Elsevier

Biophysical Chemistry

Volume 104, Issue 2, 1 June 2003, Pages 469-476
Biophysical Chemistry

On the stiffness of the natural actin filament decorated with alexa fluor tropomyosin

https://doi.org/10.1016/S0301-4622(03)00036-XGet rights and content

Abstract

Natural, phalloidin-free, actin filaments were decorated with tropomyosin made fluorescent by reaction with alexa fluor (R) 488 C5 maleimide. The elastic modulus by stretching of these filaments was then determined and found to span between 38.2 MPa and 61.48 MPa. We tried also to determine the yield strength of the same filaments in the laser light trap operated at 920 mW, the maximum power of the apparatus. Only two out of the 10 filaments tested were broken under these conditions, yield strength being 50.5 and 55 pN, respectively.

Introduction

The mechanical properties of phalloidin F-actin were extensively investigated by the Yanagida group. Tensile strength was measured and found to be 430 pN [1], to be independent of the length of tropomyosin-decorated phalloidin F-actin between 5 and 30 μm [2] but to be strongly dependent on the torsion applied to the filament (230 pN for a torsion of 360°) [1]. Surprisingly, no consideration of the influence of critical concentration on tensile strength was made. In fact, theoretical considerations predict that critical concentration (i.e. the dissociation constant of the elongation reaction) is the main determinant of the free energy of the monomer–monomer interaction [3], [4], [5]. We directly investigated this aspect by determining tensile strength of phalloidin rhodamine F-actin as a function of the critical concentration [6]. At the conventional ionic strengths, the phalloidin F-actin critical concentration is so low as to be practically not determinable. Therefore, we worked at low ionic strength, where critical concentration is easily determined. We found tensile strength to change inversely with the critical concentration and to increase directly (5.6–28.6 pN) for the increase of ionic strength from 3 to 19 mM [6]. Extrapolation of these data to 100 and 150 mM ionic strengths leads to calculated yield strengths of 165 and 247 pN, respectively, values close to that found by the Yanagida group for phalloidin F-actin at the physiological ionic strength [1], [2].

The finding that critical concentration is the main determinant of the yield strength of F-actin raised the question of how thin filament bears the forces developed in muscle contraction. The critical concentration of F-actin in 0.1 M KC1 and 2 mM MgCl2 is approximately 330 nM and even with the contribution of tropomyosin [7] and the increase of the macromolecular osmotic pressure [8], it cannot be expected to reach the values of the critical concentration of rhodamine phalloidin F-actin (30 nM) observed in our experiments. In turn, the largest yield strength experienced in our work for rhodamine phalloidin F-actin is ∼28 pN, i.e. the contribution of only three to four attached crossbridges [9].

We found that the actin filament, decorated with tetramethylrhodamine (without phalloidin) and at 0.15 M ionic strength displays a yield strength of only 3.5 pN (2 orders of magnitude lower than phalloidin F-actin) [10], With the same filament we showed also that the yield strength increases from 3.5 to 10 pN after decoration with tropomyosin and from 3.5 to 15 pN after decoration with myosin subfragment-1 [11].

In the present work we investigate the elastic properties of natural actin filaments, phalloidin-free and chemical modification free, made detectable by interaction with fluorescent tropomyosin, obtained by coupling with alexa fluor (R) 488 C5 maleimide.

Section snippets

Experimental

G-actin [12] and tropomyosin [13] were from rabbit muscle. Heavy meromyosin was obtained by chymotryptic digestion of myosin [14] and further modified by reaction with N-ethylmaleimide [15]. Alexa fluor (R) 488 C5 maleimide was purchased by Molecular Probes Europe BV, The Netherlands. Stock solutions of Alexa Fluor were prepared in dimethylformamide (Uvasol grade) and kept in the dark at −20 °C.

Protein sedimentation was performed in the Beckman TL100 rotor of the Beckman TL100 centrifuge for 10

Binding of alexa fluor tropomyosin to F-actin

Alexa fluor (R) 488 C5 maleimide is a bulky reagent. It was therefore checked whether the tropomyosin labelled with 2.9 moles of alexa fluor per mole recovered the proper conformation. In a first experiment F-actin (0.45 to 48 μM, as the monomer) and alexa fluor tropomyosin were mixed at the 7:1 molar ratio and the amount of tropomyosin cosedimenting with F-actin was determined. It was found that the fraction of tropomyosin sedimenting with actin was spanning from 0.4 to 0.7, independently on

Discussion

This is the first time that an investigation is performed on the elastic properties of natural actin filaments, phalloidin-free and chemical modification free. The filaments were made detectable by interaction with fluorescent tropomyosin, obtained by coupling with alexa fluor (R) 488 C5 maleimide.

When trying to determine the yield strength, even though the laser light trap was operated at its maximum power (920 mW), only two out of the 10 filaments tested were broken at 50.5 and 55 nN,

Acknowledgements

This work was supported by grants of the University of Ferrara and of the Fondazione della Cassa di Risparmio di Ferrara.

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