Filamentous Actin Content Research Articles

Shape oscillations of human neutrophil leukocytes: characterization and relationship to cell motility.

When neutrophil leukocytes are stimulated by chemotactic factors or by substratum contact, they change their shape. Shape changes are a prerequisite for cellular migration and typically involve the extrusion of thin, veil-like lamellipods and the development of morphological polarity. Stimulation also leads to changes in the neutrophil content of filamentous actin (F-actin), which is the major cytoskeletal component. Suspensions of human neutrophils stimulated with chemoattractants exhibit sinusoidal light-scattering oscillations with a period of approximately 8 s at 37 degrees C. These oscillations arise from periodic fluctuations in the cell body size caused by lamellipod extension and retraction cycles. The light-scattering oscillations are paralleled by corresponding oscillations in F-actin content. This raises the interesting possibility that cyclic actin polymerization constitutes the driving force for shape oscillations of suspended neutrophils. Similar periodic shape changes are present in neutrophils crawling on a surface, suggesting that shape oscillations are important for neutrophil motion. This review summarizes our present knowledge about shape oscillations in suspended and crawling neutrophils and discusses a possible role for these oscillations in neutrophil motility.

WR-1065 and Radioprotection of Vascular Endothelial Cells. II. Morphology

Although the aminothiol WR-1065 protects normal tissues, its direct effect on the damage and restoration of the vascular endothelium is not clear. In endothelial cells, WR-1065 attenuates both the DNA damage and the G1-phase arrest induced by radiation. After the destruction of nearby endothelial cells, the survivors rearrange their cytoskeleton, migrate and replicate. To determine the effect of radiation on morphology and migration, portions of bovine aortic endothelial cell cultures were denuded with a pipette tip and irradiated (137Cs gamma rays). The following observations were noted after 5 Gy: within 10 min, there was increased formation of protein-mixed disulfides including actin-mixed disulfide; after 30-min, alpha 5 beta 1, the integrin receptor for fibronectin, was up-regulated on the apical membrane surface. Within 5 h, actin-containing stress fibers reorganized, although there was no change in the total filamentous (F-)actin content within the cells. Compared to controls after 24 h, the irradiated cells had migrated 15% farther (P < 0.01), and at the leading edge covered twice the surface area (P < 0.0001). The addition of 2 mM WR-1065 for 2 h before 5 Gy inhibited the increased expression of alpha 5 beta 1, promoted retention of stress fibers and prevented the enhanced cell migration and spreading. These results indicate that WR-1065 prevents radiation-induced morphological responses. This effect appears to be mediated by an impact on both adhesion molecule expression and cytoskeletal reorganization.

Stimulation of cortical actin polymerization in the sea urchin egg cortex by NH4Cl, procaine and urethane: elevation of cytoplasmic pH is not the common mechanism of action.

Previous studies have demonstrated that the penetrating weak base NH4Cl and the anesthetics procaine and urethane disrupt the normal attachment of cortical granules to the cortex of the sea urchin egg. Hylander and Summers (1981: Dev. Biol. 86:1-11) hypothesized that this effect may be caused by a pH-induced polymerization of cortical actin. We have tested this hypothesis by measuring the intracellular pH of eggs of the sea urchins S. purpuratus and A. punctulata treated with NH4Cl, procaine, or urethane, and determining the effects of these agents on the organization of cortical actin. Intracellular pH was determined by the ratiometric measurement of the fluorescent dye BCECF, and filamentous actin organization was examined by confocal laser scanning microscopy of BODIPY-phallocidin stained eggs. Treatment of eggs with either NH4Cl or procaine resulted in a rapid and reversible increase in cytoplasmic pH of up to 1 pH unit and a dose-dependent increase in the intensity of fluorescent staining of the cortex, indicating an increase in the content of filamentous actin. While urethane also induced a dramatic polymerization of cortical actin, no effect on cytoplasmic pH could be detected. These results demonstrate that NH4Cl, procaine and urethane all induce an increase in the amount of filamentous actin in the sea urchin egg cortex that may participate in the detachment of cortical granules. However, these compounds do not share a common mechanism of action based on the elevation of cytoplasmic pH.

Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts.

The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.

ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils

Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP-ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N-formyl-Met-Leu-Phe. Regulation of these oscillations may be important for the control of neutrophil motility.

Myosin Light Chain Kinase from Vascular Smooth Muscle Inhibits the ATP-Dependent Interaction between Actin and Myosin by Binding to Actin1

Myosin light chain kinase (MLCK) was prepared from smooth muscle of bovine aorta. MLCK inhibited the ATP-dependent movement of actin filaments on a glass surface coated with smooth muscle myosin that had been phosphorylated. The inhibitory effect was abolished by calmodulin in the presence of Ca2+ (Ca-CaM). The abolition was also observed when the concentration of actin filaments was increased. The inhibitory effect and its abolition were related to the actin-binding activity of MLCK, that is antagonized by Ca-CaM.

Effects of CapG overexpression on agonist-induced motility and second messenger generation.

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control-transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca2+ signaling. These responses are consistent with enhanced PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP(2)-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.

Shape oscillations: a fundamental response of human neutrophils stimulated by chemotactic peptides?

Neutrophils undergo periodic cytoskeletal rearrangements that lead to cycles of shape change, ultimately resulting in cell translocation. Repeated stimulation of resting neutrophils with subsaturating chemoattractant doses induced transient sinusoidal oscillations in neutrophil filamentous actin content at the second and subsequent stimulations. Oscillation frequencies increased with increasing concentration of the first stimulus. In contrast, neutrophils pretreated with the phosphatidylinositol 3-kinase inhibitor (17-hydroxy)wortmannin displayed shape oscillations with the first stimulation, and the frequencies were independent of agonist type and dose. We demonstrate that oscillations in filamentous actin, which may be critical for neutrophil motility, can be induced in untreated cells by natural peptide chemoattractants.

Coagulation factor Va is an actin filament binding and cross-linking protein.

Bovine coagulation cofactor factor Va is shown to bind to filament of skeletal muscle actin with a dissociation constant of 40-50 nM in the presence of 50 mM NaCl. At saturation, approximately one molecule of factor Va was bound for every two actin molecules. The binding of factor Va to F-actin was inhibited by increasing ionic strength, being approximately 20-fold weaker at 150 mM NaCl. Addition of factor Va dramatically increased both the low-speed sedimentation and the low-shear viscosity of actin filament solutions, indicating that factor Va cross-linkis actin filaments. Factor Va bound to actin filaments saturated with myosin. The isolated 74-kilodalton light chain of factor Va displayed actin binding and cross-linking properties indistinguishable from those of intact factor Va. The procofactor factor V bound weakly to F-actin, indicating that proteolytic activation is required to uncover the actin binding sites within the light chain domain. Actin filaments had only a slight inhibitory effect on the prothombinase activity of the factor Va-factor Xa-phospholipid complex. Since high concentrations of actin filaments can be exposed to the circulation when cells are damaged, the interaction of factor Va with actin may be of physiological relevance.

Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0.30 +/- 0.03 (mean +/- SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0.49 +/- 0.03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0.5 +/- 0.07 (n = 4), indicating a 1.7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10(-6) and 10(-5) M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10(-4) M), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of cyclic AMP, calcium and filamentous actin in adenosine modulation of Fc receptor-mediated phagocytosis in human neutrophils

The role of cyclic AMP, calcium and filamentous actin (F-actin) content during adenosine modulation of Fc receptor (FcR)-mediated phagocytosis in adherent human neutrophils was investigated. Phagocytosis of IgG-opsonized yeast particles was found to be enhanced by pico- to nanomolar concentrations of adenosine or the A 1-agonist N 6-cyclopentyl-adenosine (CPA) but reduced by micromolar concentrations of adenosine or the A 2-agonist 5′- N-ethylcarboxamidoadenosine (NECA). NECA, in the presence of the cAMP-specific phosphodiesterase inhibitor Ro 20–1724, increased the intracellular content of cAMP during phagocytosis. Ro 20–1724 potentiated the NECA-induced reduction of the phagocytic capacity. These observations indicate that cAMP elevations are involved in A 2-receptor-mediated inhibition of phagocytosis. NECA, in the presence of Ro 20–1724, markedly enhanced the actin polymerization associated with adhesion to the substrate and contact with the phagocytic prey. During advanced phagocytosis, however, the F-actin content reached levels clearly below those observed in control cells. This prolonged depolymerization phase correlated with the A 2-receptor-induced cAMP elevation. Depletion of intracellular free calcium abolished the cAMP-elevating effects of NECA, and also completely abrogated the A 1- and A 2-receptor-mediated effects on phagocytosis. However, since NECA reduced the F-actin content even in Ca 2+-depleted cells, A 2-receptor-mediated inhibition of phagocytosis could not be directly coupled to changes in the overall content of F-actin. Our results indicate that adenosine modulates FcR-mediated phagocytosis in a calcium-dependent way, and does so through ‘stimulatory’ A 1 and ‘inhibitory’ A 2 receptors, and also that cAMP elevation is linked to the A 2-receptor-induced inhibition of phagocytosis.

Effects of Benzene Metabolites on Receptor-Mediated Phagocytosis and Cytoskeletal Integrity in Mouse Peritoneal Macrophages

Exposure to benzene can induce a number of hematotoxicities and decrease host resistance to microorganisms and tumors. Several studies have shown that metabolism of benzene to reactive intermediates is required for myelotoxicity. Since receptor-mediated phagocytosis by macrophages is an important host defense, we have examined the effects of benzene metabolites on receptor-mediated phagocytosis in cultured murine peritoneal macrophages. 1,4-Benzoquinone (BQ) was the most potent of the metabolites examined. Ten-minute exposures to a 12.5 μM concentration inhibited Fc and complement receptor-mediated phagocytosis by ≥90%. Macrophage viability was largely unaffected by BQ treatment. Exposure to 50 and 100 μM 1,2,4-benzenetriol (BT) inhibited Fc receptor-mediated phagocytosis by 70 and 95%, respectively. Hydroquinone (HQ) elicited a major decrease (50%) only at 100 μM. The comparative inhibitory potencies of BT and HQ correlate with previously published data on their relative facility for autooxidation to quinones at physiological pH. Catechol had no effect at the concentrations employed. Macrophages treated with BQ and BT failed to recover their Fc receptor-mediated phagocytic capacity when incubated overnight in the absence of the xenobiotics. Only small differences in the inhibition of Fc receptor-mediated phagocytosis were observed between macrophages exposed to BQ at 4 versus 37°C. BQ also had little effect on the Fc receptor binding of target cells. Fluorescent digital imaging microscopy demonstrated that BQ treatment markedly decreased the filamentous actin content of macrophages. However, BQ bound in low amounts to purified actin and did not affect its assembly. Our findings suggest that a mechanism for inhibition of Fc receptor-mediated phagocytosis by BQ is disruption of filamentous actin via an effect(s) other than the direct alkylation of actin by BQ.

Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein.

Granulocyte Deformability and F-Actin Content: Relationship to Diabetes Mellitus and Retinopathy

Capillary closure in diabetic retinopathy may be initiated by lumenal occlusion by granulocytes. To determine whether subjects with diabetes mellitus have less deformable granulocytes than healthy subjects, granulocyte deformability was measured by mean entry time into a model capillary system in 16 diabetic-nondiabetic pairs. Granulocyte F-(filamentous) actin content between groups was compared, under basal conditions and after cellular stimulation. The relationship of granulocyte deformability to several diabetes-related variables was examined. Diabetic granulocytes were only 9% +/- 22% less deformable than normal granulocytes (p = 0.16). Deformability was increased in subjects with retinopathy and those with the worst glycemic control (r = 0.61, p = 0.026); both findings were in the opposite direction from that predicted. Basal and stimulated granulocyte F-actin content did not differ between the two groups (p > 0.2 for all assays). Although granulocytes may be important in the pathogenesis of diabetic retinopathy, granulocyte deformability (measured by mean entry time) and F-actin content are not significantly different between healthy patients and those with diabetes.

Activation of the protein kinase a signal transduction pathway by granulocyte‐macrophage colony‐stimulating factor or by genetic manipulation reduces cytoskeletal organization in lewis lung carcinoma variants

Granulocyte-macrophage colony-stimulating factor (GM-CSF) that is produced by metastatic Lewis lung carcinoma (LLC-LN7) cells functions as an autocrine stimulator of tumor-cell motility through protein kinase A (PKA) signal transduction. This GM-CSF-mediated enhancement of LLC-LN7 cell motility coincides with a reduction in the level of polymerized F-actin. In contrast, non-metastatic LLC-C8 tumor cells, which have a diminished level of PKA signaling, do not produce GM-CSF and do not respond to exogenous GM-CSF, since they remain non-motile and retain a high content of filamentous actin. The capacity of PKA to regulate the cytoskeletal organization of tumor cells was further studied with the use of LLC variants that had been stably transfected to over-express the C alpha subunit of PKA (CEV cells) or to express a mutant cAMP-resistant PKA RI alpha subunit resulting in a defective PKA (REV cells). When compared with wild-type metastatic LLC-LN7 cells, in which the F-actin staining was too diffuse to be clearly visualized microscopically, the PKA-defective REV-LN7 transfectants had an increased level of F-actin. In comparison with the wild-type non-metastatic LLC-C8 cells, which had a high content of F-actin, the CEV-C8 transfectants that over-expressed PKA activity had a reduced level of F-actin. The reduced polymerization of actin in these CEV-C8 transfectants was accompanied by reduced levels of the intermediate filament protein vimentin and a shift in the distribution both of F-actin and of vimentin to the periphery of the cells. These results show reduced cytoskeletal organization in metastatic LLC-LN7 cells as compared with that of non-metastatic LLC-C8 cells, and indicate that elevation of PKA activity, either by autologous GM-CSF or by genetic manipulation, diminishes cytoskeletal organization.

Formation of liquid crystals from actin filaments.

Actin is cross-linked by actin-binding proteins in the cytoplasm to form either isotropic or highly oriented anisotropic structures. The inherent orientation among actin filaments could influence whether an isotropic or highly oriented anisotropic structure is formed. A highly oriented state can arise spontaneously through the formation of liquid crystals as predicted by polymer theory. In this study, the ability of filamentous actin to form liquid crystalline domains was detected using the anisotropic component of scattered light and by observation of birefringence. As liquid crystalline domains formed, the intensity of the anisotropic component of scattered light increased, and birefringent macroscopic oriented domains were directly observed. The formation of liquid crystalline domains was dependent on the concentration of actin filaments and on the average filament length controlled by varying the ratio of gelsolin to actin monomers. The concentration of actin filaments required to form liquid crystalline domains increased moderately as the average length was decreased. At a fixed actin concentration, orientation among the filaments attained a maximum value at a ratio of actin to gelsolin in the range from 1500 to 2000 and decreased as the ratio was increased or decreased from this range. The results are not well explained by theoretical treatments for liquid crystal formation by monodisperse, charged worm-like chains. Differences from the theoretical predictions for formation of liquid crystals are most likely due to the polydisperse filament length of actin. This phenomenon may have important effects on the structural and rheological properties of the cytoplasm in living cells.

Actin filament annealing in the presence of ATP and phalloidin.

The re-formation of actin filaments after fragmentation by sonication in the presence of phalloidin and ATP has been found to follow second-order kinetics. The data are described by a model in which the rate of actin filament annealing is proportional to the square of the number concentration of actin filaments and the rate of fragmentation is proportional to the actin polymer concentration. In the presence of 100 mM KCl, 1 mM MgCl2, and equimolar phalloidin, the second-order rate constant for annealing of actin filaments is 2.2 x 10(6) M-1 s-1 and the first-order rate constant for fragmentation is 7 x 10(-7) s-1. In addition, the observed pseudo-first-order rate constant for annealing was found to increase with increasing ionic strength. Thus, annealing may play a major part in the length redistribution phase of actin polymerization and may be important for actin filament rearrangement in the cell.

Regulation of water flow by actin-binding protein-induced actin gelatin

Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation.

Heterogeneity in filamentous actin content among individual human blood platelets

The content of filamentous actin in individual platelets was measured by flow cytometry, using a fluorescent probe specific for filamentous actin (F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin). NBD-phallacidin binding to fixed platelets was specific in that either pretreatment of platelets with unlabeled phallacidin or absorption of NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G- actin), resulted in a significant loss in the bound fluorescent probe. Mean NBD-phallacidin binding to fixed platelets varied with the agonist and paralleled the changes in F-actin reported with the DNAse I inhibition assay. (1) NBD-phallacidin binding increased with stimulation by ADP, U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape change. (2) Epinephrine did not increase NBD- phallacidin binding. (3) Platelets treated at 4 degrees C contained more F-actin than did platelets kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the increase of phallacidin binding to individual platelets stimulated by either ADP or U46619. In measurements of cytosolic free calcium concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's dose-response for actin polymerization was similar to that for calcium mobilization. As shown by flow cytometry, a tail population that had a minimal increase in F- actin upon stimulation with ADP or U46619 also contained the platelets with the least forward and right angle light scattering, which are functions of platelet size and shape. When platelets treated with NBD- phallacidin were incubated with S12-murine monoclonal antibody (a marker of alpha-granule secretion detected by phycoerythrin-conjugated antimouse IgG second antibody), phallacidin fluorescence paralleled S12 binding. Thus, human blood platelets are heterogeneous in regard to actin polymerization at rest and in association with platelet activation; different degrees of phallacidin binding may identify functionally different platelet populations.

Heterogeneity in Filamentous Actin Content Among Individual Human Blood Platelets

The content of filamentous actin in individual platelets was measured by flow cytometry, using a fluorescent probe specific for filamentous actin (F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin). NBD-phallacidin binding to fixed platelets was specific in that either pretreatment of platelets with unlabeled phallacidin or absorption of NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G-actin), resulted in a significant loss in the bound fluorescent probe. Mean NBD-phallacidin binding to fixed platelets varied with the agonist and paralleled the changes in F-actin reported with the DNAse I inhibition assay. (1) NBD-phallacidin binding increased with stimulation by ADP, U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape change. (2) Epinephrine did not increase NBD-phallacidin binding. (3) Platelets treated at 4 degrees C contained more F-actin than did platelets kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the increase of phallacidin binding to individual platelets stimulated by either ADP or U46619. In measurements of cytosolic free calcium concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's dose-response for actin polymerization was similar to that for calcium mobilization. As shown by flow cytometry, a tail population that had a minimal increase in F-actin upon stimulation with ADP or U46619 also contained the platelets with the least forward and right angle light scattering, which are functions of platelet size and shape. When platelets treated with NBD-phallacidin were incubated with S12-murine monoclonal antibody (a marker of alpha-granule secretion detected by phycoerythrin-conjugated antimouse IgG second antibody), phallacidin fluorescence paralleled S12 binding. Thus, human blood platelets are heterogeneous in regard to actin polymerization at rest and in association with platelet activation; different degrees of phallacidin binding may identify functionally different platelet populations.