Polarity of actin filaments at the initial stage of myofibril assembly in myogenic cells in vitro.

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.

Section: Discussionmentioning

confidence: 88%

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

Ishiwata

Funatsu

1985

“…These ideas are supported by ultrastructural observations of myofibril assembly which showed that some thin filaments initially extend all the way across the sarcomere. Restriction of filament length and separation of thin filaments into two half sarcomeres (as evidenced by defined H zone and M lines) occurred late in myofibril assembly, after interdigitation of thick and thin filaments (Peng et al, 1981;Shimada and Obinata, 1977;Legato, 1972;Markwald, 1973;Brooks et al, 1983).…”

Section: Discussionmentioning

confidence: 99%

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

Gregorio

Fowler

1995

100

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.,

“…For example, how actin filaments with the correct polarity form in developing skeletal muscle remains obscure (14). I know of only three cases in nonmuscle cells in which there is evidence demonstrating that the actin filaments grow from specific sites, i.e., in Mytilus sperm, in Limulus sperm, and in the formation of intestinal microvilli (the evidence has been summarized in reference 21).…”

mentioning

confidence: 99%

Polymerization of actin. V. A new organelle, the actomere, that initates the assembly of actin filaments in Thyone sperm

Lg¹

1978

Between the acrosomal vacuole and the nucleus is a cup of amorphous material (profilactin) which is transformed into filaments during the acrosomal reaction. In the center of this cup in untreated Thyone sperm is a dense material which I refer to as the actomere; it is composed of 20-25 filaments embedded in a dense matrix. To visualize the substructure of the actomere, the profilactin around it must be removed. This is achieved either by demembranating the sperm with Triton X-100 and then raising the pH to 8.0, or by adding ionophores to intact sperm at pH 8.0. Under these conditions, the actomere remains as a unit while the rest of the profilactin is solubilized or polymerized. When demembranated sperm are incubated under conditions in which the actin should polymerize, filaments grow from the end of the actomere: the actomere thus appears to behave as a nucleating body. This observation is strengthened by experiments in which untreated sperm are incubated in seawater or isotonic NaC1 at pH 7.0 and the ionophore X537A is added; in this case, only a partial polymerization of the actin occurs and the acrosomal vacuole does not fuse with the cell surface. The actin filaments that do form, however, are attached to the apical end of the actomere. In fact, the elongating filaments push their way into and frequently through the acrosomal vacuole. Thus, it appears that the sperm organizes the actin filaments by controlling their nucleation. My model is that the cell controls the amount of unbound actin such that it is slightly above the critical concentration for polymerization. Then, spontaneous nucleation is unfavored and polymerization would proceed from existing nuclei such as the actomere.KEY WORDS actin polymerization sperm nucleation 9 acrosome reaction actomere One of the most fascinating aspects in the assembly of structural proteins such as microtubules and actin filaments in cells is the control of their distribution, for ultimately the factors involved in this control regulate motility, cell shape, intracellular transport, and secretion. Although many actin filaments appear randomly distributed in cells or in large regions of cells, actin filaments are often seen attached to specific regions of the cell, i.e., to the "Z" line in skeletal muscle, to the dense bodies in smooth muscle, and to membranes in many nonmuscle ceils. Whether this attachment occurs because these sites nucleate the J. CELL BIOLOGY 9 The Rockefeller University Press 9 0021-9525/78/0501-055151.00 551