Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy.

Abstract

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397. In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region ~0.15/~m from the center of the A band.Connectin (also called titin) is a very long flexible protein of striated muscle (1-3) that is assumed to be responsible for passive tension generated upon stretch (4,5).Immunofluorescent studies have shown that connectin is mainly located in the A-I junction area in a sarcomere (6-9). In a recent paper (8) we showed that the connectin filaments are associated with the "gap filaments" that Sj6strand described in 1962 (10). Trinick and associates (3) have demonstrated that "end filaments" extruding from the isolated native thick (myosin) filaments are morphologically indistinguishable from connectin filaments (11).On the other hand, it was not clear whether the connectin filaments run through the entire I band to Z lines or not. Wang has presented the view that connectin filaments are linked to "nebulin meshwork" that is assumed to be connected with Z lines (12). Our previous study using immunoelectron microscopic observations was inconclusive with respect to this (8). Present reinvestigation has clearly shown that connectin structures are longitudinally located through the I band reaching the Z lines from the both sides of the thick filaments. MATERIALS AND METHODS Preparation of Skinned Fibers:Single muscle fibers were dissected from semitendinous muscle of the bullfrog (Rana castesbeiana), and mechanically skinned fibers were prepared in a relaxing solution (90 mM KCI, 5.2 mM MgCI2, 4.3 mM ATP, 4.0 mM EGTA, and 10 mM PIPES, pH 7.0). Partial dissociation of thick filaments in skinned fibers was performed by the extraction with a solution of ionic strength of 0.35 (relaxing solution containing 290 mM KCI) for 10 rain at 20°C at appropriate sarcomere lengths. Skinned fibers were fixed in situ for 10 rain at 20"C with the relaxing solution or the solution of ionic strength of 0.35 containing 10% formalin.Another type of skinned fiber was prepared at low ionic strength. Skinned fibers in the relaxing solution were incubated for 10 min at 5"C in a rigor solution (110 mM KCI, 1.2 mM MgC12, 4.0 mM EGTA, and 10 mM PIPES, pH 7.0). They were then immersed for 5 min at 20"C in 0.5 mM PIPES buffer, p...

Distinct roles for telethonin N‐versus C‐terminus in sarcomere assembly and maintenance

“…In mature striated muscle, it provides passive muscle elasticity (Maruyama, 1976;Maruyama et al, 1977;Mitsui et al, 1987), and has been proposed to act as a stretch sensor (Knöll et al, 2002;Miller et al, 2004;Linke, 2008). During sarcomere assembly, titin is hypothesized to act as a molecular ruler and organizational scaffold (Fü rst et al, 1988;Maruyama, 1976;Maruyama et al, 1985, Trinick, 1994. Therefore, titin must be rigidly and precisely anchored within the sarcomere, and this is accomplished in part by a small 19-kDa Z-disc protein called telethonin, also known as T-cap (Valle et al, 1997;Gregorio et al, 1998;Zou et al, 2003Zou et al, , 2006Pinotsis et al, 2006).…”

Dynamics of actin and assembly of connectin (titin) during myofibrillogenesis in embryonic chick cardiac muscle cells in vitro

“…13). In wide spaces unstained with anti-myosin where two Z bands were close together (black arrowheads in connectin filaments are believed to link myosin filaments to Z bands (Maruyama et al, 1985;Furst et al, 1988), connectin coordinately developing with myosin may play some role in integrating myosin filaments with Z bands of preformed I-Z-I brushes (NSFs).…”

Physiological Functions of the Giant Elastic Protein Titin in Mammalian Striated Muscle

“…[16,17]). Later, Maruyama and colleagues used antibodies to demonstrate the ordered alignment of titin in the sarcomere [18]. However, the first clear and direct observation of titin in situ was conducted by Funatsu and colleagues [19,20].…”