When whole muscle fibers or myofibrils of rabbit and chicken skeletal muscles are directly solubilized in hot SDS solution, a very high molecular protein called titin can be isolated by gel filtration (Wang et al. 1979). Connectin, an elastic protein of muscle (Maruyama et al. 1977), can be isolated by a similar method from thoroughly extracted muscle residues. Studies of electrophoretic mobility on 2-3% polyacrylamide gel electrophoresis, amino acid composition, and localization in myofibrils determined by the indirect immunofluorescence technique showed that titin and connectin are identical. Connectin was found to be unstable in SDS solution on storage for a few days at room temperature; the doublet band of connectin on SDS gel electrophoresis became diffuse and eventually disappeared. Connectin was concentrated around the A-I junction region of a myofibril, although it was present in an entire sarcomere except for the Z lines. On removal of myosin, the A-I junction was still fluorescent, when treated with fluorescent antibody against connectin. In the KI-extracted myofibril, materials accumulated on both sides of the Z lines were strongly stained, and there were fluorescent filaments between the neighboring Z lines, but the Z lines were not stained at all.

Two kinds of monoclonal antibodies (3B9 and SM1) against connectin, muscle elastic protein, reacted with both alpha- and beta-connectins. Immunofluorescence studies revealed that 3B9 stained both edges of the A band of chicken breast muscle myofibrils and remained as such upon stretching to a sarcomere length of 3.5 microns. On the other hand, SM1 stained the I band very close to the edges of the A band and the SM1-stained stripes moved considerably upon stretching to a sarcomere length of 3.5 microns. Immunoelectron microscopic observations with frog semitendinosus muscle revealed that three distinct stripes bound with 3B9 in the edges of the A band did not move on stretching up to 3.5 microns. On the other hand, the two stripes stained with SM1 in the I band clearly moved to the same extent as the stretching. However, when a sarcomere was stretched to 4.0 microns, all the stripes with 3B9 or SM1 disappeared and diffused deposits of the antibodies were observed. Thus it is concluded that connectin filaments in the I band region are more extensible than those at both edges of the A band.

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...

The actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs. Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton. AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro. AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins. Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers. These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin.

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