Phalloidins

Ph The phenyl group, CfiHs-. pH See hydrogen-ion concentration, phaeophorbide See chlorophyll, phaeophytin See chlorophyll, phalloidins See amanitins. 

Zigmond, 1988). The ATP-hydrolysis that accompanies actin polymerization, ATP —> ADP + Pj, and the subsequent release of the cleaved phosphate (Pj) are believed to act as a clock (Pollard et ah, 1992 Allen et ah, 1996), altering in a time-dependent manner the mechanical properties of the filament and its propensity to depolymerize. Molecular dynamics simulations suggested a so-called back door mechanism for the hydrolysis reaction ATP ADP - - Pj in which ATP enters the actin from one side, ADP leaves from the same side, but Pj leaves from the opposite side, the back door (Wriggers and Schulten, 1997b). This hypothesis can explain the effect of the toxin phalloidin which blocks the exit of the putative back door pathway and, thereby, delays Pi release as observed experimentally (Dancker and Hess, 1990). 

Dancker and Hess, 1990] Dancker, P., and Hess, L. Phalloidin reduces the release of inorganic phosphate during actin polymerization. Biochira. Biophys. Acta. 1035 (1990) 197-200... 

Phalloidin and phallacidin are cyclic peptides from the mushroom Amanita phalloides that stabilize F-actin. Phalloidin binds to residues 114-118 of an actin protomere and blocks nucleotide exchange without interfering with nucleotide hydrolysis. It enhances the rate of nucleation as well as that of elongation. It slowly penetrates the cell membrane and is used for immunocytochemical localization of F-actin. 

In contrast, jasplakinolide, a cyclodepsipeptide from the marine sponge Jaspis johnstoni, rapidly penetrates the cell membrane. It competes with phalloidin for F-actin binding and has a dissociation constant of approximately 15 nM. It induces actin polymerization and stabilizes pre-existing actin filaments. Dolastatin 11, a depsipeptide from the mollusk Dolabella auricu-laria, induces F-actin polymerization. Its binding site differs from that of phalloidin or jasplakinolide. 

Cytokinesis (cell division) in animal cells involves the progressive formation in telophase of a furrow between the two daughter cells in the equator of the mitotic spindle. Immunofluorescent staining of the cortical cytoplasm at the site of the contraction ring reveals an abundance of actin as well as myosin, a-actinin, and filamin (Fishkind and Wang, 1995). Cytokinesis is highly sensitive to actin-myosin inhibitors such as cytochalasin and phalloidin. 

Fig. 20.1. Confocal images of whole mounts of the ovijector region of A suum stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to show muscle and with an anti-RFamide antiserum coupled to fluorescein isothiocyanate (FITC) to show FaRPergic nerves. (A) Main ventral nerve cord encircles opening of ovijector where it meets the body wall and is immunopositive for FaRPs. (B) Flat-fixed preparation of the ovijector showing circular muscles and tracts of parallel FaRPergic nerves (arrows). (C) Detail of the circular muscle of ovijector and associated nerves (arrows). (D) A FaRPergic cell body is localized in the ventral nerve cord at junction with ovijector and provides innervation to ovijector muscle.

Wieland, T., et al. Identity of hepatic membrane transport systems for bile salts, phalloidin, and antamanide by photoaffinity labeling. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 5232-5236. 

Schmit A-C, Lambert A-M. Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells. Plant Cell 1990 2 129-138. 

Cleary AL. F-actin redistributions at the division site in living Tradescantia sto-matal complexes as revealed by microinjection of rhodamine-phalloidin. Protoplasma 1995 185 152-165. 

Doris FP, Steer MW. Effects of fixatives and permeabilisation buffers on pollen tubes implications for localisation of actin microfilaments using phalloidin staining, Protoplasma, 1996 195 25-36. 

When staining with any of the fluorescent phalloidins, dilute 10 pL methanolic stock solution into 400 pi PBS. To reduce nonspecific background staining with these conjugates, add 1% bovine serum albumin (BSA) to the staining solution. It may also be useful to preincubate fixed cells with PBS containing 1 % BSA. 

Apply the phalloidin-staining solution for 20 min at room temperature (generally, any temperature between 4°C and 37°C is suitable). 

Note Be aware that phalloidin labeling is incompatible with alcohol treatment. 

Labeling with fluorescent phalloidins may be combined with immunostaining. In this case, the phalloidin-staining solution can be applied in a mixture with fluores-cently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell. 

Several experimental approaches can be employed to determine the pool sizes of polymerised and non-polymerised actin. Firstly, the enzyme DNAse I is inhibited by monomeric (G) actin, but not by polymerised (F) actin. Secondly, polymerised actin can be directly visualised by use of fluorescent derivatives of phalloidin, a cyclic peptide isolated from the toadstool Amanita phalloides that selectively binds to polymerised actin with high affinity. 

Sampath P and Pollard TD [1991] Effects of cytochalasin, phalloidin and pH on the elongation of actin. Biochemistry 30 1973-1980... 

Labeled phallotoxines (phalloidins) The bicyclic peptides isolated from Amanita phalloides mushroom bind selectively to F-actin in nanomolar concentrations. They have advantages over antibodies for actin labeling... 

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