actin present, but not to either tubulin or vimentin. In addition, the recovery of hyaluronate-binding activity was dramatically enhanced (to 100% in most cases) if the cells were extracted in the presence of phalloidin, a drug that stabilizes actin filaments. However, the recovery of binding activity was dramatically decreased when whole cells were treated with cytochalasin B before extraction, and when extracted cells were treated with DNase I, which promotes the depolymerization of actin filaments. In addition, preincubating an extract of SV-40-transformed Swiss 3T3 cell membranes with DNase I caused a change in the elution profile of the receptor as judged by molecular-sieve chromatography. Presumably this decrease in the size of the receptor is due to the loss of associated actin filaments. The results of these experiments strongly suggest that the receptor for hyaluronate is associated either directly or indirectly with cytosolic actin filaments.     

Stimulus-Induced Selective Association of Actin-Associated Proteins (α-Actinin) and Protein Kinase C Isoforms with the Cytoskeleton of Human Neutrophils

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein α-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal α-actinin and actin. Increased association of PKCβI and βII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, α-actinin, and PKCβII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal α-actinin and PKCβII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 μM) completely blocked PMA-induced increases in cytoskeletal α-actinin but reduced cytoskeletal recruitment of PKCβII only by 16%. Higher concentrations of latrunculin A (4 μM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCβII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

Intracellular acidification, guanine-nucleotide binding proteins, and cytoskeletal actin

The addition of propionic acid to rabbit neutrophils causes cell acidification and increases the amount of actin associated with the cytoskeleton. Both responses are rapid, and while the cell acidification is somewhat long-lasting, the increase in cytoskeletal actin is transient. It reaches a maximum value within 15 seconds and then returns to the basal level. Unlike fMet-Leu-Phe, however, propionic acid does not cause a rise in the intracellular concentration of free calcium. Pretreatment of the cells with pertussis toxin inhibits the propionic acid-produced increase in cytoskeletal actin but not the decrease in intracellular pH. However, the rate of return to the base line of the cell acidification produced by propionic acid is diminished in cells pretreated with pertussis toxin. On the other hand, both the decrease in intracellular pH and the increase in cytoskeletal actin produced by fMet-Leu-Phe are inhibited by pertussis toxin treatment. The results presented here suggest two important points. First, while cell acidification may trigger directly or indirectly the association of actin with the cytoskeleton, it is certainly not sufficient. Second, a functional guanine-nucleotide regulatory protein is required for stimulated cytoskeletal actin. One or more components of the G-protein and/or their effects on phosphoinositide hydrolysis may increase the number of actin monomers and the availability of preexisting actin filaments to these monomers.     

Actin-associated proteins in human neutrophils: identification and reorganization upon cell activation

The neutrophil cytoskeleton, especially the actin network, is thought to play a crucial role in neutrophil migration. However, little is known on the modulation of this network by actin-associated proteins. We have demonstrated the presence of immuno-reactive forms of alpha-actinin (an actin cross-linking and bundling protein) and vinculin (a putative actin-membrane linker) in human neutrophils using specific antibodies to chicken gizzard vinculin and bovine epithelial alpha-actinin. In contrast, talin, another putative actin-membrane linker protein, could not be detected in significant amounts in human neutrophils using a polyclonal antibody raised against chicken gizzard talin, which reacted with human platelet and lymphocyte talin. We have also analyzed the vinculin and alpha-actinin content of Triton X-100 insoluble cytoskeletons, isolated from resting and activated neutrophils. A small amount of alpha-actinin was already associated with the cytoskeleton of resting cells. Addition of chemotactic peptide to the cells rapidly increased the alpha-actinin content of the cytoskeletons 1.6 to 7-fold. This rapid increase was followed by a slower decrease to a lower level which, after 30 min of stimulation, was still significantly higher than that of control cells. The time-course of the association of alpha-actinin with the cytoskeleton paralleled that of actin association. This stimulus-induced rearrangement of cellular alpha-actin may thus play an important role in determining the structure of actin networks in motile neutrophils. Vinculin in contrast could not be detected in significant amounts in the Triton X-100-insoluble neutrophil cytoskeleton, not even after prolonged stimulation of the cells by chemotactic peptide.     

Contraction of Dictyostelium ghosts reconstituted with myosin II.

Cytoskeletons, or 'Triton ghosts,' that contained mainly actin and myosin II were prepared from Dictyostelium discoideum amoebae by extraction with Triton X-100. The Triton ghosts contracted immediately upon addition of ATP. However, under high-salt conditions in the presence of ATP, they did not contract but released myosin II. Almost all of the applied myosin II became associated with ghosts when myosin-free Triton ghosts, prepared in this way, were incubated with purified actin and then with myosin II from Dictyostelium. Immunofluorescence microscopy demonstrated that the associated myosin was localized in the cortical actin layer of the ghosts. Furthermore, the ghosts reconstituted with purified myosin resumed ATP-dependent contraction. Skeletal muscle myosin could also restore contractility to ghosts from which myosin had been extracted. The amount of myosin II necessary for the contraction of the ghosts was calculated by two methods. Less than 10% of the myosin II in intact cells was necessary for the contraction. These results show that myosin II is responsible for the contraction of the Dictyostelium cytoskeleton.     

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.     

Characterization of the membrane-associated GTPase activity: effects of chemotactic factors and toxins

Membranes prepared from rabbit neutrophils exhibit GTPase activity which can be stimulated by the chemotactic factor fMet-Leu-Phe. The maximum contribution of the ATPase activities to the basal and the fMet-Leu-Phe-stimulated GTPase activities are less than 20% and 9%, respectively. The basal GTPase activity has a Vmax = 34.2 +/- 1.3 (pmol/mg protein, min) and a Km = 0.39 +/- 0.03 microM; and the fMet-Leu-Phe-stimulated has a Vmax = 52.3 +/- 2.5 (pmol/mg protein, min), and a Km = 0.29 +/- 0.02 microM. The GTPase activity can be stimulated by fMet-Leu-Phe and leukotriene B4. Unlike these two chemotactic factors, concanavalin A does not stimulate this GTPase activity. In addition, the rise in intracellular concentration of free calcium produced by concanavalin A is not inhibited by pertussis toxin treatment. Both the basal and stimulated GTPase activities are affected by pertussis toxin, cholera toxin and N-ethylmaleimide.     

Role of the actin cytoskeleton during respiratory burst in chemoattractant-stimulated neutrophils

The aim of this study was to clarify the role of the actin cytoskeleton during chemotactic peptide fMet-Leu-Phe (fMLF)-stimulated respiratory burst in human neutrophil granulocytes. Reactive oxygen species (ROS) was measured as luminol-amplified chemiluminescence (CL) and F-actin content as bodipy phallacidin fluorescence in neutrophils treated with latrunculin B or jasplakinolide, an inhibitor and activator of actin polymerization, respectively. Latrunculin B markedly decreased, whereas jasplakinolide increased, the F-actin content in neutrophils, unstimulated or stimulated with fMLF. Latrunculin B enhanced the fMLF-triggered ROS-production more than tenfold. Jasplakinolide initially inhibited the fMLF-induced CL-response, however, caused a potent second sustained phase (>400% of control). Both actin drugs triggered a substantial CL-response when added 5-25 min after fMLF. This was also valid for chemotactic doses of fMLF, where latrunculin B and jasplakinolide amplified the ROS-production 5-10 times. By using specific signal transduction inhibitors, we found that the NADPH oxidase activation triggered by destabilization of the actin cytoskeleton occurs downstream of phospholipase C and protein kinase C but is mediated by Rho GTPases and tyrosine phosphorylation. In conclusion, rearrangements of the actin cytoskeleton are a prerequisite in connecting ligand/receptor activation, generation of second messengers and assembly of the NADPH oxidase in neutrophil granulocytes.     

Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors. Effect of fMet-Leu-Phe and partial characterization of the protein kinase

Rabbit peritoneal neutrophils were stimulated with either the chemotactic factor, fMet-Leu-Phe (10(-8) M, 10 s) or the protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), (0.1 microgram/ml, 3 min) at 37 degrees C, lysed with Triton X-100 at the indicated times and the histone H4 kinase activity of the lysate measured. The histone H4 protein kinase activity was increased severalfold by fMet-Leu-Phe but not PMA. The inclusion of the potent protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (50 microM) inhibited little if any of the histone H4 protein kinase activity. The effect of fMet-Leu-Phe was transient, maximum stimulation occurring within 10 s and decaying thereafter. The soluble fraction (extract) of the Triton X-100 lysates from control and fMet-Leu-Phe-treated cells was found to contain both histone H4 protein kinase and calcium-phospholipid-activated protein kinase (protein kinase C) activities. The histone H4 protein kinase activity obtained after fMet-Leu-Phe treatment was very little affected by calcium, phospholipid, and PMA and preferred histone H4 but not H1 or H2A as its substrate. In contrast, the calcium-phospholipid-activated protein kinase activity of the extract preferred histones H1 or H2A as substrates and was strongly inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine. The histone H4 protein kinase was partially separated from kinase C by DEAE-cellulose and phenyl-Sepharose 4B chromatography. It phosphorylated mostly serine in histone H4. The results indicate that the chemotactic factor, fMet-Leu-Phe, stimulates a protein kinase with substrate specificity and biochemical properties distinct from calcium-phospholipid-activated protein kinase C.     

Cell adhesion and acquisition of detergent resistance by the cytoskeleton of cultured chick fibroblasts

About 30% of the proteins of adherent cultured chick embryo fibroblasts are not solubilized by the non-ionic detergent Triton X-100 and remain firmly attached to the substratum. The insoluble residue contains a considerable part of the cell's cytoskeleton and its major constituents are large external transformation-sensitive (LETS) protein, the heavy chain of myosin, a 52,000 molecular weight protein and actin. Kinetic studies reveal that cytoskeleton insolubility in Triton is acquired either concurrently with cell adhesion or very closely with it. Neither cell adhesion nor binding of the Triton cytoskeleton to the substratum require de novo synthesis of protein. In the attempt to assess the role of LETS protein in cytoskeleton attachment, we find that trypsin-detached cells rapidly acquire Triton-insoluble cytoskeleton although their LETS protein content is about 15--20% of its level in long-term cultures. Removal of the great majority of LETS molecules of adherent cultures by either urea or trypsin treatment does not affect the relative amount or composition of the anchored cytoskeletal proteins. Also, LETS protein of cultures exposed to cycloheximide for extended periods of time, is reduced to 10% of its maximum amount without much affecting the attachment and composition of the cytoskeleton. It is deduced that the great majority of LETS protein is not required for the attachment of the Triton cytoskeleton to the substratum.     

Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand- receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet- Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X- 100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu- Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G- protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet- Leu-Phe-associated G-protein.     

NPA binding activity is peripheral to the plasma membrane and is associated with the cytoskeleton

N-1-Naphthylphthalamic acid (NPA) binding activity is released into the supernatant when plasma membranes are subjected to high-salt treatment, indicating that this activity is peripherally associated with the membrane. Extraction of plasma membrane vesicles with Triton X-100 resulted in retention of NPA binding activity in the detergent-insoluble cytoskeletal pellet. Treatment of this pellet with KI released NPA binding activity, actin, and alpha-tubulin. Dialysis to remove KI led to the repolymerization of cytoskeletal elements and movement of NPA binding activity into an insoluble cytoskeletal pellet. NPA binding activity partitioned into the detergent-insoluble cytoskeletal pellet obtained from both zucchini and maize membranes and was released from these pellets by KI treatment. Treatment of a cytoskeletal pellet with cytochalasin B doubled NPA binding activity in the resulting supernatant. Together, these experiments indicate that NPA binding activity is peripherally associated with the plasma membrane and interacts with the cytoskeleton in vitro.     

A novel 36,000-dalton actin-binding protein purified from microfilaments in Physarum plasmodia which aggregates actin filaments and blocks actin-myosin interaction

In the plasmodia of Physarum polycephalum, which show a cyclic contraction-relaxation rhythm of the gel layer, huge aggregates of entangled actin microfilaments are formed at about the onset of the relaxation (R. Nagai, Y. Yoshimoto, and N. Kamiya. 1978. J. Cell Sci. 33:205-225). By treating the plasmodia with Triton X-100, we prepared a demembranated cytoskeleton consisting of entangled actin filaments and found that the actin filaments hardly interact with rabbit skeletal myosin. From the cytoskeleton we purified a novel actin-binding protein which binds stoichiometrically to actin and makes actin filaments curled and aggregated. It also inhibits the ATPase activity as well as the superprecipitation of reconstituted rabbit skeletal muscle actomyosin. This protein has a polypeptide molecular weight of 36,000 and binds 7 mol of actin/mol 36,000 polypeptide.     

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.     

Organization of stress fibrils in cultured fibroblasts studied by using an actin filament-fragmenting protein

Earlier we isolated a 1:1 complex of 90 kD-protein and actin from bovine brain. This complex was able to fragment actin filaments. Effects of this complex on the cytoskeleton of mouse and quail embryo fibroblasts are described. The cells were extracted with Triton X-100, and the resulting cytoskeletons were treated with the complex. Tetramethylrhodaminylphalloin and actin antibody staining failed to detect actin in preparations treated with the 90 kD protein-actin complex. Electrophoretic data confirmed actin solubilization upon this treatment. Immunofluorescent microscopy showed that actin solubilization was accompanied by extraction of vinculin and alpha-actinin from focal contacts and stress-fibers. In contrast, myosin distribution in treated cytoskeletons did not differ significantly from that in control extracted cells: in both the cases myosin was found mainly in the stress-fibers. Thus, myosin localization in stress-fibers does not depend on actin and is probably controlled by some other cytoskeletal component(s).