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At least 19 recordsLinked to original sources
PubMed2003

Growth inhibition and changes in morphology and actin distribution in Acetabularia acetabulum by phalloidin and phalloidin derivatives.

Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4-7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424-433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 microM. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 microM), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy.

Acetabularia↗
PubMed1982

Effects of phalloidin microinjection and localization of fluorescein-labeled phalloidin in living sand dollar eggs.

Effects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 microM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 microM, though the latter seemed to be more sensitive to phalloidin than the former. Fluorescein-labeled phalloidin (FL-phalloidin) was injected into eggs in order to investigate F-actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL-phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rod-like structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL-phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.

Actins↗
PubMed1982

Chemical modification of membrane proteins by brominated taurodehydrocholate in isolated hepatocytes; relationship to the uptake of cholate and of phalloidin and to the sensitivity of hepatocytes to phalloidin.

In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4'-diisothiocyano 2,2'-stilbene disulfonate (DIDS). When hepatocytes were incubated with 35S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of 35S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with 35S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).

Affinity Labels↗
PubMed1995

The effect on actin ATPase of phalloidin and tetramethylrhodamine phalloidin.

Actin polymerization has been studied in the absence of excess nucleotide. Using G-actin ATP monomers, it was shown that mechanical shearing stimulates ATP hydrolysis. The procedures used enabled the detection of differential effects of phalloidin and tetramethylrhodamine-phalloidin, on the P(i)-release step of the actin ATPase. It is concluded that tetramethylrhodamine, in contrast to phalloidin, accelerates P(i)-release from actin filaments.

Actins↗
PubMed1981

[Phalloidin counteracts the destruction of F-actin by osmic acid. II. Protection by phalloidin of F-actin crosslinked with aldehydes (author's transl)].

We have studied by viscometry and spectrophotometry the effects of glutaraldehyde, acetaldehyde, acrolein and formaldehyde on F-actin in vitro. Pretreatment with acrolein and, to a lesser extent, with glutaraldehyde, results in increased destruction of F-actin during the subsequent degradation with osmic acid. Formaldehyde alone disintegrates actin filaments. Acetaldehyde gives the best results and does not seem to damage F-actin. Phalloidin protects F-actin against destruction by osmic acid and this protection is also observed in the F-actin which has been pretreated with the four aldehydes mentioned.

Acetaldehyde↗
PubMed1996

A study on the mechanism of phalloidin-induced tension changes in skinned rabbit psoas muscle fibres.

The time course of phalloidin induced changes in isometric tension of partially activated skinned rabbit psoas fibres was studied as a function of both phalloidin concentration and time of pre-incubation with phalloidin. Upon addition of phalloidin to non-pretreated (control) fibres there was a fall in tension followed by an increase in tension. The latency of both parts of the response was inversely related to the phalloidin concentration in the range 40-130 microM phalloidin. By preincubating the fibres with phalloidin for varying periods of time it was possible to obtain responses which appeared to represent later portions of the control response. Thus after pre-treatment with 40 microM phalloidin in either rigor or relaxing solution for 5 min (the time corresponding to minimal tension in the control response) the tension response resembled that of the control, beginning from the vicinity of the minimum. The pattern of staining of the fibres by rhodamine-phalloidin was analysed by laser confocal microscopy to relate the mechanical response to phalloidin localization. If fibres were treated with rhodamine-phalloidin for 20-25 min there was a labelling of the I-Z-I segment with intense peaks of fluorescence at the Z-line and the ends of the I filaments. If fibres were pre-incubated for 5 min with phalloidin and then labelled with rhodamine-phalloidin the fluorescence at the Z-line and at the ends of the I filaments was suppressed and the peak of the fluorescence intensity was shifted toward the middle part of the I filament. The data indicate that the decrease in tension caused by phalloidin was associated with binding of phalloidin to the pointed ends of actin filament and the Z-line region, whereas the increase in tension occurred when phalloidin was bound along entire length of the actin filament.

Animals↗
PubMed1996

Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species.

We compared the kinetics and thermodynamics of rhodamine phalloidin binding to actin purified from rabbit skeletal muscle, Acanthamoeba castellanii, and Saccharomyces cerevisiae in 50 mM KCl, 1 mM MgCl2, and pH 7.0 buffer at 22 degrees C. Filaments of S. cerevisiae actin bind rhodamine phalloidin more weakly than Acanthamoeba and rabbit skeletal muscle actin filaments due to a more rapid dissociation rate in spite of a significantly faster association rate constant. The higher dissociation rate constant and lower binding affinity of rhodamine phalloidin for S. cerevisiae actin filaments provide a quantitative explanation for the inefficient staining of yeast actin filaments, compared with that of rabbit skeletal muscle actin filaments [Kron et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4466-4470]. The temperature dependence of the rate constants was interpreted according to transition state theory. There is a small enthalpic difference (delta H++) between the ground states and the transition state. Consequently, the free energy of activation (delta G++) for association and dissociation of rhodamine phalloidin is dominated by entropic changes (delta S++). At equilibrium, rhodamine phalloidin binding generates a positive entropy change (delta S0). The rates of rhodamine phalloidin binding are independent of the pH, ionic strength, and filament length. Rhodamine covalently bound decreases the association rate and affinity of phalloidin for actin. The association rate constant is low for both phalloidin and rhodamine phalloidin because the filaments must undergo conformational changes (i.e. "breathe") to expose the phalloidin binding site [De La Cruz, E. M., & Pollard, T. D. (1994) Biochemistry 33, 14387-14392]. Raising the solvent microviscosity, but not the macroviscosity, dampens these conformational fluctuations, and phalloidin binding kinetics are inhibited. Yeast actin filaments bind rhodamine phalloidin more rapidly, suggesting that perhaps they are more flexible and can breathe more easily than rabbit or Acanthamoeba actin filaments.

Acanthamoeba↗
PubMed1997

Distribution and orientation of rhodamine-phalloidin bound to thin filaments in skeletal and cardiac myofibrils.

Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned myofibrils. TR-phalloidin staining of isolated skeletal myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2-micron dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in myofibrils, minimally permeabilized (briefly skinned) myofibrils, or myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal myofibrils with sarcomere lengths greater than 3.0 microns, staining was nearly uniform all along the actin filaments. Exogeneous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825-833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal myofibrils and nebulette can block phalloidin binding to cardiac thin filaments.

Actin Cytoskeleton↗
PubMed1987

What we have learned from phalloidin.

In contrast to popular opinion phallotoxins do not play a role in poisoning with Amanita phalloides when the fungi are ingested orally. All toxic properties of this mushroom are due to amatoxins which, in contrast to the phallotoxins, are absorbed upon ingestion. Nearly all experiments on intact animals were performed by parenteral injection of phalloidin and therefore, most of these are unsuitable for practical consideration. In the present survey, however, a series of important findings are discussed, which provide insight into various functions of liver cells. When present in the blood, phalloidin and other phallotoxins are selectively taken up by hepatocytes. No other types of cells are sensitive to the toxin. No extrahepatic tissue is primarily impaired by phalloidin. Phalloidin cannot be degraded by peptidases or by proteases occurring in animals. Phalloidin is therefore a useful model substance for studies on the uptake of cyclopeptides by liver cells. The carrier system responsible for the active uptake of phalloidin can also translocate antamanide and several cyclic modifications of somatostatin. Phallotoxins bind with high affinity to microfilamentous structures, in particular to F-actin [Govindan et al., Naturwissenschaften, 59 (1972) 521-522] whereas phallotoxins are not bound to the monomer (G-actin). With respect to the strong organotropism of phallotoxins, intravenously injected phalloidin binds preferentially to microfilamentous F-actin of hepatocytes. Phalloidin is therefore a tool for inactivation of microfilamentous functions specifically in liver cells, and is suitable as a prototype of a cholestatic agent. In perfused livers arrest of bile flow is the earliest effect seen after addition of the toxin. In cells from other tissues phalloidin is only toxic when applied by intracellular microinjection. Phalloidin poisoning has been often used as a model for liver damage in the testing of hepatoprotective drugs. This substance is, however, not useful for such studies since the mechanism of phalloidin poisoning is too specific for interpretation in the sense of general liver protection.

Actin Cytoskeleton↗
PubMed1995

Phalloidin unzips nebulin from thin filaments in skeletal myofibrils.

Fluorescent phallotoxins such as rhodamine-phalloidin take hours to bind uniformly to thin filaments of skeletal myofibrils, after fast initial binding to both ends of thin filaments. Observation of this process in skeletal and cardiac myofibrils and of the resulting re-distribution of nebulin using anti-nebulin antibody showed that: (1) rhodamine-phalloidin binds uniformly to actin in cardiac myofibrils within minutes, in contrast to skeletal myofibrils; (2) overnight pre-incubation of skeletal myofibrils with phalloidin results in uniform initial binding of rhodamine-phalloidin and a changed nebulin localization; (3) pre-incubation of skeletal myofibrils with Ca(2+)-calmodulin results in uniform initial binding of rhodamine-phalloidin; (4) the binding of rhodamine-phalloidin to actin in skeletal myofibrils is unidirectional, i.e. the fluorescence of incorporated rhodamine-phalloidin moves from the pointed ends where it is bound initially toward the barbed end at the Z-band; (5) the unidirectional binding of rhodamine-phalloidin results in redistribution of nebulin, i.e. the initial fluorescent bands associated with the epitopes of bound nebulin antibody change to a single band located close to Z-line. These results indicate that nebulin inhibits rhodamine-phalloidin binding to actin and suggests that the unidirectional rhodamine-phalloidin binding may be due to cooperative competitive binding, i.e. phalloidin 'unzips' nebulin starting from the pointed ends of the thin filaments.

Actins↗
PubMed2000

Secophalloidin and phalloidin-(S)-sulfoxide as contraction modifiers for comparative study of skeletal and cardiac muscles.

Phalloidin, a toxic product of the mushroom Amanita phalloides, binds specifically to F-actin resulting in strong stabilization of F-actin structure (for review, see; Wieland, 1986). Binding to a specific site on the muscle thin filament F-actin, phalloidin modifies contraction in a tissue specific manner. Phalloidin induced changes depend on functionally important parameters (thin filament activation, cross-bridge kinetics), indicating changes in essential steps of the contractile mechanism. Moreover, there is a different action with different phalloidin derivatives. Such properties make phallotoxins (phalloidin and its derivatives) powerful modifiers for muscle research (for review, see: Bukatina, 1996). Phalloidin-induced changes vary qualitatively with muscle types. In all types of skinned skeletal muscle preparations that have been studied (fast and slow muscles from evolutionarily distant animals), the most general effect of phalloidin is to cause a decrease in tension (Bukatina, Morozov, 1979; Alievskaya et al., 1987; Bukatina et al., 1993). In mammalian skeletal muscles, this decrease in tension may be followed by a slowly developing increase in tension. The resulting tension may considerably exceed the tension before phalloidin administration. In contrast, skinned cardiac muscle responds to phalloidin only by increasing isometric tension from the onset of the response. Moreover, the phalloidin response is completed in approximately one-tenth the time in cardiac muscle that it takes in skeletal muscle. These phalloidin effects in cardiac muscle result in an enhanced Ca2+ responsiveness (Boels, Pfitzer, 1992) with an increase in both the force at maximum Ca2+ activation and the Ca2+ sensitivity (Bukatina et al., 1995).

Animals↗
PubMed1975

[Phalloidin antagonists 4th communication: Thioctic acid, SH-compounds, rifampicin, choleretics, dexamethasone, estradiol, unspecific inhibitors, and ineffective compounds (author's transl)].

1. Thioctic acid used clinically in poisoning by A. phalloides, protected perfused livers and also isolated hepatocytes against phalloidin, when given in high concentrations. 2. Some SH-compounds like coenzyme A, dimercaprol, cysteine and cysteamine were found to be protective in different concentrations. 3. Rifampicin protects mice against lethal doses of phalloidin, and inhibits poisoning of isolated hepatocytes at low concentrations. 4. Some choleretic drugs like dehydrocholate, temoebilin (extr. cucumae xanth.), ethacrynic acid influenced phalloidin poisoning by inhibition of binding. 5. Doses of 0.2 to 4.0 mg dexamethasone added to 100 ml of perfusion medium did not protect perfused rat livers against 0.5 mg phalloidin. 6. Pretreatment of female rats with estrogens effected protection against phalloidin in vivo. The same procedure resulted in moderate decrease of phalloidin effects when the livers of pretreated animals were poisoned in vitro. In male rats estrogen pretreatment was less effective. Castration did not augment the protective effect. 7. Secophalloidin, a biologically inactive derivative, did not influence phalloidin poisoning in perfused livers, even when applied in excessive concentrations. 8. Concanavalin A, probably bound in the neighborhood of binding sites for phalloidin, did not protect perfused livers against phalloidin. 9 Diethyldithiocarbamate, a compound protecting livers against carbon tetrachloride and halothane, was ineffective in phalloidin poisoning. 10. Further protective actions of Evans blue, of some phenanthrolines and of EDTA are discussed. 11. Pretreatment of animals with hepatotoxic compounds (CCl4, CHCl3, cinchophen) decreased the toxicity of phalloidin in vivo. Possible mechanisms are discussed.

Animals↗
PubMed1993

Probing the phalloidin binding site of actin.

Phallotoxins form tight complexes with filamentous actin and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol-capturing moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys-374 next to the C-terminus and Cys-10, close to the N-terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP-G-actin. Phalloidin bound to Cys-374 stabilizes filaments against shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C-terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F-actin, however, the subsequent formation of a covalent linkage with Cys-374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization of filaments by phalloidin linked to Cys-374 a destabilizing effect was observed when phalloidin was attached to Cys-10. It appears that phalloidin located close to the N-terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxin is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phalloidin binding site of actin is distant from the N-terminus, but close to the C-terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C-terminus.

Actins↗
PubMed1992

Characterization of tetramethylrhodaminyl-phalloidin binding to cellular F-actin.

Fluorescent derivatives of phalloidin are widely used to measure filamentous actin (F-actin) levels and to stabilize F-actin. We have characterized the kinetics and affinity of binding of tetramethylrhodaminyl (TRITC)-phalloidin to rabbit skeletal muscle F-actin and to F-actin in lysates of rabbit polymorphonuclear leukocytes (PMNs). We have defined conditions where TRITC-phalloidin can be used to inhibit F-actin depolymerization and to quantify F-actin without prior fixation. By equilibrium measurements, the affinity of TRITC-phalloidin binding to rabbit skeletal muscle F-actin (pyrene labeled) or to PMN lysate F-actin was 1-4 x 10(-7) M. In both cases, the stoichiometry of binding was approximately 1:1. Kinetic measurements of TRITC-phalloidin binding to PMN lysate F-actin resulted in an association rate constant of 420 +/- 120 M-1 sec-1 and a dissociation rate constant of 8.3 +/- 0.9 x 10(-5) sec-1. The affinity calculated from the kinetic measurements (2 +/- 1 x 10(-7) M) agreed well with that obtained by equilibrium measurements. The rate with which 0.6 microM TRITC-phalloidin inhibited 0.1 microM pyrenyl F-actin depolymerization (90% inhibition in 10 sec) was much faster than the rate of binding to pyrenyl F-actin (less than 1% bound in 10 sec), suggesting that phalloidin binds to filament ends more rapidly than to the rest of the filament. We show that TRITC-phalloidin can be used to measure F-actin levels in cell lysates when G-actin is also present (i.e., in cell lysates at high concentrations) if DNase I is included to prevent phalloidin-induced polymerization.

Actins↗
PubMed1994

Effect of phalloidin on the ATPase activity of striated muscle myofibrils.

Phalloidin was shown to increase the ATPase activity and Ca2+ sensitivity of both bovine cardiac and rabbit psoas myofibrils when assayed in a solution containing 50 mM KCl, 100 mM MOPS (pH 7.0), 2 mM MgCl2, 1 mM ATP, 2 mM EGTA, and varying concentrations of Ca2+ (temperature 21-22 degrees C). The phalloidin effect in cardiac myofibrils developed over a time course of several minutes in the presence of 50 microM phalloidin. Relative increase of ATPase activity was maximal at pCa 8 and decreased with decrease in pCa. In cardiac myofibrils the increase was about 70% at pCa 8 and 20% at pCa 4 following 20-30 min pre-incubation with 2 microM or 50 microM phalloidin. The effect persisted after excess phalloidin was washed out. The increase in Ca2+ sensitivity was approximately 0.15 pCa units. For skeletal myofibrils treated with 2 microM phalloidin all changes were considerably less than those seen with cardiac myofibrils and the changes were even less when the myofibrils were exposed to 50 microM phalloidin. These results show that when specifically bound to actin, phalloidin can change the kinetic parameters of the cross-bridge cycle and may also alter the Ca2+ sensitivity of the contractile system. The effects of phalloidin seem to vary with muscle type.

Actins↗