phalloidine and pyrene

The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy.

Topics: Actin Cytoskeleton; Cell Movement; Cytokinesis; Cytoplasm; Endocytosis; Humans; Kinetics; Microscopy, Fluorescence; Molecular Biology; Phalloidine; Polymerization; Pyrenes

Proteolytic cleavage of actin between Gly(42) and Val(43) within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess. The most significant effect of the cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration at saturating concentrations of S1, is strong inhibition of association of G-actin-S1 complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln(41) indicate substantial inhibition of the initial association of G-actin-S1 into longitudinal dimers. The data provide experimental evidence for the critical role of D-loop conformation in both longitudinal and lateral, cross-strand actin-actin contact formation in the nucleation reaction. Electron microscopic analysis of the changes in filament-length distribution during polymerization of actin by S1(A1) and S1(A2) suggests that the mechanism of S1-induced polymerization is not substantially different from the nucleation-elongation scheme of spontaneous actin polymerization.

Topics: Actins; Adenosine Triphosphate; Animals; Biophysics; Cadaverine; Deoxyribonuclease I; Dimerization; Dose-Response Relationship, Drug; Glycine; Kinetics; Light; Magnesium; Microscopy, Electron; Myosin Subfragments; Phalloidine; Polymers; Protein Binding; Protein Conformation; Protein Isoforms; Protein Structure, Tertiary; Proteins; Pyrenes; Rabbits; Scattering, Radiation; Spectrometry, Fluorescence; Temperature; Time Factors; Valine

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Amino Acid Sequence; Animals; Ca(2+) Mg(2+)-ATPase; Fluorescent Dyes; Models, Molecular; Molecular Sequence Data; Myosin Type I; Nucleotides; Phalloidine; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrenes; Rats; Recombinant Fusion Proteins; Sequence Alignment

The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is <<1 microM at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a Kd below 0.1 microM. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for Kd's as low as 10 nM and we demonstrate that the Kd's can be estimated using only microgram quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.

Topics: Actins; Actomyosin; Adenosine Triphosphate; Animals; Binding, Competitive; Dose-Response Relationship, Drug; Fluorometry; Myosin Subfragments; Myosins; Phalloidine; Pyrenes; Rabbits

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