Electron Microscopic Particle Length of F-Actin Polymerized in Vitro

Kawamura, Masaru; Maruyama, Kosçak

doi:10.1093/oxfordjournals.jbchem.a129267

Cited by 145 publications

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“…Fig. 4 also shows the relaxation spectrum predicted for actin at 6 mg/ml and 220C by assuming the length distribution offilaments ofKawamura and Maruyama (29) and that it behaves according to a rigid-rod model for isotropic (nonliquid crystal, nondomain) polymers (28). As shown, the viscoelastic behavior of actin at 6 mg/ml is significantly different than that predicted by this rigid-rod model.…”

Section: Resultsmentioning

confidence: 94%

Liquid crystal domains and thixotropy of filamentous actin suspensions.

Kerst

Chmielewski

Livesay

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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The thixotropic properties of filamentous actin suspensions were examined by a step-function shearing protocol. Samples of purified framentous actin were sheared at 0.2 sec' in a cone and plate rheometer. We noted a sharp stress overshoot upon the initiation of shear, indicative of a gel state, and a nearly instantaneous drop to zero stress upon cessation of shear. Stress-overshoot recovery was almost complete after 5 min of "rest" before samples were again sheared at 0.2 sec1.Overshoot recovery increased linearly with the square root of rest time, suggesting that gel-state recovery is diffusion limited. Actin suspensions subjected to oscillatory shearing at frequencies from 0.003 to 30 radians/sec confirmed the existence of a 5-min time scale in the gel, similar to that for stress-overshoot recovery. Flow of filamentous actin was visualized by polarized light observations. Actin from 6 mg/ml to 20 mg/ml showed the "polycrystalline" texture of birefringence typical for liquid crystal structure. At shear rates <1 sec', flow occurred by the relative movement of irregular, roughly ellipsoidal actin domains 40-140 ,um long; the appearance was similar to moving ice floes. At shear rates >1 sect, domains decreased in size, possibly by frictional interactions among domains. Eventually domains flow in a "river" of actin aligned by the flow. Our observations confirm our previous domain-friction model for actin rheology. The similarities between the unusual flow properties of actin and cytoplasm argue that cytoplasm also may flow as domains.Cytoplasm and suspensions of actin have unusual and complex fluid behaviors that must play a role in such phenomena as cytoplasmic streaming, amoeboid movement, movement of vesicles through the cytoplasm, and bulk flow of polymer. Both cytoplasm and actin solutions are shear thinning; their viscosity decreases with increasing shear rate (1-8). Shear thinning of actin differs from most polymer solutions in the absence of a Newtonian viscosity plateau at very low shear rates (1-4, 7). Further, cytoplasm and cytoskeletal suspensions show a constant shear stress (force) for shear rates between 0.001 and 1 sect (1, 5). Flow rate, therefore, is not fixed by the force; no flow occurs at forces less than the constant and fluid velocity is limited by inertia alone at forces higher than the constant. We (1) called this "flow indeterminacy," which, for example, helps explain the capacity of Physarum to support very rapid cytoplasmic streaming within "walls" of cytoplasm that do not shear away: High flow rates do not necessarily require large forces and a small difference in filament concentration could account for the difference between flowing and nonflowing cytoplasm. Cytoplasm and actin suspensions are also thixotropic (3, 4, 8-10)-i.e., show time-dependent decreases in viscosity at steady shear rate and subsequent recovery when the flow is discontinued (11). Thixotropy is frequently discussed in terms of gel --sol transitions familiar to biologists and has been among the most durable ...

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Section: Resultsmentioning

confidence: 94%

Liquid crystal domains and thixotropy of filamentous actin suspensions.

Kerst

Chmielewski

Livesay

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast, the length distribution of thin filaments reconstructed in vitro, where a dynamic binding equilibrium exists between the filaments and free actin molecules, is very broad ( 13,21,22). This suggests that there is a mechanism for size determination in vivo.…”

Section: Discussionmentioning

confidence: 94%

“…Although there is no way at present to measure exactly the number of myosin and actin molecules constituting filaments, it seems reasonable to suppose that the number is strictly determined. On the other hand, if a binding equilibrium between the protein molecules operates, one would expect the length distribution of the filaments to be as broad as in an in vitro system (13,21,22). Thus, the question arises, what kind of mechanism does exist in vivo for constructing and maintaining the weU-organized structure and fixed size of filaments?…”

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confidence: 99%

Does actin bind to the ends of thin filaments in skeletal muscle?

Ishiwata

Funatsu

1985

The Journal of Cell Biology

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We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ionic strength, marked binding of actin to the free ends, i.e., the pointed ends, of thin filaments was observed when actin was added at an early phase of polymerization. As the polymerization of actin proceeded, the binding efficiency decreased. The critical actin concentration for this binding was higher than that for polymerization in solution. The binding of G-actin was not observed at low ionic strength. On the basis of these results, we suggest that a particular structure suppressing the binding of actin is present at the free ends of thin filaments in intact myofibrils and that a part of the end structure population is eliminated or modified at high ionic strength so that further binding of actin becomes possible. The myofibril and I-Z-I brush appear to be useful systems for studies aimed at elucidating the organizational mechanisms of actin filaments in vivo.

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“…Annealing is calculated to contribute minimally to filament elongation during the initial stages of self-assembly. However, the rapid rate of annealing of sonicated fixed filaments observed in vitro suggests that it may be an efficient mechanism for repairing breaks in filaments and that annealing together with polymer-severing mechanisms may contribute significantly to the dynamics and function of actin filaments in vivo.steady state in vitro, preparations of actin filaments (Nakaoka and Kasai, 1969;Kawamura and Maruyama, 1970) and microtubules (Mitchison and Kirschner, 1984) grow longer and become progressively fewer in number. While monomer exchange at polymer ends is clearly an important factor in determining the growth and stability of polymers of actin and tubulin, it is likely that polymer fragmentation and annealing are also involved in determining the lengths and numbers of polymers at steady state.…”

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confidence: 99%

“…steady state in vitro, preparations of actin filaments (Nakaoka and Kasai, 1969;Kawamura and Maruyama, 1970) and microtubules (Mitchison and Kirschner, 1984) grow longer and become progressively fewer in number. While monomer exchange at polymer ends is clearly an important factor in determining the growth and stability of polymers of actin and tubulin, it is likely that polymer fragmentation and annealing are also involved in determining the lengths and numbers of polymers at steady state.…”

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confidence: 99%

Direct demonstration of actin filament annealing in vitro.

Murphy

Grasser

et al. 1988

The Journal of Cell Biology

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Abstract. Direct electron microscopic examination confirms that short actin filaments rapidly anneal endto-end in vitro, leading over time to an increase in filament length at steady state. During annealing of mixtures of native unlabeled filaments and glutaraldehyde-fixed filaments labeled with myosin subfragment-1, the structural polarity within heteropolymers is conserved absolutely. Annealing does not appear to require either ATP hydrolysis or the presence of exogenous actin monomers, suggesting that joining occurs through the direct association of filament ends. During recovery from sonication the initial rate of annealing is consistent with a second-order reaction involving the collision of two filament ends with an apparent annealing rate constant of 107 M-Is -I. This rapid phase lasts <10 s and is followed by a slow phase lasting minutes to hours. Annealing is calculated to contribute minimally to filament elongation during the initial stages of self-assembly. However, the rapid rate of annealing of sonicated fixed filaments observed in vitro suggests that it may be an efficient mechanism for repairing breaks in filaments and that annealing together with polymer-severing mechanisms may contribute significantly to the dynamics and function of actin filaments in vivo.steady state in vitro, preparations of actin filaments (Nakaoka and Kasai, 1969;Kawamura and Maruyama, 1970) and microtubules (Mitchison and Kirschner, 1984) grow longer and become progressively fewer in number. While monomer exchange at polymer ends is clearly an important factor in determining the growth and stability of polymers of actin and tubulin, it is likely that polymer fragmentation and annealing are also involved in determining the lengths and numbers of polymers at steady state. In recent studies of microtubule assembly, we demonstrated that microtubule polymers can rapidly join end-to-end (anneal) (Rothwell et al., 1986) and that, under certain conditions, mechanisms of polymer annealing and monomer exchange both play important roles in determining microtubule dynamics in vitro (Rothwell et al., 1987). In the present paper, we examine the role of annealing in the assembly of actin filaments.There are divided opinions regarding the existence of both fragmentation and annealing during actin filament assembly. There is irrefutable evidence that actin filaments can break into smaller pieces when subjected to strong mechanical forces (Asakura, 1961), although it is less certain that thermal energy alone is strong enough to break filaments in samples that have not been stirred. Mathematical models for the spontaneous polymerization of actin fit the time course of assembly better when a term for spontaneous fragmentation is included (Wegner, 1982;Wegner and Savko, 1982;Cooper et al., 1983). However, the contribution of fragmentation and annealing to filament assembly are thought to be complex (Frieden and Goddette, 1983). In these papers, a fragmentation term was not required for all polymerization conditions and there were contradiction...

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Electron Microscopic Particle Length of F-Actin Polymerized in Vitro

Cited by 145 publications

References 0 publications

Liquid crystal domains and thixotropy of filamentous actin suspensions.

Liquid crystal domains and thixotropy of filamentous actin suspensions.

Does actin bind to the ends of thin filaments in skeletal muscle?

Direct demonstration of actin filament annealing in vitro.

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