Myofibrillar interaction of blot immunoaffinity-purified antibodies against native titin as studied by direct immunofluorescence and immunogold staining

Abstract

Titin (also called connectin), a major but so far highly elusive myofibrillar component in striated muscle was purified from glycerinated chicken breast muscle in its native state by use of a similar purification procedure as recently introduced for purification of native titin from rabbit psoas muscle. Low-angle rotary shadowing reveals highly convoluted, long and slender strands, sometimes more extended and with nodules, but also an aggregation into filamentous bundles and reticular networks. Antisera were raised against the purified native molecule and monospecific titin antibodies prepared by a rapid nitrocellulose blot immunoaffinity-purification procedure. Titin antibodies bound to the nitrocellulose immobilized native antigen were directly conjugated with fluorescein isothiocyanate. Titin specifity of purified antibodies was checked by immunoblotting. Direct immunofluorescence of glycerinated myofibrils revealed a uniform doublet staining pattern within the sarcomeres by labelling the region of the A-I junctions and some diffuse staining in the region of the myosin filaments. The same myofibrils examined by indirect immunoelectron microscopy revealed the gold particles highly concentrated at the A-I junctions with considerable labelling within the A-bands, except in their centers. Residual I-bands and Z-lines are free of label. In overstretched myofibrils immunogold staining labelled the gap filaments in the space between I- and A-bands. Isolated native thick filaments showed gold labelling of coiled superthin filaments at the ends of the thick filaments (end-filaments) and at their sides, respectively. The colloidal gold technique in combination with an affinity-purified titin antibody raised against the native molecule adds further evidence for the existence and distribution of an endosarcomeric superthin cytoskeletal filament lattice with titin as a major component.