Detection of actin assembly by fluorescence energy transfer.

Abstract

A fundamental problem in cell motility is to define the state of assembly and interaction of the major cytoskeletal and contractile proteins both in vitro and in vivo . One example is the role played by actin assembly and disassembly during motile events . To date, actin assembly has usually been assayed indirectly both in vitro and in vivo. Experiments using viscosity, light scattering, and electron microscopy yield results that could be interpreted in several ways (2,10,12,13,20,44; see Taylor and Condeelis [32] and Uyemura and Spudich [37] for reviews) . Furthermore, more direct measurements of assembly using sedimentation assays (3,25) can only be applied in vitro. Therefore, techniques must be developed that would yield direct evidence for the state of actin assembly and interaction with specific molecules even in living cells.Fluorescence spectroscopy has already generated some information in vitro on the assembly of actin (4, 6, 40), the structure of actin (14,15,19,21,31), and the interaction of actin with associated proteins (6,23,24).I Recently, the technique of molecular cytochemistry (33,40; see Taylor and Wang [39] for a review) has opened the possibility of making spectroscopic measurements of fluorescently labeled actin in living cells. Actin labeled with 5-iodoacetamidofluorescein has been studied initially, because the site of labeling is known, the extinction coefficient is high, and it is excited by a relatively long wavelength of light (495 nm), which minimizes autofluorescence and cellular damage (40) .Dr. P. Detmers kindly provided D. L. Taylor with a copy of her manuscript (6) before publication. ABSTRACTFluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide) . Donor-labeled actin and acceptor-labeled actin were coassembled . The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is -35 A, which means that this site is located near the outer surface of the filament . The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.Fluorescence energy transfer (7, 28) is a powerful technique for studying the structure and dynamics of molecular...

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

“…1b) of a residue in the actin filament (15). In the present study, FRET experiments were carried out to determine the radial coordinate of Cys 374 at 22°C.…”

“…In these calculations it was also assumed that the efficiency of transfer occurring between the donor and acceptors located in more distant monomers was negligible. Accordingly, if we have a radial coordinate of r, the individual distances (R n ) can be calculated as follows (15).…”

“…1b). In earlier studies this method was applied to determine the radial coordinate of a number of labeled amino acids in the actin filament (15)(16)(17)(18)(19)(20)(21)(22). FRET spectroscopy was also * This work was supported by the Hungarian Academy of Sciences, National Research Foundation OTKA Grants T032700, T023209, D32813, and F020174, Ministry of Education FKFP Grant 0463/99, and the Ministry of Health (202/99).…”

“…1b). In earlier studies this method was applied to determine the radial coordinate of a number of labeled amino acids in the actin filament (15)(16)(17)(18)(19)(20)(21)(22). FRET spectroscopy was also used to characterize the flexibility of the protein matrix between a donor and an acceptor molecule (23) and to study the dynamic properties of the actin filament (24).…”

“…Accordingly, the application of fluorescence spectroscopy seems to be reasonable for better understanding the biological importance of the nucleotide dependence of filament flexibility. A fluorescence resonance energy transfer (FRET) 1 method first described by Taylor et al (15) allows one to determine the radial coordinate (which is defined as the distance between the residue and the longitudinal filament axis) of a labeled side chain within the filament (Fig. 1b).…”