Substructure of nebulin filaments: localization and characterization of subfragments produced by 0.1 mM CaCl2

The locations of five kinds of nebulin subfragments in chicken myofibrils were studied by indirect immunofluorescence microscopy and immunoelectron microscopy. The subfragments were produced by treatment of the myofibrils with a solution containing 0.1 mM CaCl2. Antinebulin-subfragment antibodies displayed five stripes from the Z-disk to the distal end of thin filaments in each half sarcomere. Anti-40-kDa subfragment antibodies provided a wide stripe near the Z-disk. Anti-33-kDa subfragment antibodies displayed three stripes in the I-band. Anti-23-kDa subfragment antibodies displayed three stripes, whose positions could not be distinguished from those of three stripes provided by anti-33-kDa subfragment antibodies. Anti-180-kDa subfragment antibodies provided fluorescence at the A-I junction region. Anti-200-kDa subfragment antibodies displayed a single stripe at the distal end region of thin filaments. The location of these stripes corresponded well to that of the mother protein, nebulin. On the basis of these results, we propose a model for the substructure of chicken nebulin filaments. All the nebulin subfragments possessed the property of binding to F-actin, indicating that nebulin filaments bind to thin filaments along their entire length in situ. There is a possibility that nebulin filaments are anchored at the Z-disk through interaction with some other Z-disk constituents than alpha-actinin, because the binding site for alpha-actinin exists on the distal end region of nebulin filaments.