On the elastic properties of tetramethylrhodamine F-actin

doi:10.1016/S0301-4622(01)00198-3

Biophysical Chemistry

Volume 92, Issue 3, 18 September 2001, Pages 201-207

https://doi.org/10.1016/S0301-4622(01)00198-3 Get rights and content

Abstract

(Iodoacetamido)tetramethylrhodamine disrupts F-actin. At the 1:1 fluorophore to actin (as monomer) ratio approximately 80% of the protein becomes non-sedimentable. The fluorescent, non-sedimentable actin copolymerizes with G-actin to yield fluorescent filaments. The tensile strength of these filaments changes with the ratio of the fluorescent non-sedimentable actin to the G-actin, being 1.6 pN, 2.9 pN and 3.6 pN at the 1/4, 2/3 and 1/1 ratios, respectively. These tensile strengths are approximately two orders of magnitude lower than those obtained by decoration of F-actin with phalloidin.

Introduction

The laser trap is an excellent technique which allows in vitro measurements of picoNewton forces and of nanometer steps. When applied to the study of contractile events, phalloidin F-actin (rhodamine phalloidin F-actin) is used as a substitute for the actin filament [1], [2], [3], [4], [5], [6], [7]. Unfortunately rhodamine phalloidin F-actin is a very poor substitute for native F-actin because it displays a much lower critical concentration, a higher tensile strength and different osmotic properties. The critical concentration of phalloidin F-actin is at least one order of magnitude lower than that of F-actin [8]. Critical concentration (i.e. the dissociation constant of the elongation reaction) is the main determinant of the free energy of the monomer–monomer interaction, thus of the tensile strength of the actin filament. As a matter of fact, critical concentration and tensile strength are inversely related [9], and tensile strength of phalloidin F-actin is much larger than of F-actin. Furthermore, phalloidin F-actin and F-actin display distinctly different osmotic properties [10]. It thus appears quite unreasonable to use phalloidin F-actin to study the mechano-elastic properties of F-actin. With the aim to overcome this drawback, we now address our attention to tetramethylrhodamine-F-actin as a possible fluorescent, bona fide substitute for F-actin. We report here that fluorescent actin filaments are obtained by copolymerization of tetramethylrhodamine-labeled, non-sedimentable actin with G-actin. Under comparable ionic strength conditions, these tetramethylrhodamine-labeled actin filaments display a tensile strength approximately two orders of magnitude lower than that displayed by rhodamine phalloidin F-actin, furthermore, their tensile strength depends on the fluorophore to actin ratio.

Section snippets

Materials and methods

Globular(G)-actin was prepared from rabbit muscle [11]. Molar concentrations were calculated on the basis of molecular masses of 42 kDa [12] and of an absorption coefficient A^1%₂₉₀ of 6.2 [13].

Tetramethylrhodamine-5-iodoacetamide was purchased from Molecular Probes Europe BV, Leiden, The Netherlands. The compound was dissolved in dimethylformamide to prepare 6 mM stock solutions.

The viscosity was measured with Ostwald viscosimeters (water flow time 100 s at 20°C) maintained at 22°C.

Protein

The viscosity of rhodamine F-actin

Coupling F-actin (1 mg/ml) with (iodoacetamido)tetramethylrhodamine decreases the specific viscosity of the polymer from 0.85, in the absence of the fluorophore, to either 0.82 or 0.05 M the presence of the fluorophore at the 1/10 and 2/1 fluorophore to actin (as monomer) ratios, respectively. Essentially the same results are obtained by labeling F-actin either for 17 h at 22°C or for 72 it at 2°C (Fig. 1).

The sedimentation of rhodamine F-actin

Sedimentation of F-actin (1 mg/ml) in the absence of tetramethylrhodamine-iodoacetamide

Discussion

Treatment with (iodoacetamido)tetramethylrhodamine disrupts the actin filament. This phenomenon- was never mentioned specifically for rhodamine, however, modification at the actin C-terminus by substitution or truncation was previously reported to affect filament stability [18], [19]. Apparently the yield, as polymeric actin, of the F-actin labeled with tetramethylrhodamine iodoacetamide was never tested [16], [20], [21], [22], [23]. The disruption of the polymer increases with the increase of

Acknowledgements

This work was supported by grants from the University of Ferrara.

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On the elastic properties of tetramethylrhodamine F-actin

Abstract

Introduction

Section snippets

Materials and methods

The viscosity of rhodamine F-actin

The sedimentation of rhodamine F-actin

Discussion

Acknowledgements

J. Mol. Biol.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Communs.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

Archiv. Biochem. Biophys.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Cell