Phallotoxin and actin binding assay by fluorescence enhancement.

The fluorescence of five fluorophores conjugated to phallotoxins was found to be specifically enhanced upon binding to F-actin in a polymerizing buffer. Rhodamine phalloidin had the greatest fluorescence enhancement of ninefold. The fluorescence titration of rhodamine phalloidin by actin was shown to be consistent with stoichiometric binding. The fluorescence enhancement of rhodamine phalloidin at 5 microM is linearly related to F-actin concentrations up to 2 microM and therefore can be used as an easy means of F-actin quantitation. In a competition assay, other phallotoxins reduce the fluorescence enhancement that results from the binding of rhodamine phalloidin to polymerized actin. This reduction also permits a convenient measurement of the binding constants of any competing phallotoxins.     

Localization of actin in normal human hepatocytes using fluorescent phallotoxins and immunohistochemical amplification.

Two different methods, fluorescent phallotoxins and immunohistochemical amplification systems were used to visualize actin in normal human hepatocytes. With fluorescent phallotoxins (NBD-phallacidin or rhodamine phalloidin), F-actin was distributed along the plasma membranes and at the bile canaliculi. With immunohistochemical methods (biotin-avidin, biotin-streptavidin, silver enhancement), actin was found at the same level, however a cytoplasmic staining was observed and discussed as G-actin localization.     

Comparison of cryofixation and aldehyde fixation for plant actin immunocytochemistry: aldehydes do not destroy F-actin

For walled plant cells, the immunolocalization of actin microfilaments, also known as F-actin, has proved to be much trickier than that of microtubules. These difficulties are commonly attributed to the high sensitivity of F-actin to aldehyde fixatives. Therefore, most plant studies have been accomplished using fluorescent phallotoxins in fresh tissues. Nevertheless, concerns regarding the questionable ability of phallotoxins to bind the whole complement of F-actin necessitate further optimization of actin immunofluorescence methods. We have compared two procedures: (1) formaldehyde fixation and (2) rapid freezing and freeze substitution (cryofixation), both followed by embedding in low-melting polyester wax. Actin immunofluorescence in sections of garden cress (Lepidium sativum L.) root gave similar results with both methods. The compatibility of aldehydes with actin immunodetection was further confirmed by the freeze-shattering technique that does not require embedding after aldehyde fixation. It appears that rather than aldehyde fixation, some further steps in the procedures used for actin visualization are critical for preserving F-actin. Wax embedding, combined with formaldehyde fixation, has proved to be also suitable for the detection of a wide range of other antigens.     

Phallotoxins as an instrument for studying the intramyofibrillar regulation of muscle contraction

The contraction of myofibrils is regulated by the coordinated action of Ca2+ and myosin cross-bridges. Phallotoxins induce a variety of specific changes in myofibrillar functioning and are therefore a potentially valuable tool for muscle research. There are two greatly differing classes of drugs among phallotoxins: (1) recently discovered secophalloidin and its derivatives. The unique property of secophalloidin is muscle activation without Ca2+, possibly by direct influence on actomyosin interaction. These drugs seem to be especially useful for studying the role of cross-bridges in muscle regulation; (2) the phalloidin group, which includes the majority of phallotoxins. When binding to the phalloidin site on F-actin, they cause muscle-specific changes. In cardiac muscle they work as Ca2+ sensitizers, increasing both the maximal force and Ca2+ sensitivity. An advantage of these drugs is that the target site is known, which allows one to unravel the sequence of molecular events leading to increased contractile function. Presumably, the complex effect of phalloidin in skeletal muscle is related to the disturbance of the actin-nebulin interaction, which may help to clarify the role of nebulin in the regulation contraction.     

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).     

Analogs of viroidin. Synthesis of four virotoxin-like F-actin binding heptapeptides with one less hydroxyl group in the dihydroxy-proline ring

The vitroxins, toxic cyclic heptapeptides from Amanita virosa fungi, contain a 3,4-dihydroxy-L-proline, an imino acid scarcely accessible in greater amounts. Therefore in the syntheses of the analogs described this imino acid was replaced by 4-cis-hydroxy-L-proline, a component of the analogously toxic phallotoxins. The following syntheses are described: 1a = cyclo (L-alanyl-D-threonyl-D-seryl-L-4-cis-hydroxy-L-prolyl-L-alanyl-2- methylthio-L-tryptophyl-L-leucyl), 1b = 1a but with 2-methylsulfonyl-L-tryptophan in position 6, 2a = cyclic (L-valyl-D-threonyl-D-seryl-4-cis-hydroxy-L-prolyl-L-alanyl-2- methylthio-L-tryptophyl-hydroxyl-L-leucyl), and 2b = 2a, but with 2-methylsulfonyl-L-tryptophan in position 6. In a test for binding strength to rabbit muscle F-actin 2b and 2a exhibited about 40% of that of demethylphalloin. Analogs 1a and 2b have not been tested.     

Tryptathionine bridges in peptide synthesis

The tryptathionine linkage is a crosslink formed between tryptophan and cysteine. This feature is characteristic of the bicyclic peptides: the phallotoxins and the amatoxins. These peptides both bind to protein folds of their respective targets (F-actin and RNA pol II, respectively) with extremely high affinities. Studies on these peptides have shown that the tryptathionine crosslink is essential for this binding affinity. Tryptathionines have been investigated for many years and several syntheses exist for their formation. In this review, we report on the various methodologies employed in tryptathionine synthesis, and discuss some of the advantages and disadvantages associated with each of them.     

A protein-binding assay for phallotoxins using muscle actin

The high affinity of phallotoxins to filamentous actin (K_D = 3.6 × 10⁻⁸m) has been used to assay small amounts of phallotoxins. The limit of detection was as low as 160 ng of phallotoxins. This is 50 times lower than the limit of all other procedures known so far. The assay was successfully applied to the analysis of phallotoxins in crude extracts of Amanita pholloides mushrooms.     

Muscle development in dendrobranchiate shrimp, with comparison with Artemia

The muscle pattern of malacostracan and entomostracan crustacean nauplius larvae was compared using fluorescent phallotoxins. In the dendrobranchiate malacostracan Sicyonia ingentis, F-actin staining was first detected in limb setae at 12 h, likely within sensory nerves. Staining of F-actin was detected in the trunk at 15 h and grew into the naupliar limbs. Sarcomeres were detected at 19 h, identifying the structures as extrinsic limb muscles. The extrinsic limb muscles enlarged but retained their general pattern during the later nauplius stages. Longitudinal trunk muscles and circumferential visceral muscle (VM) developed in the post-naupliar region during nauplius instars 4 and 5, at the time when the gut also formed. In the anostracan branchiopod Artemia salina, the newly hatched nauplius contained an extensive system of extrinsic and intrinsic limb muscles. The gut was almost complete at hatching, along with its associated circumferential VM. Muscles similar in position and structure could be identified in nauplii from the two taxa, but different anatomical origins of extrinsic muscles were evident. Whether the naupliar limb muscles are homologous in malacostracans and branchiopods remains an open question. The strong musculature of the dendrobranchiate naupliar limbs correlates with the use of all three pairs of limbs for swimming.     

Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures

Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis. We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis. Uptake of phallotoxins and [14C]cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin. Uptake of bovine lactoperoxidase strictly depends on external Ca2+, was insensitive to A23197 and was not inhibited by monensin. No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes. However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase. Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up. In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes. The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization. As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.     

The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.     

Inorganic phosphate regulates the binding of cofilin to actin filaments

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers. These changes transform the nucleotide cleft of F-actin to G-actin-like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility.     

Some perspectives on the viscosity of actin filaments

Measurements of the dynamic viscosity of various actin filament preparations under conditions of low and controlled shear: (a) confirm the shear rate dependence of F-actin viscosities and show that this dependence obeys the power law relationship observed for entangled synthetic polymers; (b) permit estimation of the extent to which shear artifact amplifies changes in the apparent viscosity of F-actin measured in a falling ball viscometer; (c) show that gel-filtration chromatography of actin and the addition of cytochalasin B to F-actin bring about small (20-40%) changes in the viscosity of the F-actin solutions. These variations are consistent with alterations in the actin-binding protein concentrations required for incipient gelation, a parameter inversely related to average filament length. Therefore: (a) changes in the viscosity of F-actin can be magnified by use of the falling ball viscometer, and may exaggerate their biological importance; (b) chromatography of actin may not be required to obtain meaningful information about the rheology of actin filaments; (c) changes in actin filament length can satisfactorily explain alterations in F-actin viscosity exerted by cytochalasin B and by chromatography, obviating the need to postulate specific interfilament interactions.     

我国28种鹅膏菌主要肽类毒素的检测分析*

利用高效液相色谱(HPLC)技术对产于我国的28种鹅膏菌的主要肽类毒素(鹅膏毒肽和鬼笔毒肽)进行了检测分析,并和采于欧洲(德国)的毒鹅膏Amanita phalloides作对照,结果表明,3种东亚所特有的鹅膏菌(灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种)和欧洲毒鹅膏所含毒素种类多、含量高,其子实体菌盖部位主要毒素总量分别达到12583.7μg/g、8152.6μg/g、1058.2μg/g、7456.2μg/g干重子实体,这4种鹅膏菌可称之为剧毒鹅膏菌。其它25种鹅膏菌中有10种检测出含有微量鹅膏毒肽,含量在19.5μg/g-151.2μg/g之间。在4种剧毒鹅膏菌中,子实体组织部位不同,毒素含量以及鹅膏毒肽和鬼笔毒肽在其中的分布也不一样,菌盖中的毒素含量最高,菌柄的毒素含量次之,菌托中的毒素含量最低;对于灰花纹鹅膏、致命鹅膏和黄盖鹅膏白色变种,无论在菌盖、菌柄和菌托中,鹅膏毒肽类毒素的含量都高于鬼笔毒肽类毒素,尤其以α-amanitin的相对含量最高;而在欧洲毒鹅膏中,菌盖、菌柄和菌托中都以鬼笔毒肽为主,尤其以phallacidin的相对含量最高,并且从菌盖至菌柄到菌托,鬼笔毒肽的相对含量依次增加。     

Levels of filamentous and globular actin in Chinese hamster ovary cells throughout the cell cycle

Synchronous Chinese hamster ovary (CHO) cells were obtained by mitotic selection and the levels of globular (G) actin, filamentous (F) actin, and cytoskeletal-associated F-actin were determined as cells progressed through the cell cycle. Total actin levels remained quite constant when expressed as a percent of the total protein. An increase in F-actin occurred upon plating the mitotic cells, but this increase was shown to be a result of attachment to the substratum, since cells which remained attached during the second mitosis failed to show these changes. No large variation in the levels of either F-actin or cytoskeletal-associated F-actin occurred throughout the cell cycle. Therefore, changes in the morphology of the CHO cells which are accompanied by a reorganization of actin-containing microfilaments during the cell cycle are not accompanied by significant changes in the size of the monomeric actin pool.     

Interaction of the enzymes of cellular energy support with the F-actin of the thin filaments of muscle fiber. I. The binding of lactate dehydrogenase with F-actin induces changes in the structural state of the components of the complex

A study was made of changes in F-actin conformation occurring in a myosin-free single ghost fibre induced by the binding of glycolytic enzyme lactate dehydrogenase (LDG) to F-actin. The formation of the complex between LDG and F-actin induces changes in the parameters of the intrinsic (tryptophan) and extrinsic (rodominil--phalloin) polarized fluorescence of F-actin of the ghost muscle fibre. It is found that LDG stimulates Mg2+-ATPase of actomyosin in solution. It is assumed that the coupling of energy-providing mechanism with that of muscle contraction may be accomplished through the conformation changes in F-actin.     

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin

F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.     

The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin

The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.     

Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.     

Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8–10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.