phalloidine and tetramethylrhodamine

The actin cytoskeleton is a dynamic filamentous network whose formation and remodeling underlies the fundamental processes of cell motility and shape determination. To serve these roles, different compartments of the actin cytoskeleton engage in forming specific coupling sites between neighbouring cells and with the underlying matrix, which themselves serve signal transducing functions. In this review, we focus on methods used to visualise the actin cytoskeleton and its dynamics, embracing the use of proteins tagged with conventional fluorophores and green fluorescent protein. Included also is a comparison of cooled CCD technology, confocal and 2-photon fluorescence microscopy of living and fixed cells, as well as a critique of current procedures for electron microscopy.

Topics: Actins; Animals; Cells, Cultured; Chickens; Cytoskeleton; Fishes; Fluorescent Dyes; Freezing; Green Fluorescent Proteins; Immunohistochemistry; Keratinocytes; Luminescent Proteins; Microscopy; Phalloidine; Rhodamines; Staining and Labeling; Structure-Activity Relationship; Tissue Fixation

The general morphology of cuticle-lined internal genitalia and oviduct is analyzed in intact females of the phytophagous mites, Loboquintus subsquamatus and Trisetacus cf bagdasariani (Acari: Eriophyoidea) using tetramethylrhodamine B isothiocyanate-phalloidin, three anaesthetics (magnesium sulphate, lidocaine and CO2-enriched water) and confocal laser scanning microscopy (CLSM). This is the first protocol adopted for CLSM studying musculature of mites. Revision of the previous terminology of eriophyoid internal genitalia from Nuzzaci and Alberti (Eriophyoid mites: their biology, natural enemies and control. World crop pests 6. Elsevier, Amsterdam, pp 101-150, 1996) resulted in the refinement of the terms "distal oviduct", "genital chamber" and "spermatheca". Relative position of the elements of cuticle-lined internal genitalia is discussed and a generalized 3D model and animation (available on-line as supplementary material) of eriophyoid genital apparatus are provided. The wall of eriophyoid oviduct contains strong longitudinal muscles attached to the cuticle genital chamber with folded walls. When the egg is being extruded by contraction of the oviduct muscles, it forms lobes corresponding to the internal topography of the oviduct and genital chamber; these lobes invaginate inward from the gonopore, resulting in the "flower-shaped" figures rarely observed in slide-mounted mites. Gnathosomal muscles (cheliceral muscles and extrinsic muscles of palps) and opisthosomal muscles D1 of Loboquintus mites are attached to the three posterior depressions near the rear prodorsal shield margin. Prospects of CLSM approach for studying different aspects of mite morphology are briefly discussed.

Topics: Animals; Carbon Dioxide; Female; Lidocaine; Magnesium Sulfate; Microscopy, Confocal; Mites; Oviducts; Phalloidine; Rhodamines

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.

Topics: Actins; Adenosine Triphosphatases; Animals; Filtration; Hydrolysis; Kinetics; Magnesium Chloride; Phalloidine; Phosphates; Potassium Chloride; Rabbits; Rhodamines

High concentrations of Tris are effective in dissociating actin-containing complexes, such as the red cell membrane cytoskeleton. A preparative procedure for red cell actin is based on the dissociation of the membrane skeletal complex in a buffer containing 1 M Tris hydrochloride, followed by gel filtration chromatography in the same medium. The actin is recovered as the monomer and is fully native, as judged by its critical concentration of polymerization, inhibition of DNase I, stimulation of myosin ATPase, and the appearance in the electron microscope of filaments, both bare and decorated with heavy meromyosin, and of magnesium ion-induced paracrystals. The Tris solution causes rapid depolymerization of F-actin with no denaturation, and the solution of monomeric actin in this medium is stable for many weeks in the cold; concentrated Tris is more reliable than guanidinium chloride for the depolymerization of F-actin in the estimation of total actin concentration by the DNase I inhibition assay.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Biophysical Phenomena; Biophysics; Cations, Divalent; Chromatography; Cytoskeleton; Deoxyribonuclease I; DNA; Erythrocytes; Fluorometry; Guanidine; Guanidines; Humans; In Vitro Techniques; Microscopy, Electron; Muscle, Skeletal; Myosin Subfragments; Nucleotides; Phalloidine; Polymers; Pyrenes; Rabbits; Rhodamines; Tromethamine

We describe an assay for measuring both the extent and kinetics of the severing of F-actin, based on the enhanced fluorescence emission of tetramethylrhodamine-phalloidin bound to F-actin. The enhanced fluorescence is lost after exposure to active gelsolin by displacement of the phalloidin from actin during severing. This assay requires small amounts of actin and gelsolin, can be used to measure reaction times ranging from 1 to 10(3) s, and does not require covalent modification of either protein. The rate of fluorescence loss is linearly related to the concentrations of both actin and gelsolin. However, the apparent rate constant of the reaction is highly dependent on the divalent cation concentration, varying between 10(4) and 10(6) M-1 s-1 when the [Ca2+] varies between 20 and 200 microM. Addition of Mg2+ increases the apparent rate constant at equivalent Ca2+ concentration. These results suggest that in vitro the rate-limiting step in the severing process is the activation of gelsolin by the binding of Ca2+ and Mg2+ to several low affinity (Kd approximately 100 microM) sites on gelsolin. While activation of gelsolin by Ca2+ is a slow process, the binding and severing of actin occurs at a rate approaching the diffusion limit.

Topics: Actins; Animals; Calcium; Coloring Agents; Gelsolin; Kinetics; Magnesium; Phalloidine; Rabbits; Rhodamines; Spectrometry, Fluorescence

When rat peritoneal mast cells were treated with the potent histamine releaser compound 48/80 in the presence of tetramethylrhodamine-labeled G-actin, the fluorescent G-actin particles were bound to the surface of extruded granules and to the cell surface. When rhodamine-phalloidin was incorporated into permeabilized rat mast cells in a Ca2+-free medium, rhodamine fluorescence was observed on the cell surface accompanied by serpentine ridges which appeared in the resting state. After perfusion with a cytosol-like solution containing Ca2+, rhodamine fluorescence appeared on the cell surface as a distinct network formation. In some cases, circular fluorescences which appeared to surround the extruded pores were observed in the cell membrane. These findings indicate the existence of actin filaments in the cell membrane and/or subplasmalemmal network. In whole-mount preparations, the granules were surrounded very densely with microfilaments of various widths. After exposure to compound 48/80, granules protruding through the cell membrane were wrapped in many filaments. The extruded granules located in the periphery of the cells were connected by many filamentous structures and disruptions in the middle of these connections were occasionally observed. In some cases, circular configurations of microfilaments were observed at the bottom of the extruded granules and in others dense gatherings of microfilaments were seen just beneath the granules, as if the latter were being pushed up and out of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actin Cytoskeleton; Actins; Animals; Cytoskeleton; Exocytosis; Fluorescent Dyes; Macromolecular Substances; Male; Mast Cells; Microscopy, Electron, Scanning; p-Methoxy-N-methylphenethylamine; Peritoneal Cavity; Phalloidine; Rats; Rats, Inbred Strains; Rhodamines

A fluorescent phallotoxin with high photostability, tetramethylrhodaminyl-phalloidin (Rh-phalloidin), has been prepared. The affinity of this compound to rabbit muscle actin has been determined to be about 6 times lower than that of phalloidin. In freshly isolated hepatocytes the internalized fluorescent toxin stains the cellular actin. In contrary, there is no actin staining visible in cultured hepatocytes. Short-term cultured hepatocytes (5 h of culturing) incorporate the toxin by endocytosis; it is kept sealed in the endocytotic vesicles, which are usually found accumulated at the sites where cells touch after reaggregation.

Topics: Actins; Animals; Cells, Cultured; Endocytosis; Liver; Male; Oligopeptides; Phalloidine; Rats; Rats, Inbred Strains; Rhodamines