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

Ishiwata, Shin’ichi; Funatsu, Takashi

doi:10.1083/jcb.100.1.282

Cited by 46 publications

(37 citation statements)

References 32 publications

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“…Similar in vitro findings have been reported [Ishiwata and Funatsu, 1985;Sanger et al, 19841. When fluorescence was observed, it was seen either around the periphery of the myofibril or as doublets on either side of the H-zone (similar to what is shown in Fig.…”

Section: Incorporation Of Actin Is Atp Dependentsupporting

confidence: 91%

Post‐translational incorporation of actin into myofibrils in vitro: Evidence for isoform specificity

Peng

Fischman

1991

Cell Motil. Cytoskeleton

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The incorporation of actin into myofibrils has been examined in a cell-free system [Bouché et al.: Journal of Cell Biology 107:587-596, 1988; Goldfine et al.: Cellular and Molecular Biology of Muscle Development, 1989]. Actin was translated in a reticulocyte lysate in the presence of 35S-methionine (35S-actin) or purified from muscle and labeled with fluorescein-5-isothiocyanate (FITC-actin). Myofibrils were incubated with either 35S-actin or FITC-actin and then analyzed by gel electrophoresis or fluorescence microscopy. When myofibrils were incubated with FITC-actin monomer in the reticulocyte lysate buffer, strong fluorescent labeling was observed in Z-band regions and less so in I-bands. No fluorescence was detected in non-overlap regions of A-bands. Confocal microscopic analysis of these myofibrils indicated that FITC-actin was distributed evenly across the diameter of the myofibrils. These observations suggest that actin incorporation in the reticulocyte lysate buffer occurred at sites in the sarcomere which contain actin. In contrast, FITC-actin showed a variety of non-physiological incorporation patterns when incubated with myofibrils in the presence of an isotonic buffer (I-buffer). However, when ATP was added to I-buffer, FITC-actin showed a pattern of incorporation into myofibrils similar to that seen in the reticulocyte lysate buffer. Immunoblots indicated that actin of native size was released from myofibrils during incubation in the reticulocyte lysate buffer. No actin release was detected when the myofibrils were incubated in I-buffer lacking ATP. We used this system to compare the incorporation of actin isoforms into myofibrils. Both alpha- and beta-actins exhibited incorporation into the myofibrils but there was a three-fold greater incorporation of the alpha isoform. We propose that the differential affinities of actin isoforms for myofibrils and other cytoskeletal structures could provide a mechanism for actin isoform targeting within the cytoplasm.

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Section: Incorporation Of Actin Is Atp Dependentsupporting

confidence: 91%

Post‐translational incorporation of actin into myofibrils in vitro: Evidence for isoform specificity

Peng

Fischman

1991

Cell Motil. Cytoskeleton

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“…Myofibrils were prepared as described previously (38). Briefly, bundles of psoas muscle fibers were obtained from white rabbits (2-3 kg) anesthetized by sodium pentobarbital (25 mg/kg) into an ear vein.…”

Section: Methodsmentioning

confidence: 99%

Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch

Shimamoto

Suzuki

Mikhailenko

et al. 2009

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

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The force generation and motion of muscle are produced by the collective work of thousands of sarcomeres, the basic structural units of striated muscle. Based on their series connection to form a myofibril, it is expected that sarcomeres are mechanically and/or structurally coupled to each other. However, the behavior of individual sarcomeres and the coupling dynamics between sarcomeres remain elusive, because muscle mechanics has so far been investigated mainly by analyzing the averaged behavior of thousands of sarcomeres in muscle fibers. In this study, we directly measured the length-responses of individual sarcomeres to quick stretch at partial activation, using micromanipulation of skeletal myofibrils under a phase-contrast microscope. The experiments were performed at ADPactivation (1 mM MgATP and 2 mM MgADP in the absence of Ca 2؉ ) and also at Ca 2؉ -activation (1 mM MgATP at pCa 6.3) conditions. We show that under these activation conditions, sarcomeres exhibit 2 distinct types of responses, either ''resisting'' or ''yielding,'' which are clearly distinguished by the lengthening distance of single sarcomeres in response to stretch. These 2 types of sarcomeres tended to coexist within the myofibril, and the sarcomere ''yielding'' occurred in clusters composed of several adjacent sarcomeres. The labeling of Z-line with anti-␣-actinin antibody significantly suppressed the clustered sarcomere ''yielding.'' These results strongly suggest that the contractile system of muscle possesses the mechanism of structure-based inter-sarcomere coordination.myofibril ͉ sarcomere ''yielding'' ͉ Z-line ͉ partial activation T he periodic architecture of striated muscle relies on the series connection of the basic contractile units, called sarcomeres, which are connected through the Z-line to form a myofibril. Each sarcomere is composed of a bipolar array of myofilaments, i.e., the thick (myosin) and thin (actin) filaments. The cyclic interaction of myosin with actin coupled to ATP hydrolysis produces force and motion along the long axis of the myofibril (1, 2). Such characteristics of striated muscle imply that individual sarcomeres are structurally and mechanically interconnected and interact with each other. Tension and length responses of sarcomeres to the external perturbations have revealed various mechanochemical properties of striated muscle, particularly those relating to the mechanism of force generation (3-6) and to the attachment/detachment kinetics of cross-bridges (7-9). On the other hand, the interaction between sarcomeres remains unclear, primarily because the widely used technique for measuring sarcomere response is laser light diffraction, where the individual sarcomere dynamics are obscured by the ensemble averaging of thousands of sarcomeres connected in parallel and in series in muscle fibers.Recent developments in microscopic analysis using skeletal and cardiac myofibrils revealed various dynamic properties of individual sarcomeres (or even half-sarcomeres) upon force generation and relaxation of stria...

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“…"Ghost myofihrils" were prepared by extraction of thick filaments in high salt buffer (0.5 M KCI, 5 mM MgCI2, 10 mM K-pyrophosphate, 1 mM EGTA, 10 mM Tris-HCl, pH 6.5, 0.2 mg/ml -l saponin) for 10 min at room temperature (Huxley and Hanson, 1957;Ishiwata and Funatsu, 1985). The ghost myofibrils were then incubated in buffer containing 20 mM KCI, 5 mM Tris-HCl, pH 6.8, 0.1 mM CaCI2, 0.1 mM ATP, 0.2 mg/ml -I saponin for '~15 s.…”

Section: Reconstitution Of Tropomodulin Onto Thin Filamentsmentioning

confidence: 99%

Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly.

Gregorio

Fowler

1995

The Journal of Cell Biology

100

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Abstract. Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420;Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994. J. Cell Biol. 127:1627-1635. To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, ~actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (,,o39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin illaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity, We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.SEMBLV of myofibrils during striated muscle differentiation is a complex process that requires coordinate expression of the constituent proteins and their association into highly organized sarcomeres. Multiple proteins appear to be responsible for specifically regulating the length, polarity, stability and spatial organization of thin filaments during myofibrillogenesis: properties which are required for efficient contraction. Proteins associated with the barbed (fast-growing) ends of muscle thin filaments at the Z disk assemble early during myofibrillogenesis; for example, the actin filament barbed end capping protein, capZ and the actin cross-linking protein, c¢-actinin (for example, see Sanger et al., 1986;Schultheiss et al., 1990;Schafer et al.,

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

Cited by 46 publications

References 32 publications

Post‐translational incorporation of actin into myofibrils in vitro: Evidence for isoform specificity

Post‐translational incorporation of actin into myofibrils in vitro: Evidence for isoform specificity

Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch

Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly.

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