Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

Abstract

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alphaactinin, and tropomyosin, In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin . Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands . When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands . Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i .e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the Aband which lacks cross-bridges . In the presence of 0.1-0.2 mM ATP, no actin binding occurred . When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added, fluorescence occurred not in a doublet pattern but along the entire length of the myofibril . Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the I-band . Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band . These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments . Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin . These observations are discussed in terms of the structure and in vivo assembly of myofibrils.The myofibrils of cross-striated muscles are contractile complexes of thick myosin filaments, that interdigitate and interact with thin tropomyosin-coated actin filaments (16). These thin filaments are attached or embedded in a Z-band composed of alpha-actinin and several other proteins (4) . Although thick and thin filaments can self-assemble in vitro from their constituent molecules, the synthetic filaments are not uniform in length, as they are in vivo (21,23,24). Furthermore, we do not know how newly synthesized thick and thin filaments become oriented to form functional contractile units in muscle cells . Studies that have attempted to identify which contractile proteins are synthesized first in developing muscle cells have concluded that there is coordi-THE JOURNAL Of CELL BIOLOGY " VOLUME 98 MARCH 1984 $25-833 © The Rockefeller University Press~0021-9525/84/0310825109 $1 .00 nate synthesis of actin, myosin, tropomyosin, and alphaactinin during myogenesis (5) . Immunocytochemical ( I S) and electron microscopic data (7) suggest that thick and thin filaments are formed concurrently in the cytoplasm and subsequently...

The Three Mouse Actin-depolymerizing Factor/Cofilins Evolved to Fulfill Cell-Type–specific Requirements for Actin Dynamics

“…However, all ADF/cofilins disassemble platelet actin more efficiently than muscle actin (Figure 6). This difference has not been reported previously, but it agrees with the finding that muscle actin filaments are less dynamic than actin in other cells (Sanger et al, 1984).…”

“…However, all ADF/cofilins disassemble platelet actin more efficiently than muscle actin ( Figure 6). This difference has not been reported previously, but it agrees with the finding that muscle actin filaments are less dynamic than actin in other cells (Sanger et al, 1984). The three ADF/cofilins in mice are biochemically distinct: the muscle-specific cofilin-2 is less efficient at actin disassembly than cofilin-1 and ADF (Figure 6).…”

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

“…After the submission of our manuscript, Sanger et al (26) reported results similar to ours (compare Fig. 4, a-e of their paper and Fig.…”

How can cells sense the elasticity of a substrate? An analysis using a cell tensegrity model

“…Topologically, the acto-myosin units are located at the FACs, which pin the AFs to the matrix. Thus, the actomyosin contraction force is transmitted by the AFs (under tension) partially to the rest of the cell (prestressed, under compression) and partially to the matrix (under traction) (Gordon and Bushnell, 1979;Sanger et al, 1984;Boal, 2002;Kumar et al, 2006). It is also known that microtubules (MTs) are compressed in the cell because they appear highly curved despite their long persistent length (Brangwynne et al, 2006).…”