Journal ArticleDOI
Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization.
TLDR
It is established that pyrene actin fluorescence is a valid assay for actin polymerization that is more sensitive than any other current assay.Abstract:
The fluorescence of pyrene-labelled actin is much higher after polymerization. We have characterized in detail the polymerization properties of pyrene actin and report that native and pyrene actin are identical using the following criteria: (1) the time course of polymerization; (2) the elongation rate constants; (3) the intrinsic viscosity; and (4) the critical concentration. Native and pyrene actin copolymerize. Fluorescence of polymerized pyrene actin is 7-10 times higher than monomer. The fluorescent signal is proportional to polymer weight concentration and is insensitive to filament length distribution. Bleaching can be minimized by appropriate filters to allow continuous monitoring of signal. Measurements do not influence polymerization kinetics. This establishes that pyrene actin fluorescence is a valid assay for actin polymerization that is more sensitive than any other current assay.read more
Citations
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Journal ArticleDOI
Lifeact: a versatile marker to visualize F-actin
Julia Riedl,Alvaro H. Crevenna,Kai Kessenbrock,Jerry Haochen Yu,Dorothee Neukirchen,Michal Bista,Frank Bradke,Dieter E. Jenne,Tad A. Holak,Zena Werb,Michael Sixt,Roland Wedlich-Söldner +11 more
TL;DR: Lifeact, a 17-amino-acid peptide, is described, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.
Journal ArticleDOI
Mechanism of Blebbistatin Inhibition of Myosin II
TL;DR: The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myOSin II isoforms, whereas the stabilization of a specificMyosin intermediate confers a great potential in structural studies.
Journal ArticleDOI
Antagonism between Ena/VASP Proteins and Actin Filament Capping Regulates Fibroblast Motility
James E. Bear,Tatyana Svitkina,Matthias Krause,Dorothy A. Schafer,Joseph Loureiro,Geraldine A. Strasser,Ivan V. Maly,Oleg Y. Chaga,John A. Cooper,Gary G. Borisy,Frank B. Gertler +10 more
TL;DR: It is concluded that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia.
Journal ArticleDOI
Fluorogenic probes for live-cell imaging of the cytoskeleton.
Gražvydas Lukinavičius,Luc Reymond,Elisa D’Este,Anastasiya Masharina,Fabian Göttfert,Haisen Ta,Angelika Güther,Mathias Fournier,Stefano Rizzo,Herbert Waldmann,Claudia Blaukopf,Christoph Sommer,Daniel W. Gerlich,Hans-Dieter Arndt,Stefan W. Hell,Kai Johnsson +15 more
TL;DR: Far-red, fluorogenic probes are introduced that reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.
Journal ArticleDOI
Mechanism of N-Wasp Activation by Cdc42 and Phosphatidylinositol 4,5-Bisphosphate
TL;DR: A signaling pathway in Xenopus extracts leading from PI(4,5)P2 to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex is delineated and it is found that the previously described physical interaction between the NH2- terminal domain and the COOH-terminal effector domain is a regulatory interaction.
References
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Journal Article
Protein Measurement with the Folin Phenol Reagent
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
Journal ArticleDOI
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
Journal ArticleDOI
Fluorimetry Study of N‐(1‐Pyrenyl)iodoacetamide‐Labelled F‐Actin
Tsutomu Kouyama,Koshin Mihashi +1 more
TL;DR: The results suggest that binding of heavy meromyosin to the protomer of F-actin alters the local structure of the protomers towards a G-Actin-like one.
Journal ArticleDOI
Head to tail polymerization of actin.
TL;DR: It is found that the net result of four association and four dissociation steps is a lengthening of the filament by one protomer at one end and a corresponding shortening at the other.
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