The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study

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Abstract
Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 μm, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 μm, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.