amanitins and 7-nitrobenz-2-oxa-1-3-diazole-phallacidin

Topics: Actins; Amanitins; Animals; Cell Adhesion; Cell Movement; Cell Transformation, Neoplastic; Contractile Proteins; Cytoskeleton; Endothelium; Fibroblasts; Fluorescent Antibody Technique; Humans; Microscopy, Electron; Morphogenesis

Tobacco smoking is a major risk factor in the incidence and severity of periodontal diseases. Alterations of neutrophil function by short-term high levels of smoke during the act of smoking (acute smoke exposure) as well as long-term exposure to lower levels of tobacco substances in the bloodstream (chronic smoke exposure) may play a role in the pathogenesis of periodontal diseases in smokers. The polymerization and depolymerization of f-actin in response to infectious agents or inflammatory mediators is a critical process in a variety of neutrophil functions. In this study, we examined the effects of in vitro smoke exposure on neutrophils from smokers and non-smokers (which may be comparable to in vivo acute smoke exposure) and neutrophils from smokers not exposed to further in vitro smoke (which may be comparable to chronic smoke exposure) on f-actin kinetics. Peripheral neutrophils were isolated from seven healthy smoking subjects and seven healthy age-matched non-smoking subjects and exposed to 1-5 min of acute smoke in a smoke box system or not exposed to further smoke (baseline controls). Selected aliquots of neutrophils from control and 5-min exposures of acute smoke were then stimulated with the chemotactic peptide F-met-leu-phe at 10(-7) M for an additional 30-360 s. Cells were fixed and permeabilized, stained for f-actin with NBD phallacidin, and analyzed by flow cytometry. From baseline to 5 min of in vitro smoke exposure, there was a 38% decline in f-actin stain in non-smokers and a 30% decline in f-actin stain in smokers (p > 0.05) with f-actin values slightly higher in smokers than-non-smokers (p > 0.05). With F-met-leu-phe stimulation, both smokers and-non-smokers demonstrated a characteristic rise in f-actin stain from 0 to 120 s with a subsequent decline to baseline at 360 s and no significant differences in f-actin levels at any time of stimulation between groups. After preincubation with 5 min of in vitro smoke, the magnitude of rise in f-actin was less in both smokers and non-smokers when compared to cells not incubated with 5 min of smoke (p < 0.05 at 120 s for both smokers and non-smokers). F-actin values in smokers were higher than-non-smokers from 30 to 360 s of F-met-leu-phe exposure (p > 0.05). These results demonstrate that in vitro smoke exposure may impair normal f-actin kinetics. These alterations in f-actin kinetics may in turn affect other neutrophil functions which may impact on the pathogenesis of periodontal diseases in

Topics: Actins; Adult; Amanitins; Analysis of Variance; Case-Control Studies; Chemotactic Factors; Female; Flow Cytometry; Fluorescent Dyes; Humans; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nicotiana; Periodontal Diseases; Smoke; Smoking; Statistics as Topic; Time Factors

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca2+]i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca2+ -depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes.

Topics: Actins; Amanitins; Benzopyrans; Calcium; Chelating Agents; Chemokine CCL5; Chemotactic Factors; Chemotaxis, Leukocyte; Complement C5a; Egtazic Acid; Eosinophils; Flow Cytometry; Fluorescent Dyes; Humans; Neutrophils

Microfilaments in epithelial cells are important for the structural and functional integrity of tight junctions. In the present study, we examined the relationship between microfilaments and tight junctions in hepatocytes of rat liver following common bile duct ligation (CBDL) for up to 2 weeks. Actin filaments and tight junctions were studied by fluorescence microscopy using 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph) and an anti-ZO-1 antibody, respectively. Double-stained sections were examined with confocal laser scanning microscopy (CLSM). Electron microscopy was applied for the assessment of structural alterations in microfilaments and in tight junctions with detergent-extraction and freeze-fracture preparations. Our results showed that F-actin was present at the entire plasma membrane of hepatocytes in control liver, whereas CBDL increased the amount of F-actin mainly at the bile canalicular and lateral plasma membranes. Simultaneously, the immunofluorescence of ZO-1 underwent striking changes, i.e., from a uniform to an irregular staining pattern with various fluorescence intensities. CLSM demonstrated a colocalization of ZO-1 and F-actin in control liver and its deterioration in CBDL liver. Electron microscopy showed marked alterations of microfilaments and tight junctions due to CBDL. It is concluded that actin filaments are intimately associated with tight junctions in normal hepatocytes. CBDL impairs this association by progressively diminishing the structural interaction between F-actin and ZO-1, which may in turn lead to functional disturbances of tight junctions.

Topics: Actin Cytoskeleton; Actins; Amanitins; Animals; Antibody Specificity; Cholestasis, Extrahepatic; Common Bile Duct; Fluorescent Antibody Technique; Fluorescent Dyes; Freeze Fracturing; Ligation; Liver; Male; Membrane Proteins; Microscopy, Confocal; Microscopy, Electron; Phosphoproteins; Rats; Rats, Wistar; Tight Junctions; Zonula Occludens-1 Protein

This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP-120- cells are not as large or thick as those produced by ABP-120+ cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120- cells when compared to pseudopods of ABP-120+ cells. The actin filaments found in areas of pseudopods in ABP-120+ cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120- cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP-120- cells compared to ABP-120+ cells and is consistent with the role of ABP-120 in regulating pseudopod extension through its cross-linking of actin filaments.

Topics: Actin Cytoskeleton; Actins; Amanitins; Animals; Carrier Proteins; Cyclic AMP; Cytoskeleton; Dictyostelium; Gene Deletion; Microfilament Proteins; Microscopy, Confocal; Microscopy, Electron; Microscopy, Fluorescence; Models, Biological; Phagocytes; Polyethylene Glycols; Pseudopodia

alpha-Thrombin interacts with confluent bovine pulmonary artery endothelial cells to produce two types of actin microfilament rearrangements: (1) The loss of cortical actin correlates with interruption of barrier integrity, evident from increased permeability to 125I-albumin; and (2) an increase in actin stress fibers results in greater adherence of the cells to their extracellular substrate. Use of phallatoxin compounds that stabilize actin filaments and prevent their depolymerization prevents both the cytoarchitectural and functional changes resulting from thrombin challenge.

Topics: Actins; Amanitins; Animals; Cattle; Cell Adhesion; Cell Division; Cell Survival; Cells, Cultured; Endothelium, Vascular; Fluorescent Dyes; Microscopy, Fluorescence; Peptides, Cyclic; Thrombin

Previous work has shown that H2O2 causes an increase in polymerized actin (F-actin) inside cells. To test the hypothesis that increased polymerization resulted from a mechanism involving increased actin nucleation activity, we employed methods utilizing pyrene-labeled actin to quantify the actin nucleation activity of cell lysates and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallacidin binding assays to quantify the amount of F-actin in P388D1 cells. H2O2 increased polymerized actin (NBD-phallacidin assay) in a dose-dependent manner with an effective dose giving 50% response (ED50) approximately 1 mM. Five millimolar H2O2 caused a 1.6-fold increase in NBD-phallacidin staining. In contrast, actin nucleation activity decreased in a dose-dependent manner with a similar ED50. Five millimolar H2O2 caused a 30-40% decrease in actin nucleation activity. The effect was rapid, occurring within 5 min of H2O2 addition. The results indicate that H2O2 causes cytoskeletal changes that enhance NBD-phallacidin binding without increasing actin nucleation activity. Fractionation studies showed that the nucleation activity in H2O2-treated cells and controls sedimented with the Triton X-100-insoluble cytoskeleton, and the cytosolic fraction appeared to contain an inhibitor of actin polymerization.

Topics: Actins; Amanitins; Animals; Cell Line, Transformed; Cytosol; Fluorescent Dyes; Hydrogen Peroxide; Kinetics; Macromolecular Substances; Macrophages; Mice

Human polymorphonuclear neutrophils undergo characteristic shape changes that are critical to their ability to move and ingest their targets. We present here the construction of a simple shape vector, calculated from the coordinates of the cell perimeter, that can identify critical forms that a neutrophil assumes during the course of ameboid movement. The vector can be used to find neutrophils of a specific shape from the image analyzer data produced during a typical neutrophil tracking experiment.

Topics: Amanitins; Cell Size; Chemotaxis, Leukocyte; Fluorescent Dyes; Humans; Mathematics; Microscopy, Fluorescence; Models, Biological; Neutrophils; Xanthenes

In this paper we report the positive staining of epididymal spermatozoa and testicular cells (late spermatids and spermatozoa) with fluorescent phallotoxins. Staining is most obvious with rhodamine phalloidin, but is also detectible with NBD-phallacidin. Specific fluorescence is emitted as a linear tract along the dorsal curvature of the head and as an inverted V-shaped structure in what appears to be the anterior aspect of the post-acrosomal region. We conclude that filamentous actin occurs in the heads of rat spermatozoa. Moreover, we speculate that this filamentous actin is concentrated in two regions of the perinuclear theca; in the subacrosomal space along the dorsal curvature of the nucleus, and in the post-acrosomal region in an area termed the ventral spur.

Topics: Actins; Amanitins; Animals; Epididymis; Fluorescent Dyes; Male; Rats; Rats, Sprague-Dawley; Rhodamines; Sperm Head; Spermatids; Spermatozoa

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve

Topics: Actins; Amanitins; Calcium-Binding Proteins; Cell Compartmentation; Cytoskeleton; Gelsolin; Humans; Microfilament Proteins; Neutrophils; Polymers

We studied the effect of cytochalasins (B, D, and E) on the F-actin content in human neutrophils and lymphocytes using NBD-phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration- and time-dependent increase in the F-actin content in both cell types. The order of potency was cytochalasin D greater than E greater than B. The increase in F-actin content was accompanied by a decrease in the G-actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin-binding or sequestering proteins. 2-deoxyglucose (20 mM) caused a decrease in the basal F-actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.

Topics: Actin Cytoskeleton; Actins; Amanitins; Antimetabolites; Cytochalasins; Flow Cytometry; Humans; Lymphocytes; Neutrophils; Polymers

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.

Topics: Actins; Adenosine Diphosphate; Amanitins; Animals; Blood Platelets; Calcium; Collagen; Cytochalasin D; Cytoplasmic Granules; Epinephrine; Flow Cytometry; Fluorescent Dyes; Humans; Light; Prostaglandin Endoperoxides, Synthetic; Rabbits; Scattering, Radiation

T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with high affinity thus stabilizing the F-actin cytoskeleton by preventing depolymerization, an event necessary for dynamic reordering of actin microfilaments. NBD-phallicidin loading is not cytotoxic as assessed by lactic dehydrogenase release, protein synthesis, transepithelial resistance, and the ability of the loaded cells to pump Na+ in an absorptive direction in response to the apical addition of a Na+ ionophore. However, cAMP-elicited redistribution of F-actin and the cAMP-elicited Cl- secretory response are both markedly impaired in NBD-phallicidin preloaded T84 cells. In contrast, the carbachol-elicited Cl- secretory response (Ca++ mediated) is not attenuated by NBD-phallicidin preloading nor is it accompanied by redistribution of F-actin. These findings suggest that the cAMP-elicited cytoskeletal redistribution we describe is an integral part of cAMP-elicited Cl- secretion in T84 cells.

Topics: Actins; Amanitins; Carbachol; Cell Line; Chlorides; Cyclic AMP; Cytoskeleton; Electric Conductivity; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Intestinal Mucosa; Secretory Rate

The distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD-phallacidin-positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD-phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.

Topics: Acrosome; Actins; Amanitins; Animals; Calcimycin; Epididymis; Female; Fertilization; Immunohistochemistry; Male; Muridae; Spermatids; Spermatozoa; Testis

This effect of parasympathectomy was assessed by fluorescent microscopy using nitrobenzoxadiazole-phallacidin, which is known for its specific binding to actin filaments. Resection of the chorda tympani within 48 h after birth inhibited the normal formation of actin in the myoepithelial cells and the maturation of myoepithelial cells. The effect of denervation decreased progressively if the operation was delayed. Denervation 30 days after birth had no effect on development and differentiation of myoepithelial cells. Moreover, the procedure produced no histochemical changes in the myoepithelial cells in mature rats, even two months later. These findings suggest that the parasympathetic innervation is closely involved in the maturation of myoepithelial cells. Further, its involvement is limited to the first 48 h after birth, during which it exerts its neurotrophic effect on the myoepithelial cells present in all the acinar buds.

Topics: Actin Cytoskeleton; Actins; Age Factors; Amanitins; Animals; Cell Differentiation; Chorda Tympani Nerve; Epithelial Cells; Female; Fluorescent Dyes; Male; Microscopy, Fluorescence; Muscles; Organ Size; Rats; Rats, Inbred Strains; Sublingual Gland

Topics: Actins; Amanitins; Cyclic AMP; Cytoskeleton; Dictyostelium; Fluorescent Dyes; Kinetics; Macromolecular Substances; Spectrometry, Fluorescence

Vasopressin (AVP) induces the rapid fusion of water channel-containing vesicles with the luminal membrane of its target cell. We have carried out a quantitative study of the F-actin content of toad bladder epithelial cells, using the rhodamine phalloidin binding assay. As early as 1 min after AVP stimulation, there is a significant 15% reduction of cellular F-actin, which remains reduced by 20-30% for the duration of action of AVP. Comparable reductions were seen following 8-bromoadenosine 3',5'-cyclic monophosphate, 1-desamino-8-D-arginine vasopressin, and forskolin. F-actin content rose to and then exceeded that of control bladders after AVP washout. Inhibition of prostaglandin synthesis enhanced both water flow and the decrease of F-actin. In the living cell, stabilization of F-actin with NBD-phallacidin selectively inhibited water flow. In view of the rapidity of the response, we conclude that AVP shifts the equilibrium between F-actin and G-actin monomers, and this depolymerization may be required for vesicle fusion.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Actins; Amanitins; Animals; Arginine Vasopressin; Bufo marinus; Deamino Arginine Vasopressin; Epithelium; Female; In Vitro Techniques; Macromolecular Substances; Microscopy, Electron; Rana catesbeiana; Urinary Bladder

The possible role of tyrosine phosphorylation in the activation of granulocytic HL60 cells was examined using vanadate, a phosphotyrosine phosphatase inhibitor. Treatment of permeabilized cells with micromolar concentrations of vanadate resulted in a substantial accumulation of tyrosine-phosphorylated proteins, detected by immunoblotting. At comparable concentrations, vanadate was also found to elicit an NADPH-dependent burst of oxygen utilization. Actin assembly, studied using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, was similarly stimulated by vanadate, though considerably higher concentrations were required to observe this effect. In contrast with these responses, the secretion of lysozyme was not stimulated by vanadate, nor did vanadate affect calcium-induced secretion. Therefore, accumulation of tyrosine-phosphorylated proteins is associated with stimulation of some, but not all, of the responses characteristic of granulocytic cell activation. This indicates that the effects of vanadate are selective and suggests divergence of the signalling pathways leading to the individual effectors.

Topics: Actins; Adenosine Triphosphate; Amanitins; Calcium; Cell Membrane Permeability; Exocytosis; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoblotting; Leukemia, Promyelocytic, Acute; Muramidase; NADP; Neutrophils; Oxygen Consumption; Phosphotyrosine; Polymers; Signal Transduction; Thionucleotides; Tumor Cells, Cultured; Tyrosine; Vanadates

Serial sections containing neurosecretory cells from chicken hypothalamus were cut after fixation in formaldehyde and embedding in paraffin. Sections were exposed to NBD-Ph (nitrobenzoxadiazole-phallacidin) and showed evidence of containing actin. By using a medium with sodium borohydride, non-specific fluorescence could be excluded.

Topics: Actins; Amanitins; Animals; Chickens; Fixatives; Fluorescent Dyes; Hypothalamus; Neurosecretory Systems

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.

Topics: Actins; Amanitins; Animals; Calcium; Cytoplasm; Deoxyribonuclease I; Exocytosis; Molecular Weight; Neutrophils; Oxidoreductases; Phagocytosis; Rats; Receptors, Formyl Peptide; Receptors, Immunologic

Capping of Concanavalin A (Con A) receptors can be inhibited in Dictyostelium by treatment of amoebae with the microtubular drug tubulozole. In cells that were incubated with Con A or with fluorescent Con A conjugate the capping process was completed in 30 min as could be demonstrated by fluorescence microscopy and Con A peroxidase labeling. In the presence of 10(-5) M tubulozole redistribution of the receptors did not proceed beyond a stage that can be characterized as patching. The effect of the drug on microtubule integrity was checked by electron microscopy and immunofluorescence of tubulin. Treatment resulted in shortening of the peripheral parts of the microtubules, in agreement with results described by other authors. Electron microscopy confirmed that the Con A receptor complexes remained on the plasma membrane and were not internalized. The distribution of F-actin in Con A-treated cells showed a pattern closely resembling that of Con A. Cells that were also treated with tubulozole remained spherical and did not resume significant directional movement until tubulozole was removed from the medium. It is concluded that microtubules are involved in the rearrangement of the microfilament network in moving cells.

Topics: Actins; Amanitins; Dictyostelium; Dioxolanes; Dioxoles; Fluorescent Antibody Technique; Fluorescent Dyes; Membrane Proteins; Microscopy, Electron; Microtubules; Receptor Aggregation; Receptors, Concanavalin A

Studies in our laboratory have shown that polymorphonuclear leucocytes (PMNL) from chronic myeloid leukemia (CML) patients are defective in chemotaxis towards a synthetic peptide, n-formyl-methionyl-leucyl-phenylalanine (FMLP), during the active phases of the disease and in remission. Actin plays a major role in cellular movements and binding of chemo-attractant to cells induces polymerization of G-actin to F-actin. We have, therefore, compared polymerization of actin in FMLP stimulated PMNL from CML patients with those from normal subjects by fluorescence microscopy and flow cytometry, using F-actin specific probe, NBD-phallacidin. Our results show that binding of FMLP to normal PMNL induces rapid conversion of G-actin to F-actin followed by depolymerization to some extent. In CML PMNL, such a biphasic response is not seen. Conversion of G-actin to F-actin is slower and F-actin content is significantly lower than that in normal PMNL. Moreover, organization of F-actin is different in CML PMNL as compared to that in normal PMNL.

Topics: Actins; Amanitins; Flow Cytometry; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Microscopy, Fluorescence; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Polymers

Biochemical, immunological, and electron microscopic methods have been used to provide semi-quantitative estimates and to localize actin in membranes of boar spermatozoa. Immunoblots, using a monoclonal antibody raised against actin from chicken gizzard, detected the protein in caput and cauda sperm plasma membranes. Immunoassay indicated that approximately 1% of the total plasma membrane protein was actin. Monomeric actin accounted for more than one-half of the membrane actin. Approximately 30-40% of plasma membrane actin was insoluble in Triton X-100, and approximately 10% of the total actin remained insoluble after treatment with guanidine hydrochloride. The presence of F-actin in sperm plasma membranes and in plasma membrane detergent-insoluble proteins was detected by fluorescence microscopy using the specific probe NBD phallacidin. When S1 myosin subfragments attached to colloidal gold were used to localize F-actin by electron microscopy, the label was restricted to the outer acrosomal membrane of intact epididymal and ejaculated sperm. Filaments appeared in short arrays along the anterior region of the membrane. S1/gold labeled detergent-insoluble plasma membrane fractions but did not label the plasma membrane in intact sperm. Filaments were least prominent in intact caput spermatozoa and most prominent in ejaculated spermatozoa. We conclude that most actin associated with sperm membranes is in monomeric form in boar spermatozoa, but that actin filaments or protofilaments are components of the outer acrosomal membrane. These filaments may also associate with the plasma membrane overlying the acrosome.

Topics: Acrosome; Actins; Amanitins; Animals; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Male; Membrane Proteins; Myosin Subfragments; Octoxynol; Polyethylene Glycols; Sodium Chloride; Solubility; Spectrometry, Fluorescence; Sperm Capacitation; Spermatozoa; Swine

Muscle cells of the ascidian larva originate from three different lines of progenitor cells, the B-line, A-line and b-line. Experiments with 8-cell embryos have indicated that isolated blastomeres of the B-line (primary) muscle lineage show autonomous development of a muscle-specific enzyme, whereas blastomeres of the A-line and b-line (secondary) muscle lineage rarely develop the enzyme in isolation. In order to study the mechanisms by which different lines of progenitors are determined to give rise to muscle, blastomeres were isolated from embryos of Halocynthia roretzi at the later cleavage stages when conspicuous restriction of the developmental fate of blastomeres had already occurred. Partial embryos derived from B-line muscle-lineage cells of the 64-cell embryo (B7.4, B7.5 and B7.8) showed autonomous expression of specific features of muscle cells (acetylcholinesterase, filamentous actin and muscle-specific antigen). In contrast, b-line muscle-lineage cells, even those isolated from the 110-cell embryo (b8.17 and b8.19), did not express any muscle-specific features, even though their developmental fate was mainly restricted to generation of muscle. Isolated A-line cells from the 64-cell embryos (A7.8) did not show any features of muscle differentiation, whereas some isolated A-line cells from the 110-cell embryos (A8.16) developed all three above-mentioned features of muscle cells. This transition was shown to occur during the eighth cell cycle. These results suggest that the mechanism involved in the process of determination of the secondary-lineage muscle cells differs from that of the primary-lineage muscle cells. Interaction with cells of other lineages may be required for the determination of secondary precursors to muscle cells. The presumptive b-line and A-line muscle cells that failed to express muscle-specific features in isolation did not develop into epidermal cells. Thus, although interactions between cells may be required for muscle determination in secondary lineages, the process may represent a permissive type of induction and may differ from the processes of induction of mesoderm in amphibian embryos.

Topics: Acetylcholinesterase; Actins; Amanitins; Animals; Antibodies, Monoclonal; Blastomeres; Cell Communication; Cell Differentiation; Cells, Cultured; Embryonic Induction; Immunohistochemistry; Mesoderm; Mitochondria, Muscle; Muscles; Stem Cells; Time Factors; Urochordata

The process of spermiation and sperm transport was studied using specific inhibitors of cytoskeletal elements. Within 12-24 hr after the intratesticular injection of taxol, a compound that acts to stabilize microtubules and inhibit microtubule-related processes, an unusually large number of microtubules was seen within the body of the Sertoli cell. At the same time, transport of elements within the seminiferous epithelium was affected. At the end of stage VI of the cycle, step 19 spermatids were maintained in the deep recesses of the Sertoli cell and not transported to the rim of the seminiferous tubule lumen. At stage VIII, residual bodies remained at, or near, the rim of the tubule and were not transported to the base of the tubule. They underwent only partial degradation at this site, indicating that there may have been two phases involved in their dissolution--one autophagic and one phagocytic, but the latter did not occur since the residual bodies were not transported to Sertoli lysosomes at the base of the tubule. The observations suggest that microtubules are involved in transport processes within the seminiferous epithelium. Within 1-12 hr after the intratesticular injection of 500 microM cytochalasin D, a compound which interferes with actin-related processes, normal appearing tubulobulbar complexes were not present. The tubular portion (distal tube) of the complex did not initiate development. It was assumed that filaments (which were identified as such using NBD-phallacidin and the S-1 fragment of myosin) played an important role in the development of this portion of the complex. Cells did not eliminate cytoplasm normally, as evidenced by an enlarged cytoplasmic droplet, further emphasizing the published role for tubulobulbar complexes in cytoplasmic elimination. Although sperm were released normally from stage VIII tubules, many remained within the tubular lumen and did not traverse the duct system. Cytochalasin did not inhibit fluid secretion by the Sertoli cell, as demonstrated by efferent duct ligation, but did alter myoid cell actin cytoskeletal organization, suggesting that myoid cell contractility is primarily responsible for transport of sperm. Overall, the observations suggest that cytoskeletal activity of the Sertoli cell is important for several aspects of the spermiation process as well as sperm transport.

Topics: Actins; Alkaloids; Amanitins; Animals; Cytochalasin D; Cytoskeleton; Injections; Male; Microscopy, Electron; Microtubules; Myosin Subfragments; Paclitaxel; Rats; Rats, Inbred Strains; Sertoli Cells; Sperm Transport; Staining and Labeling; Testis

The morphology and function of actin in cultured bovine retinal pigment epithelial (RPE) cells were studied. Filamentous actin was identified with a fluorescent mushroom toxin, nitrobenzoxadiazole (NBD)-phallacidin, specific for actin. Dark-field microscopy of cultured RPE cells revealed numerous pigment granules; fluorescent microscopy identified scattered lipofuscin granules. One-dimensional SDS polyacrylamide gel electrophoresis of urea-soluble proteins extracted from RPE cells showed a 46,000-dalton protein band which comigrated with authentic muscle actin. Densitometric scanning showed that this protein band comprised 7.6% of the total urea-soluble proteins. An actin-activated skeletal-muscle myosin Mg-ATPase assay, using skeletal-muscle heavy meromyosin as enzyme and [gamma-32P]-ATP as substrate, demonstrated functional actin in RPE cell extracts after DEAE-cellulose anion exchange chromatography. The actin-containing protein fractions were eluted at ionic strengths between 0.19 and 0.36 M KCl. The activation of myosin ATPase by actin in RPE cells provides a molecular basis for the phagocytic activity which is important in maintaining the integrity of retinal photoreceptor cells.

Topics: Actins; Amanitins; Animals; Cattle; Cells, Cultured; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fluorescent Dyes; Muscles; Myosins; Pigment Epithelium of Eye

These studies evaluate the effects of atrial natriuretic peptide (ANP) and nitroprusside (NP) on cultured mesangial cells (MC) grown on a flexible silicone rubber surface. The basal tone of the MC produced wrinkles on the silicone rubber surface. A decrease in number or magnitude of wrinkles was considered to represent cell relaxation, whereas an increase represented cell contraction. ANP (10(-9) M) produced a relaxation in greater than 60% of the cells by 10 min. The percentage of cells showing a decrease of wrinkles was significantly higher (P less than 0.05 at 5 min and P less than 0.001 at 10 min) during the ANP-treated period than during the control period. NP (10(-5) M) caused a decrease of wrinkles in greater than 80% of cells (P less than 0.02 at 5 min and P less than 0.01 at 10 min) compared with a 5% decrease in the control period. Dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP; 10(-4) M) also produced a decrease of wrinkles in 81% of the cells (P less than 0.02) compared with a 9% decrease in the control period. MC treated with ANP, NP, or DBcGMP and then labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-phallacidin did not show obvious alteration of morphology of actin filaments compared with untreated cells. ANP could inhibit as well as partially reverse the agonist (angiotensin II)-induced contractile response. ANP (10(-10)-10(-8) M) as well as NP (10(-5) M) increased intracellular cGMP content of MC (P less than 0.005) compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amanitins; Angiotensin II; Animals; Atrial Natriuretic Factor; Cells, Cultured; Cyclic GMP; Dibutyryl Cyclic GMP; Ferricyanides; Fluorescent Dyes; Glomerular Mesangium; Methylene Blue; Microscopy, Fluorescence; Nitroprusside; Rats

Neutrophil activation by a variety of stimuli is accompanied by an intracellular acidification, which has been postulated to mediate actin polymerization (Yuli and Oplatka, Science 1987, 235, 340). This hypothesis was tested using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin staining and flow cytometry, or right angle light scattering to study actin assembly in intact and electrically permeabilized human neutrophils. Intracellular pH was measured fluorimetrically using a pH sensitive dye. In cells stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) at 21 degrees C, actin assembly clearly preceded the intracellular acidification in response to fMLP. Moreover, actin polymerization persisted in cells where intracellular pH was clamped near the resting (unstimulated) level using nigericin/K+. Finally, fMLP induced a significant increase in F-actin content in electropermeabilized neutrophils equilibrated with an extracellular medium containing up to 50 mM HEPES. These observations indicate that fMLP-stimulated F-actin assembly is not mediated by a decrease in intracellular pH and suggest that changes in transmembrane potential and ionic gradients are unlikely to mediate actin polymerization.

Topics: Actins; Amanitins; Cell Membrane Permeability; Electricity; Flow Cytometry; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Light; Macromolecular Substances; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Scattering, Radiation

Changes in myoepithelial cells (MECs) during perinatal development were examined by using the fluorescent probe for actin, nitrobenzoxadiazole (NBD)-phallacidin. By the twentieth day of gestation, there was no distinct fluorescent pattern suggestive of MECs. In newborn and 1-day-old rats, cells with diffuse fluorescence occurred around the acini, representing incipient MECs. Between 3 and 4 days after birth, actin staining was concentrated in strands which were arranged parallel to the long axis of the cell processes. MECs had developed further by the tenth day after birth with an increased number and thickness of their processes. Fully developed MECs were found between the thirtieth and fortieth day. These were stellate and encompassed individual acini.

Topics: Amanitins; Animals; Cell Differentiation; Female; Fluorescent Dyes; Male; Microscopy, Fluorescence; Rats; Rats, Inbred Strains; Sublingual Gland

Mesangial cells are contractile and are believed to play a role in the regulation of glomerular filtration. Actin filaments constitute an integral part of the cytoskeleton of these cells. To visualize actin filaments in both living and fixed acetone-extracted (nonliving) mesangial cells, we have used the fluorophore nitrobenzoxadiazole-phallacidin. Under the fluorescence microscope, mesangial cells displayed continuous staining. Distinct linear structures running from one end to the other were actin filaments or stress fibers (SF). SF were rather feathery and of slightly curvilinear appearance in living versus fixed, acetone-extracted cells. Formation of SF started a 4 h, and the majority of the cells had well developed SF within 24 h. To find out whether adhesion to a substrate was necessary to SF formation, we plated mesangial cells on variable thickness of poly-HEMA(2-hydroxyethyl methacrylate). This plastic surface at a high concentration (10(-1) M) produces poor adherence of mesangial cells. At 10(-1) M of poly-HEMA, the cells were rounded even after 48 h and did not develop any SF. In contrast, at a lower concentration (10(-4) M), mesangial cells were well spread, and SF were readily observed. These data indicated that spreading as well as formation of SF were directly related to the adhesion properties of the substrate. To evaluate the role of SF in contraction and relaxation of mesangial cells, we have first treated these cells with angiotensin II (5 x 10(-7) M) or dibutyryl cyclic adenosine monophosphate (5 x 10(-4) M) and then labeled them with nitrobenzoxadiazole phallacidin. Dibutyryl cyclic adenosine monophosphate caused dissolution of SF.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Amanitins; Angiotensin II; Animals; Bucladesine; Cell Adhesion; Cells, Cultured; Glass; Glomerular Mesangium; Polyhydroxyethyl Methacrylate; Rats

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.

Topics: Actins; Amanitins; Cell Membrane; Cell Movement; Chemotaxis; Cytoskeleton; Dictyostelium; Fluorescent Antibody Technique; Fluorescent Dyes; Microfilament Proteins; Microscopy, Electron; Polymers; Pseudopodia; Spectrophotometry

We studied the effect of tumor necrosis factor (TNF) and gamma interferon (IFN-gamma), alone and in combination, on the expression of chemotactic peptide receptors, stimulus-induced actin polymerization, hydrogen peroxide production (H2O2), and expression of nonspecific esterase (NSE) positivity in human promyelocytic leukemic cell line HL-60. These parameters were analyzed following a five-day culture with the cytokines. Chemotactic peptide receptor expression was studied using the fluoresceinated hexapeptide, formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine and flow cytometry. Actin polymerization, an important event required for chemotaxis and phagocytosis, was studied using NBD-phallacidin labeling, following stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). TNF increased the expression of chemotactic peptide receptors in a dose-dependent fashion, and there was good correlation between the receptor expression, stimulus-induced actin polymerization, H2O2 production, and NSE positivity. IFN-gamma was less potent in inducing all the parameters studied but exerted a positive cooperative effect when combined with TNF. IFN-gamma at high concentrations induced chemotactic peptide receptors comparable in magnitude to that seen with TNF but failed to prime these cells to undergo actin polymerization in response to FMLP or PMA. Undifferentiated HL-60 cells showed a decrease in F-actin content on stimulation with PMA. This suggests that protein kinase C might have a negative regulatory role in stimulus-induced actin polymerization. The observations reported here indicate that appropriate combinations of different inducing agents with different modes of action might be necessary to duplicate the functional abilities of mature phagocytic cells.

Topics: Actins; Amanitins; Cell Differentiation; Cell Line; Drug Synergism; Fluorescent Dyes; Free Radicals; Humans; Hydrogen Peroxide; Interferon-gamma; Monocytes; Oligopeptides; Receptors, Formyl Peptide; Receptors, Immunologic; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

The identification of microfilaments contained within Sertoli cell ectoplasmic specializations (ES) in intact rat testes is reported. In order to determine the presence and configuration of ES during the cycle of the seminiferous epithelium, frozen sections of testes were prepared and stained with NBD-phallicidin (NBDP). Results revealed that Sertoli cell ES become most prominent immediately adjacent to acrosomal caps of spermatids, once these begin their elongation phase of maturation. Significant association of ES with spermatogenic cells earlier than round spermatids was not detected with NBDP. Intense staining of the ES continued up to the final stages preceding sperm release, and was followed by dissipation of the fluorescence. These results indicate that the disappearance of ES, as detected with NBDP, does not correlate precisely with sperm release.

Topics: Actin Cytoskeleton; Actins; Amanitins; Animals; Cytoskeleton; Histocytochemistry; Male; Microscopy, Electron; Rats; Rats, Inbred Strains; Sertoli Cells

Nitrobenzoxadiazol (NBD) phallacidin, an active fluorescent derivative of the actin-binding mushroom toxin phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeleton structures. Topographical fluorescent microscopy images of lymphoid cells obtained with NBD-phallacidin staining reveal that the major feature of the cellular cytoskeleton characterized by actin are mainly associated with cell membrane, a pattern that correlates strikingly with their DNAse 1 inhibition. Such actin pools may thus be involved in a membrane-associated protein network controlling membrane viscoplastic deformation and cell motility.

Topics: Actins; Amanitins; Cell Membrane; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Humans; Lymphocytes; Polymers

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150-500 microM CD; 0.5-12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture exper

Topics: Actin Cytoskeleton; Actins; Amanitins; Animals; Blood-Testis Barrier; Cell Membrane Permeability; Cytochalasin D; Cytochalasins; Intercellular Junctions; Male; Microscopy, Electron; Rats; Rats, Inbred Strains; Sertoli Cells

Aggregation-competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells. Changes in cell behavior were correlated with F-actin content by using an NBD-phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D-sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F-actin assembled by 60 sec is localized in these new pseudopods. These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.

Topics: Actins; Amanitins; Animals; Chemotactic Factors; Cytochalasin D; Cytochalasins; Dictyostelium; Fluorescent Dyes; Kinetics; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Polymers; Pseudopodia

The actin cytoskeleton of rod photoreceptors and glial cells in toad retina has been directly viewed using fluorescence microscopy of cells labeled with a potent phallotoxin that specifically binds to F-actin. The three-dimensional organization of this cytoskeletal protein consists of actin filaments, which course through the inner segment and end at the tips of the calycal processes surrounding the base of the outer segment. A transverse layer of actin staining is also observed at the base of rod outer segments in the region where new discs are formed. At the level of the external limiting membrane, evidence has been found for rings of actin within the glial cells that surround the photoreceptors. These actin rings form a structural meshwork in which photoreceptor cells are embedded.

Topics: Actins; Amanitins; Animals; Bufo marinus; Cytoskeleton; Female; Fluorescent Dyes; Histocytochemistry; Male; Microscopy, Fluorescence; Neuroglia; Phalloidine; Photoreceptor Cells; Rhodamines

The distribution of F-actin and vinculin in chicken embryo fibroblasts has been examined by nitrobenzoxadiazol (NBD)-phallacidin and indirect immunofluorescent staining, respectively, and related to the process of focal contact formation by recording the motility of the cell with differential interference contrast (DIC) or interference reflection microscopy (IRM) before fixation for staining. Linear cytoplasmic precursors of the focal contact, present within unattached lamellipodia, stained intensely with NBD-phallacidin. Without exception new focal contacts, 8 s and older at fixation, were associated with either a longer F-actin rib in the lamellipodium or, in older contacts, an F-actin structure of similar dimensions to the contact. This change in distribution of F-actin over the new contacts was accounted for by the segregation of the structural precursor into an attached part over the focal contact and a separate motile part. These results show that F-actin accumulates in the precursor adjacent to areas of the membrane competent to form the focal contact, and are consistent with the interpretation that this F-actin contributes to the initial adhesion plaque associated with the new contact. Vinculin was essentially absent from motile lamellipodia, showed no preferential association with F-actin rich precursors or very young focal contacts, but accumulated over new contacts during a 90-s period. Therefore, the association of F-actin with the membrane that precedes and persists in the initial focal contact is independent of vinculin, and the role of vinculin in development of the focal contact remains unclear.

Topics: Actins; Amanitins; Animals; Cell Adhesion; Cell Membrane; Cells, Cultured; Chick Embryo; Fibroblasts; Fluorescent Antibody Technique; Fluorescent Dyes; Muscle Proteins; Vinculin

The distribution of F-actin in the rabbit corneal endothelial cells was studied in vivo and in culture using nitrobenzoxadiazole-conjugated phallacidin. In the normal cornea, the fluorescence showing the presence of F-actin was observed along the membrane of the endothelial cells, but little fluorescence was seen in the cytoplasm. During wound-healing processes after transcorneal freezing, the endothelial cells migrating to the wound area showed abundant fiber-like fluorescence in the cytoplasm. In about 28 days after the injury, the endothelial cells recovered normal shape and the pattern of actin localization became normal, ie, fiber-like fluorescence localization along the cell membrane. The endothelial cells were cultured for about one week and the explants were removed. After further culture for about two weeks the cultured cells became confluent forming a monolayer. At the center of this monolayer, a small wound was made, and changes in the cell shape and actin distribution were studied. The actin distribution in the undisturbed monolayer cells was similar to that seen in vivo, ie, fiber-like fluorescence along the cell membrane. After the wound production, many cells were seen to migrate toward the wound center, and abundant fluorescent fiber-like structures were observed throughout the cytoplasm. Addition of cytochalasin B to the culture medium suppressed cell migration in a dose-dependent manner. At a high cytochalasin B concentration the fiber-like fluorescence was not formed and scattered fluorescent speckles were observed. Further culture in cytochalasin B-free medium after exposure to this agent permitted a recovery of cell migration and formation of the fiber-like actin fluorescence. It was suggested that polymerization of actin filaments is activated in the migrating cells during wound-healing, and that cytochalasin B reversibly blocks this polymerization, thereby suppressing cell migration. Actin filament polymerization would constitute a significant part of the mechanism underlying cell migration and wound-healing.

Topics: Actins; Amanitins; Animals; Cells, Cultured; Cytochalasin B; Endothelium, Corneal; Female; Male; Microscopy, Fluorescence; Rabbits; Time Factors; Wound Healing

The effect of calcium on oxytocin-induced contraction of myoepithelial cells was visualized with NBD-phallacidin, a fluorescent stain for filamentous actin. In the absence of oxytocin, the cells appeared relaxed; long, branching processes radiated from the cell bodies. In the presence of 50 nM-oxytocin, myoepithelial cells contracted into smaller spoke-shaped bodies in which the arms were shorter and thicker. Electron microscopy confirmed the morphological differences between oxytocin-treated and untreated myoepithelium. To determine a role for extracellular calcium, tissue was incubated in EGTA, then exposed to oxytocin, with or without added calcium. Contraction occurred in the presence of oxytocin plus additional calcium but not in the absence of calcium. When the tissue was incubated with the calmodulin antagonist trifluoperazine (TFP) in calcium-containing medium, oxytocin did not induce myoepithelial cell contraction. These data support previous results obtained with a myosin light-chain phosphorylation assay implicating calcium and calmodulin in oxytocin-induced contraction. Furthermore, NBD-phallacidin visualization of myoepithelial cells demonstrates that the effect of calcium on contraction is physiologically significant.

Topics: Amanitins; Animals; Calcium; Epithelial Cells; Epithelium; Female; Fluorescent Dyes; In Vitro Techniques; Mammary Glands, Animal; Mice; Microscopy, Electron; Microscopy, Fluorescence; Oxytocin

In this study, we describe the distribution of actin filaments in and around spermatids that are mechanically dissociated, in the presence and absence of exogenous trypsin, from the seminiferous epithelium of the rat. NBD-phallacidin and subfragment 1 of the myosin molecule (S-1) are used as probes for filamentous actin at the light and electron microscopic levels, respectively. The fluorescence associated with spermatids mechanically dissociated in the absence of trypsin is due to actin both in the spermatogenic cells themselves and in attached Sertoli cell ectoplasmic specializations. Fluorescence generated by labelled actin in ectoplasmic specializations occurs in linear tracts that follow the outer contour of spermatid heads. The residual fluorescence seen when trypsin is used to detach Sertoli cell fragments is diffuse and due to f-actin in the subacrosomal space. Electron microscopic data agree with the fluorescence results. This study conclusively demonstrates that Sertoli cell ectoplasmic specializations remain attached to spermatids mechanically dissociated from the seminiferous epithelium. This observation may be useful when attempting to isolate ectoplasmic specializations for biochemical analyses.

Topics: Actins; Amanitins; Animals; Fluorescent Dyes; Histocytochemistry; Male; Microscopy, Electron; Rats; Rats, Inbred Strains; Sertoli Cells; Spermatids; Trypsin

We have investigated the possibility that the complex patterns of fluorescence associated with spermatids of the ground squirrel labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) are due to the presence of filamentous actin within the spermatids themselves rather than to actin in attached Sertoli cell ectoplasmic specializations, as previously reported (J. Cell Biol., 100:814-825). Enzymatic treatments (trypsin, DNAase 1) freed Sertoli cell ectoplasmic specializations from spermatids and resulted in a loss, from the spermatids, of the complex fluorescence patterns, suggesting that the latter were generated by labeled actin in ectoplasmic specializations. Moreover, ectoplasmic specializations that were detached enzymatically from spermatids demonstrated the same fluorescence patterns as those emitted from spermatids in the intact or mechanically fragmented seminiferous epithelium. Most spermatids, however, do display a weak and diffuse pattern of fluorescence that changes during spermatogenesis and that is localized between the acrosomal cap and nucleus. S-1 decoration confirmed this subacrosomal localization and further demonstrated that the actin in adjacent Sertoli cell ectoplasmic specializations is arranged in a unipolar fashion. We conclude that the complex patterns of actin fluorescence associated with mechanically isolated spermatids are a superimposition of both Sertoli cell and germ cell actin; however, the latter is either poorly detected or not detected at all when Sertoli cell ectoplasmic specializations overlie the germ cells.

Topics: Actins; Amanitins; Animals; Cell Membrane; Detergents; Endoplasmic Reticulum; Histological Techniques; Male; Myosin Subfragments; Peptide Fragments; Sciuridae; Sertoli Cells; Spermatids; Testis; Tissue Distribution

Using NBD-phallacidin, which specifically binds to F-actin, we investigated changes in the localization of actin during the differentiation of ameloblasts, related epithelial cells and odontoblasts in rat incisors. In cryosections treated with NBD-phallacidin, intense fluorescence was observed in undifferentiated epithelial cells in the apical loop and at the proximal extremity of undifferentiated inner enamel epithelial cells. During differentiation, the distal extremity began to exhibit strong fluorescence. In cross-sections of secretory ameloblasts, the fluorescence took the form of polygons of uniform intensity at the proximal end, and of rectangles of non-uniform intensity at the distal end. At the distal end, the fluorescence was more intense at right angles to the long axis of the incisor. At the distal end, this pattern was established just before the appearance of the enamel layer. These patterns were maintained during the secretory stage of ameloblasts. The location, pattern and time of appearance of these sites were identical to those of the terminal webs in ameloblasts. NBD-phallacidin weakly labelled the peripheral cytoplasm of the cell body of ameloblasts, and also labelled Tomes' process. The cells forming the stratum intermedium were mainly labelled at their periphery (i.e. forming larger polygons), while the overlying epithelial cells exhibited labelling throughout their cytoplasm. Except for the terminal webs, the cell bodies of odontoblasts were weakly labelled throughout the period of differentiation. Young odontoblasts secreting predentin were first labelled on the terminal web, with the fluorescence becoming gradually more intense as the thickness of the dentin increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Amanitins; Ameloblasts; Animals; Cell Differentiation; Dentin; Epithelium; Fluorescent Dyes; Frozen Sections; Incisor; Male; Odontoblasts; Rats; Rats, Inbred Strains

Many cellular functions involve the complex network of actin filaments, microtubules, and intermediate filaments collectively known as the cytoskeleton. Stereo transmission electron microscopic observations of whole cynomolgus monkey trabecular cells, which were extracted, S-1 labeled, and critical-point dried, were employed to simultaneously identify these three major cytoskeletal systems and visualize their three-dimensional nature. A double fluorescence technique for actin and microtubules was used to provide a broad view of cytoskeletal relationships within the cell. Actin microfilaments were the most prominent elements of the cytoskeleton. They appeared as bundles in stress fibers. Between stress fiber bundles, a continuous meshwork of microfilaments and intermediate filaments could be seen. Numerous microtubules radiated from the centriole region to the cell periphery. This comprehensive overview of the cytoskeleton of the cynomolgus monkey trabecular cell can be used to understand structure-function relationships of the trabecular cell cytoskeleton and its influence on outflow facility.

Topics: Actins; Amanitins; Animals; Cytoskeleton; Fluorescent Dyes; Intermediate Filaments; Macaca fascicularis; Microscopy; Microscopy, Electron; Microscopy, Fluorescence; Microtubules; Trabecular Meshwork

We have studied changes in myoepithelial cell size and shape during different stages of mouse mammary gland differentiation by using the fluorescent probe for actin NBD-phallacidin. Pieces of mammary tissue were fixed, mounted on slides, permeabilized with cold acetone (-20 degrees C), and then treated with nitrobenzoxadiazole-phallacidin. Myoepithelial cells lining ducts of glands at all stages of development are spindle-shaped structures oriented parallel to the long axis of the duct at the base of the luminal epithelium. In virgin animals, myoepithelial cells also occur as linear tracts oriented parallel to the long axis of small projections along the sides of ducts and terminal end buds. In early pregnancy, small stellate-shaped cells begin to appear around presumptive secretory units. By late pregnancy, larger star-shaped units of intense fluorescence appear at the base of alveoli. During lactation, both cell bodies and cell processes further enlarge as these interlacing stellate-shaped cells encompass the expanded alveoli. In regressing glands, cell size decreases and the processes appear to retract. Although alveoli are virtually absent in the multipartate resting gland, myoepithelial cells remain around lateral buds of ducts. These myoepithelial cells have two distinct shapes: small star-shaped cells capping the buds and spindle-shaped cells oriented parallel to the long axis of the buds. A comparison of myoepithelial cell shape in virgin mice and nulliparous women indicates a more developed cell in the human gland at this stage of development. Intact segments of mammary gland combined with NBD-phallacidin as a probe for actin provide an ideal system for future studies of the control of myoepithelial cell size and shape and their influence on cell functions.

Topics: Adult; Amanitins; Animals; Cell Differentiation; Epithelial Cells; Female; Fluorescent Dyes; Humans; Mammary Glands, Animal; Mice; Microscopy, Electron; Pregnancy

We have localized actin in gametes of Chlamydomonas reinhardi by two approaches: (1) indirect immunofluorescence with an affinity-purified antibody and (2) staining with NBD-phallacidin, a fluorescent reagent that binds only to F-actin [Barak et al, 1980, Proc Natl Acad Sci, 77:980-984]. Staining of either mating type "plus" (mt+) or "minus" (mt-) gametes with antiactin antibody resulted in similar fluorescent images: most of the actin was located peripherally along the lateral and posterior aspects of the cells. There was diffuse staining centrally, but the flagella did not stain. No brightly stained spot was observed near the mt+ mating structure, the site where the fertilization tubule elongates with concomitant polymerization of actin [Detmers et al, 1983, J Cell Biol, 97:522-532]. Gametes stained prior to mating with NBD-phallacidin showed no fluorescence above background, indicating that there were no concentrations of F-actin in these cells. This suggested that the cytoplasmic staining observed with antiactin represented primarily a nonfilamentous form of the protein. In mating gametes staining with NBD-phallacidin was detected only in the fertilization tubule, indicating that this was the only dense accumulation of filamentous actin within the cells. Mating gametes stained with antiactin antibody exhibited cytoplasmic fluorescence that was slightly more punctate than prior to mating, and the fertilization tubule was brightly stained. Our observations suggest that the site-specific polymerization of actin within the fertilization tubule occurs in the absence of a concentrated pool of actin subjacent to the mating structure.

Topics: Actins; Amanitins; Chlamydomonas; Electrophoresis, Polyacrylamide Gel; Flagella; Fluorescent Antibody Technique; Immune Sera; Staining and Labeling; Tubulin

The effect of an antidiuretic hormone (ADH, vasopressin) on the microfilament system of the toad urinary bladder lumenal epithelium was investigated using NBD-phallacidin (NBD-ph). The latter material is a specific fluorescent label for F actin. In the presence of an osmotic gradient, both ADH and cyclic adenosine monophosphate (cAMP) appear to induce the polymerization of monomeric actin into F actin-containing microfilaments. The latter may then be involved in the morphological changes, including the formation of lateral intercellular lakes, associated with the typical hydroosmotic response.

Topics: Actins; Amanitins; Animals; Arginine Vasopressin; Bufo marinus; Cyclic AMP; Cytoskeleton; Epithelium; Female; Microscopy, Fluorescence; Peptides, Cyclic; Thionucleotides; Urinary Bladder

Actin was located in tachyzoites of Toxoplasma gondii by indirect immunofluorescence using anti-actin antibodies. Fluorescence was seen in the region of the conoid of almost all cells. In about 75% of the cells, the whole anterior region showed fluorescence and in about 25% of the cells fluorescence was seen in the posterior region. No fluorescence was seen when permeabilized parasites were incubated in the presence of NBD-phallacidin which binds to F-actin. This observation, associated to the fact that no microfilaments were seen in transmission electron micrographs of Triton X-100 extracted, tannic acid-glutaraldehyde (TA-GA) fixed cells, indicate that in tachyzoites of T. gondii actin is predominantly in the monomeric form (G-actin). No fluorescence was observed when permeabilized parasites were incubated in the presence of antibodies specific to desmin, vimentin and keratin. Examination of Triton X-100 extracted, TA-GA fixed parasites showed that the outer membrane was partially removed while the inner membrane complex was not, but had a corrugated aspect. Connections between the sub-pellicular microtubules and the inner membrane complex and between the latter and the outer membrane were seen.

Topics: Actins; Amanitins; Animals; Cytoskeletal Proteins; Cytoskeleton; Detergents; Fluorescent Antibody Technique; Microscopy, Electron; Toxoplasma

We have studied the distribution of actin, using NBD-phallacidin as a probe, in isolated sheets of seminiferous epithelia and denuded tubule walls of the rat. Sheets of intact seminiferous epithelia were separated from tubule walls using EDTA in PBS. The isolated epithelia and denuded tubule walls were fixed, mounted on slides, made permeable with cold acetone (-20 degrees C), and then treated with NBD-phallacidin. Actin was observed in myoid cells, in ectoplasmic specializations of Sertoli cells, and in Sertoli cell regions adjacent to tubulobulbar processes of late spermatids. In myoid cells, filament bundles course in circular and longitudinal directions relative to the tubule wall. In Sertoli cells viewed at an angle perpendicular to the epithelial base, actin filaments in ectoplasmic specializations adjacent to junctional complexes circumscribe the bases of the cells. Filament bundles in ectoplasmic specializations adjacent to germ cells closely follow the contour of and are arranged parallel to the long axis of the developing acrosome. Sertoli cell regions adjacent to tubulobulbar processes of late spermatids stain intensely with NBD-phallacidin. Isolated seminiferous epithelia, combined with NBD-phallacidin as a probe for actin, provide an ideal model system in which to study further the contractile properties of Sertoli cell ectoplasmic specializations and the possible involvement of these structures in events that occur during spermatogenesis.

Topics: Actins; Amanitins; Animals; Epithelial Cells; Epithelium; Male; Microscopy, Electron; Microscopy, Fluorescence; Rats; Rats, Inbred Strains; Seminiferous Tubules; Sertoli Cells; Testis; Tissue Distribution

The studies presented here characterize a simple, quantitative NBDphallacidin extraction assay for determining the F-actin content of fMLP-activated neutrophils. The NBDphallacidin extraction assay is based upon the specificity of NBDphallacidin binding to F-actin and the solubility of NBDphallacidin in methanol. Cells are fixed, permeabilized, and stained with NBDphallacidin; the cells are then pelleted, the bound NBDphallacidin is extracted into methanol, and the RFI (excite 465; emit 535) of the solution is determined. Binding of NBDphallacidin to neutrophils is saturable and 90% of bound NBDphallacidin is displaced by nonfluorescent phalloidin. The extraction of bound NBDphallacidin into methanol is complete and the excitation/emission characteristics of NBDphallacidin are not altered by extraction. The assay is relatively inexpensive, applicable to the study of cells in suspension or on substratum, allows kinetic studies with 5-10s time resolution, and is not affected by the shape of the cell or the distribution of the probe. We used the NBDphallacidin extraction assay to study the kinetics of fMLP-induced change in the F-actin content of neutrophils and the effect of tBOC peptide, an inhibitor of fMLP binding, on these changes. The extraction assay reveals a rapid, sequential fMLP-induced increase followed by a decrease in F-actin content. The tBOC peptide inhibits fMLP-induced actin polymerization. Addition of tBOC during fMLP-induced polymerization or at times when F-actin content is maximal enhances F-actin depolymerization. The rate of F-actin depolymerization is greater than or equal to fourfold faster in the presence than in the absence of tBOC. The results show that The NBDphallacidin extraction assay is useful for studying the kinetics of change in F-actin content of nonmuscle cells; fMLP receptor occupancy is required for fMLP-dependent polymerization but not depolymerization; and both the actin polymerizing and depolymerizing processes are active in the cell within 5 s after fMLP stimulation. Implications of these observations for understanding the observed increase and, then, decrease in F-actin content of fMLP-activated cells are discussed.

Topics: Actins; Amanitins; Chemotaxis, Leukocyte; Cytoskeleton; Methanol; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Polymers; Spectrometry, Fluorescence

To determine the relationship between the state of actin polymerization in neutrophils and the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced changes in the locomotive behavior of neutrophils, the mean rate of locomotion (mROL), the percent G-actin, and the relative F-actin content of neutrophils were determined. The mROL was quantified by analysis of the locomotion of individual cells; the percentage of total actin as G-actin was measured by DNase I inhibition; and the F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained neutrophils. Neutrophils stimulated with fMLP exhibit a change in their mROL that is biphasic and dose dependent. The mROL of neutrophils exposed to 10(-8) M fMLP, the KD, is 11.9 +/- 2.0 micron/min (baseline control 6.2 +/- 1.0 micron/min). At 10(-6) M fMLP, the mROL returns to baseline levels. Stimulation of neutrophils with fMLP also induces action polymerization. Evidence for actin polymerization includes a 26.5% reduction in G-actin and a twofold increase in the amount of NBD-phallacidin staining of cells as determined by FACS analysis. The NBD-phallacidin staining is not due to phagocytosis, is inhibited by phalloidin, requires cell permeabilization, and is saturable at NBD-phallacidin concentrations greater than 10(-7)M. The fMLP-induced increase in NBD-phallacidin staining occurs rapidly (less than 2 min), is temperature dependent, and is not due to cell aggregation. Since NBD-phallacidin binds specifically to F-actin, the increase in fluorescent staining of cells likely reflects an increase in the F-actin content of fMLP-stimulated cells. FACS analysis of NBD-phallacidin-stained cells shows that the relative F-actin content of neutrophils stimulated with 10(-11)-10(-8)M fMLP increases twofold and remains increased at concentrations greater than 10(-8)M fMLP. Therefore, the fMLP-induced increase in F-actin content of neutrophils as determined by FACS analysis of NBD-phallacidin-stained cells coincides with a decrease in G-actin and correlates with increased mROL of neutrophils under some (10(-11)-10(-8)M fMLP) but not all (greater than 10(-8)M fMLP) conditions of stimulation. Quantification of the F-actin content of nonmuscle cells by FACS analysis of NBD-phallacidin-stained cells may allow rapid assessment of the state of actin polymerization and correlation of that state with the motile behavior of nonmuscle cells.

Topics: Actins; Amanitins; Cell Movement; Chemotaxis, Leukocyte; Deoxyribonuclease I; Endodeoxyribonucleases; Fluorescent Dyes; Humans; Macromolecular Substances; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

One rapid response of starfish oocytes to the maturation-inducing hormone, 1-methyladenine (1-MA), is the formation of transient actin-filled spikes on the cell surface. The presence and distribution of G- and F-actin and several actin-associated proteins were examined in cortices isolated from oocytes before, during, and after spike formation by using antibodies and the F-actin-specific stain, NBD-phallacidin. Before 1-MA addition, staining with antiactin and NBD-phallacidin indicates that most of the actin in the cortex is either G-actin or oligomeric actin, but rather little is F-actin. Application of the hormone results in the conversion and redistribution of this cortical actin into large bundles of F-actin which form the cores of spikes. When the spikes recede, F-actin disappears, and the amount of all forms of actin bound in the cortex appears to decrease. Antibodies to sea urchin egg myosin, fascin and a 220-kDa protein were used to examine these actin-associated proteins during the times that the organization of actin changes. Myosin and the 220-kDa protein are bound to the cortex and uniformly distributed before 1-MA application while fascin appears to be unbound. When spikes appear after 1-MA addition, fascin and the 220-kDa protein are localized coincidently with the spikes, whereas myosin remains uniformly distributed throughout the cortex and is excluded from the spikes. After spike resorption, fascin and the 220-kDa protein appear to lose their cortical binding while myosin retains its localization unchanged. These results indicate that actin, fascin and the 220-kDa protein undergo major organizational changes in the cortex in response to 1-MA.

Topics: Actins; Adenine; Amanitins; Animals; Carrier Proteins; Egg Proteins; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Macromolecular Substances; Microfilament Proteins; Myosins; Oocytes; Starfish

We report further characterization of the physical and immunologic properties, mechanism of action, and intracellular localization of Acanthamoeba castellanii capping protein, an actin regulatory protein discovered by Isenberg (Isenberg, G., U. Aebi, and T. D. Pollard, 1980, Nature (Lond.) 288:455-459). The native molecular weight calculated from measurements of Stokes' radius (3.8 nm by gel filtration chromatography) and sedimentation coefficient (4.8 S by sucrose gradient velocity sedimentation) was 74,000 daltons. The subunit molecular weights were 31,000 and 28,000 daltons, so the native molecule is a heterodimer. The two subunits did not immunologically cross-react with each other or with any other proteins from Acanthamoeba or several other organisms. In studies of the mechanism of action, Isenberg (see above reference) found that capping protein blocked polymerization from the barbed end of actin filaments and sedimented with actin filaments. We confirmed that capping protein binds to actin filaments with a gel filtration assay. Capping protein decreased the length distribution and high shear viscosity of actin filaments. Capping protein did not bundle or cross-link actin filaments. Low concentrations of capping protein increased the critical concentration for muscle and ameba actin polymerization from 0.1 to 0.6 microM in Mg++ and EGTA. Increasing amounts of capping protein did not increase the critical concentration further. In Ca++ capping protein did not change the critical concentration for muscle actin, but did increase the critical concentration for ameba actin. Ca++ had no effect on the ability of capping protein to decrease the low or high shear viscosity of actin filaments. By indirect fluorescent antibody staining, capping protein was localized to the cell cortex, an area rich in actin filaments. During subcellular fractionation of homogenates, about 1/3 of cellular capping protein banded with a crude membrane fraction. The other 2/3 of cellular capping protein was soluble, with a Stokes' radius equal to that of the purified protein. The molar ratio of capping protein to actin in the cell was 1:150.

Topics: Actin Depolymerizing Factors; Actins; Amanitins; Amoeba; Animals; Calcium; Chickens; Cross Reactions; Destrin; Egtazic Acid; Fluorescent Antibody Technique; Magnesium; Microfilament Proteins; Molecular Weight; Proteins; Subcellular Fractions; Tissue Distribution; Viscosity

The molecular probe NBD phallacidin (7-nitro-benz-2-oxa-1, 3-diazolylphallacidin), which reacts specifically with filamentous actin (f-actin), was used to study the distribution of polymerized actin oligmers in normal and migrating rabbit corneal epithelial cells. In the normal cornea, the majority of the NBD phallacidin fluorescence was localized to the cortical or sub-plasma membrane area of the superficial and wing epithelial cells. Following full thickness corneal trephination injury, migrating corneal epithelial cell exhibited a marked increase in the cortical NBD phallacidin fluorescence. Transmission electron microscopy, using fixation techniques which revealed bundles of fine filaments (6nm underlying the plasma membrane of migrating epithelial cells, thus supporting the fluorescent results. These findings suggest that corneal re-epithelialization is characterized by a marked increase in the amount of filamentous actin within the migrating epithelial cells. We conclude that NBD phallacidin may be of value in analyzing changes in actin polymerization during wound healing.

Topics: Actins; Amanitins; Animals; Cell Movement; Cornea; Epithelium; Microscopy, Electron; Microscopy, Fluorescence; Rabbits; Wound Healing

The effect of the chemotatic peptide, N-formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM cytochalasin B or by a t-BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.

Topics: Actins; Amanitins; Chemotaxis, Leukocyte; Cytochalasin B; Flow Cytometry; Fluorescent Dyes; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Conformation

Application of ATP to cryosections of plasmodial strands from Physarum polycephalum leads to an isotonic contraction of the cytoplasmic actomyosin fibrils: when the fibrils are labelled with NBD-phallacidin, their contraction can be observed in the fluorescence microscope. While performing contraction, the fibrils separate into many small units and the formerly continuous fibrils exhibit the appearance of beaded chains. The possibility of visualizing directly the contraction of cytoplasmic actomyosin fibrils in the fluorescence microscope represents a favourable condition for the study of their physiological contraction mechanism, because this new and convenient cell-free model offers in situ contractile structures that are non-denatured and non-extracted.

Topics: Actomyosin; Adenosine Triphosphate; Amanitins; Cell-Free System; Cytoskeleton; Microscopy, Fluorescence; Muscle Contraction; Physarum

Fluorescently labeled phallacidin, a F-actin specific drug, was used to demonstrate the morphological variety in the cytoskeletal actin pattern of thin-spread plasmodia of the acellular slime mould Physarum polycephalum. The patterns observed in phallacidin-stained specimens consisted of a polygonal network in the anterior region, and of longitudinal as well as helically twisted fibrils in plasmodial strands of the posterior region. These observations are in complete accordance with our recent results obtained on comparable plasmodia by immunofluorescence microscopy using specific antibodies against actin.

Topics: Actins; Amanitins; Cytoskeleton; Physarum; Staining and Labeling

The presence and possible roles of cytoplasmic contractile elements in glomerular epithelial podocytes and the glomerular mesangium are briefly discussed. Glomerular podocytes contain actinlike filaments distributed throughout their cytoplasm and more concentrated filamentous material within their foot processes. Antibody labeling and nitrobenzoxadiazole-phallacidin labeling have confirmed the presence of especially high concentrations of actin within podocyte foot processes. Studies with cytochalasins have suggested that contraction of actin within podocytes leads to a flattening of foot processes and a loss of the intervening filtration slits. Conversely, relaxation of these contractile elements leads to a narrowing of the bases of foot processes and an increase in the number of fully open intervening filtration slits. These observations have led to the proposal that glomerular podocytes have the potential to regulate the glomerular filtration rate by changing the shapes of their foot processes and thereby decreasing or increasing the number of filtration slits available for solute efflux across the glomerular wall. There is also evidence to indicate the presence of contractile elements within the glomerular mesangium. These contractile elements may be represented in part by bundles of fine filaments associated with electron-dense bodies that are present within many processes that radiate from these cells. In vitro studies in particular have suggested that these cells may possess contractile properties. It has been proposed that contraction of the mesangial cells may lead to a shunting of blood within the glomerulus or to a decrease in glomerular size.

Topics: Actins; Amanitins; Animals; Cytoskeleton; Kidney Glomerulus; Microscopy, Electron; Microscopy, Fluorescence; Rats

The distribution of actin in dividing endothelial cells of the rat cornea was studied by fluorescence microscopy by means of the nitrobenzoxadiazole conjugated derivative of the actin-binding toxin phallacidin (NBD-Ph). In normal noninjured tissue, fluorescence is limited to an area at or near the plasma membrane. Twenty-four hours after a corneal freeze injury, stress fibers are detected but only in those cells that are migrating into the wound area. By 48 h post-injury, cells in various stages of mitosis can be identified. During metaphase, anaphase, and telophase, diffuse cytoplasmic staining is observed, although the spindle region remains free of fluorescence. At various sites along the plasma membrane, fluorescence appears stronger compared to other regions. During the latter two stages of proliferation, NBD-Ph positive material can be seen within cell processes. In addition, a band of this material is observed within the region that corresponds to the cleavage furrow. As the daughter cells separate, actin can be detected within the cytoplasmic bridge. The results indicate that NBD-Ph can be used to study the distribution of actin in cells that were proliferating in vivo, and these patterns appear similar to those obtained with immunological methods on cultured cells.

Topics: Actins; Amanitins; Animals; Cell Division; Cornea; Corneal Injuries; Endothelium; Female; Histocytochemistry; Male; Microscopy, Fluorescence; Rats; Rats, Inbred Strains

The fluorescent derivative of the actin-binding toxin phallacidin, 7-nitrobenz-2-oxa-1,3 diazole phallacidin, has been used to cytologically demonstrate the presence of actin in lens epithelium, corneal endothelium, and retinal pigment epithelium. In these noninjured tissues, no stress fibers are observed and fluorescence is confined mainly to an area at or near the cell membrane, although some diffuse cytoplasmic staining can also be seen. However, following injury to either the lens epithelium or corneal endothelium of rats and frogs, stress fibers are detected, but only in those cells that migrate into the wound area. Cells on the periphery of each tissue do not partake in would repair and thus maintain their normal appearance. After the tissue has regenerated, stress fibers disappear, and those cells involved in the injury response return to their normal morphology. When rabbit corneal endothelium is placed in tissue culture, stress fibers are observed as the cells migrate away from the initial explant. Upon reaching confluency, these cells spread out and each is surrounded by thick actin-containing bands. Furthermore, they exhibit some stress cables within their cytoplasm. This is in contrast to their appearance in vivo where stress fibers are absent and fluorescence is limited to a region near the cell membrane.

Topics: Actins; Amanitins; Animals; Cell Movement; Cornea; Corneal Injuries; Cytoplasm; Cytoskeleton; Endothelium; Epithelium; Lens, Crystalline; Microscopy, Fluorescence; Pigment Epithelium of Eye; Rana pipiens; Rats; Rats, Inbred Strains

The blue light-excited fluorescent phallotoxin derivative nitrobenzoxadiazole phallacidin (NBD-Ph) was used to stain entire tissue culture monolayers of live L6 mouse cells and other mammalian cell lines without the aid of permeabilization treatment. Although cells tend to exclude the fluorescent toxin, reducing the internal concentration by approximately 1,000 times, some of it enters the cells, probably by pinocytosis, and stains actin structures at low intracellular NBD-Ph concentrations (approximately 5-15 nM), where cell toxicity was negligible or at least not detectable by phase-contrast microscopy. Protracted treatments with NBD-Ph did induce pharmacological responses similar to those of phalloidin. The dissociation constant for NBD-Ph with F-actin in fixed and extracted L6 cells was determined, from staining intensity measurements at various NBD-Ph concentrations, to be 1.5-2.5 x 10(-8) M.

Topics: Actins; Amanitins; Animals; Cell Compartmentation; Cells, Cultured; Cytoskeleton; Mice; Muscles; Oxadiazoles; Staining and Labeling

The distribution and polymerization state of actin in metaphase rat kangaroo cells was studied by fluorescence microscopy. Formaldehyde-fixed, acetone-extracted cells were labeled with either of two types of actin probes. The first, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin, has high affinity for F actin and does not bind monomeric G actin. The second was a conjugate of DNase I labeled with either tetramethylrhodamine or fluorescein. DNase binds with high affinity to G actin and with lesser affinity to F actin. The polymerization state of actin was deduced by comparing the fluorescence distribution of the phallacidin derivative with that of the fluorescent DNase. The results indicate that the pole-to-chromosome region of the metaphase spindle contains G actin but little if any conventional F actin. F actin is found concentrated in a diffuse distribution outside the spindle region in metaphase cells and returns to the interzone area between the chromosomes by early telophase. These results exclude spindle models for chromosomal movement that require more than about five F actin filaments per chromosome, support the hypothesis that F actin is involved in force generation for cell cleavage, and are not inconsistent with the possibility that actin outside the spindle may be involved in chromosomal movement.

Topics: Actins; Amanitins; Animals; Cell Line; Chromosomes; Deoxyribonuclease I; Deoxyribonucleases; Dipodomys; Endonucleases; Fluorescent Dyes; Kinetics; Metaphase; Microscopy, Fluorescence; Mitosis

Topics: Actins; Amanitins; Animals; Chromatography; Fluorescent Dyes; Macropodidae; Peptides, Cyclic; Spectrometry, Fluorescence