Myofibrillar M-band proteins represent constituents of native thick filaments, frayed filaments and bare zone assemblages

Abstract

A-segments, native thick filaments, frayed filaments, bare zone assemblages, as well as completely disassembled and reassembled thick filaments from chicken pectoralis major were investigated for the presence of M-band proteins by the colloidal gold labelling technique. Specific polyclonal antibodies against the three M-band proteins identified to date, MM-creatine kinase, M-protein (165 kDa) and a 185 kDa protein myomesin, were prepared. Incubation with anti-M-protein and anti-myomesin antibodies resulted in heavy labelling of all thick filament types mentioned above, with the exception of the completely disassembled and reassembled thick filaments. In that case no labelling was detected with either antibody. In contrast, MM-creatine kinase which is an integral component of the intact M-band structure was detectable on isolated native thick filaments with lower frequency and to a variable extent. Also, bare zone assemblages were only rarely labelled by anti-MM-creatine kinase antibodies. This study shows that the 'cuff-like' additional material which had previously been observed in the middle of the bare zone of isolated thick filaments represent remnants of all three M-band proteins, whereas the extra material in intact bare zone assemblages mainly consists of myomesin and M-protein, but not of MM-creatine kinase. Myomesin and M-line protein may be important for the assembly and structural maintenance of thick filaments as well as for anchoring of additional M-band proteins, e.g. MM-creatine kinase which is bound less tightly to thick filaments and, in accordance with earlier results, seems to represent within the M-band some of the prominent bridge-forming structures.