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Motor-Driven Dynamics in Actin-Myosin Networks Loïc Le Goff, François Amblard, Eric Furst

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Motor-Driven Dynamics in Actin-Myosin Networks Loïc Le Goff, François Amblard, Eric Furst Motor-Driven Dynamics in Actin-Myosin Networks Loïc Le Goff, François Amblard, Eric Furst To cite this version: Loïc Le Goff, François Amblard, Eric Furst. Motor-Driven Dynamics in Actin-Myosin Networks. Physical Review Letters, American Physical Society, 2001, 88 (1), pp.018101. 10.1103/phys- revlett.88.018101. hal-02415553 HAL Id: hal-02415553 https://hal.archives-ouvertes.fr/hal-02415553 Submitted on 19 Dec 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. VOLUME 88, NUMBER 1PHYSICALREVIEWLETTERS7JANUARY 2002 Motor-Driven Dynamics in Actin-Myosin Networks Loïc Le Goff, François Amblard,* and Eric M. Furst† Institut Curie, Physico-Chimie Curie, UMR CNRS/IC 168, 26 rue d’Ulm, 75248 Paris Cedex 05, France (Received 29 May 2001; published 14 December 2001) The effect of myosin motor protein activity on the filamentous actin (F-actin) rheological response is studied using diffusing wave spectroscopy. Under conditions of saturating motor activity, we find an enhancement of longitudinal filament fluctuations corresponding to a scaling of the viscoelastic shear 7͞8 modulus Gd͑v͒ϳv . As the adenosine tri-phosphate reservoir sustaining motor activity is depleted, we find an abrupt transient to a passive, “rigor state” and a return to dissipation dominated by transverse filament modes. Single-filament measurements of the apparent persistence length support the notion that motor activity leads to an increase in the effective temperature for tangential motion. DOI: 10.1103/PhysRevLett.88.018101 PACS numbers: 87.16.Ka, 83.60.Bc, 83.85.Ei, 87.15.La The globular protein actin plays an important role in cel- The actin-myosin cycle forms the basis of many cellular lular mechanics, motility, and morphology by polymeriz- motility mechanisms, including the contraction of skeletal ing into filaments of diameter d Ӎ 7 nm to form networks and smooth muscle. Motor activity may have profound and higher-order structures. Because of their high bend- effects on actin filaments and networks. Recently, it ing rigidity k, long persistence length Lp ෇ k͞kbT Ӎ was shown that double-headed myosin fluidizes actin 10 15 mm, and ease of visualization, actin filaments are solutions by an enhancement of the slow relaxation of also important models for semiflexible polymer physics. stress [12]. In addition, theoretical work suggests that Significant advances have been made in the understanding motor activity introduces an active temperature Tact of actin rheology and its consequences for polymer theory, for tangential motion of filaments, which may enhance cellular organization, intracellular transport, and cell sig- longitudinal modes [13]. Similar effects of activity have naling [1– 5]. To date, however, rheological and dynamical been reported for the dynamics of biological membranes studies of F-actin have relied on probing passive reconsti- [14], the two-dimensional analog of semiflexible fila- tuted systems through either mechanical rheometry, light ments [15]. scattering, or microrheological techniques [3,6,7]. In this Letter, we report the rheology of F-actin using In frequency regimes governed by single polymer re- tracer particle microrheology with diffusing wave spec- laxation, the linear viscoelastic storage G0͑v͒ and loss troscopy (DWS) [16] in the presence of single-headed 3͞4 G00͑v͒ shear moduli scale as G0͑v͒ϳG00͑v͒ϳv ,a myosin motor protein. We measure the linear viscoelas- direct consequence of the dominant stress relaxation by ticity of “active” F-actin solutions on time scales that end-to-end response upon affine deformation of the poly- probe the relaxation of individual filaments from motor- mer [8,9]. This is accounted for by summing up transverse driven modes. In addition, our experiments address a modes after assuming the incompressibility of the filament; number of other important issues of actomyosin. For ex- however, if one could perturb the filament locally, the ef- ample, it is suggested that filament mechanics are altered fects of finite propagation velocity of tension should give by motor activity [17]. Such changes should modify the rise to a longitudinal response that scales as v7͞8 [4]. Such spectrum of fluctuations of the filaments, and could be would be the case when pulling one end of the filament, identified through changes in the measured viscoelastic- or following the motion of a reticulation point between ity. In addition, we discuss the implications of our work cross-linked filaments. for the myosin power-stroke time scale and mechanism. The mechanochemical activity of myosin molecular We will first summarize our experimental methods. motors offers a unique opportunity to perturb F-actin Next, we present the dependence of the actomyosin filaments from their equilibrium thermal fluctuations both viscoelastic response on ATP consumption, and examine locally and anisotropically to study the resulting rheologi- the scaling behavior during motor activity. We conclude cal response and relaxation mechanisms. In a reaction by deriving an expression for the active temperature and that consumes one adenosine tri-phosphate (ATP) per discussing the time scale and mechanism of the myosin cycle, myosin binds to actin, produces a force through power stroke. a mechanical “power stroke” by rotating a lever-arm Protein preparation and characterization.—Actin is domain parallel to the filament axis, then unbinds [10]. purified from chicken skeletal muscle acetone powder The actomyosin cycle proceeds perpetually as long using the method of Pardee and Spudich [18] (actin purity as ATP is present. In the absence of ATP, myosin greater than 99% based on SDS-PAGE). Monomeric remains attached to filaments with a low dissociation actin (G-actin) is stored at 270 ±C in G-buffer [5 mM Kd ෇ 500 nM in what is known as the rigor state [11]. Tris-HCl pH 7.8, 5 mM ATP, 100 mM CaCl2, 1 mM 018101-1 0031-9007͞02͞88(1)͞018101(4)$15.00 © 2001 The American Physical Society 018101-1 VOLUME 88, NUMBER 1PHYSICALREVIEWLETTERS7JANUARY 2002 dithiothreitol (DTT), 0.01% NaN3]. Prior to experiments, During DWS measurements at high S1 concentration, we thaw the actin rapidly until completely melted and we observe two dynamical regimes: an active steady dialyze against 100 volumes of fresh G-buffer overnight state associated with ATP hydrolysis by actin-myosin at 4 ±C. Sample-to-sample variation in the micro- and a final steady state due to myosin rigor (Fig. 1). rheological response of the actin when subjected to one At 5 mM S1, myosin activity during the active steady freeze-thaw cycle is minimal provided a sufficient amount state dramatically alters the scaling behavior of the of DTT is present [19]. Myosin subfragment 1 (S1) is a modulus, shown in Fig. 1 by the rotation downward single-headed motor consisting of the motor, converter, in Gd ͑v͒ and the corresponding shift upward in d͑v͒ and lever-arm domains. To characterize the kinetics relative to the actin-only sample. By contrast, the rigor and rate of mechanochemical events in our system, we state is marked by a nearly identical response for d͑v͒ measure ATP hydrolysis by the same F-actin (13 mM) and when compared to actin alone, indicating a dominance of myosin S1 (6.75 mM) in high-ATP (14.5 mM) F-buffer passive dissipation by the semiflexible filaments. More- by absorbance at 650 nm of molybdenum blue. over, there is a uniform increase in Gd͑v͒ by a factor of 2 Microrheology.—The mean-squared displacement over actin only that can be accounted for from the increase ͗Dr2͑t͒͘ of particles embedded in an F-actin sample of the filament hydrodynamic radius to 40 nm due to the is measured by DWS using a transmission geometry attachment of rigor myosin, since there is no difference 1 2 and plane-wave Ar laser source [20]. From ͗Dr ͑t͒͘, in Lp (Fig. 2). we calculate the shear modulus amplitude Gd͑v͒ and The onset time of the transient from the active steady phase angle d͑v͒ of the F-actin [16]. Note that Gd͑v͒ state to the rigor state is consistent with the depletion represents the total resistance to deformation, while rate of the ATP reservoir by myosin activity. Our d͑v͒ characterizes whether the resistance is in-phase direct measurement of ATP hydrolysis gives a consump- (viscous) or out-of-phase (elastic) with the rate of strain tion rate of 6 mM͞s for 6.7 mM S1, in agreement with [21]. They are related to the storage and loss moduli by id͑v͒ G0͑v͒ 1 iG00͑v͒ ෇ Gd͑v͒e . Within a small cor- rection G ͑v͒ϳ͗Dr2͑t͒͘21 and d͑v͒ϳ͓d lnG ͞ d t෇2p͞v d (a) d lnt͔t෇2p͞v, which essentially gives the scaling of Gd͑v͒ 10 0 0 with respect to v [22]. Particles (0.98 mm carboxylated polystyrene, Bangs rigor Laboratories) are washed to exchange surfactant and 10 0 (Pa) impurities. We incubate tracer particles for 2 hours in d G the G-actin solution to protein coat the bead surfaces by nonspecific adsorption. Immediately prior to polymerizing 10 active the actin, myosin S1 is added to a final concentration between 0.01 and 10 mM and Mg2ATP to 14.3 mM. Each 1.6 sample is polymerized in glass cuvettes (L ෇ 0.5 cm) by (b) raising the KCl concentration to 50 mM and MgCl2 to 1 mM. 1.4 active Single-filament microscopy.—A 5 mM solution of F-actin fluorescently labeled and stabilized with (rad) 1.2 rhodamine-phalloidin in F-buffer (25 mM imidazole, δ rigor / passive 2 mM MgCl2, 0.5 mM ATP, 50 mM KCl, 0.5 mM DTT, 0.01% NaN3) is diluted 500-fold with F-buffer 1.0 and 0.05 mg͞ml glucose oxidase, 0.01 mg͞ml catalase, 2.3 mg͞ml glucose, and 2% b-mercaptoethanol added 3 4 5 6 to reduce photobleaching.
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