Assembly of connectin (titin) in relation to myosin and alpha-actinin in cultured cardiac myocytes.

By using polyclonal and monoclonal antibodies against connectin (titin) which stain the A-I junctional area and the A-band domain (polyclonal anti-connectin and monoclonal 4C9) and the I-band domain (monoclonal SM1), the developmental relationship of this elastic protein with sarcomeric proteins, especially and alpha-actin, was examined in embryonic chick cardiac myocytes in vitro under fluorescence microscopy. During premyofibril stages, I-Z-I proteins were detected first (alpha-actinin dots and diffuse actin [phalloidin and anti-troponin C] staining), and later in these areas connectin and myosin dots appeared with nearly identical distribution. Somewhat later, phalloidin-positive nonstriated fibrils were observed in a straight course. They were always reactive with antibodies against alpha-actinin and troponin C, but unreactive or only weakly reactive with anticonnectin and anti-myosin. Initially, alpha-actinin dots were aligned along these fibrils but did not form striations. As they aggregated to form Z-bands, connectin and myosin started to exhibit typical striation ('doublets' and A-bands, respectively). No difference in the staining pattern was observed with two kinds of monoclonal antibodies against different domains of connectin filaments (4C9 and SM1) at early phases. As myosin staining began to show clear A-bands, connectin epitopes became arranged in polarized positions. We conclude that primitive I-Z-I complexes appear prior to the assembly of connectin and myosin filaments and then connectin filaments, developing intimately and coordinately with myosin, become associated with the alpha-actinin lines. Thus it appears that the putative elastic protein connectin plays some role in integrating myosin filaments with the preexisting I-Z-I brushes. The occasional absence of connectin and A-bands between two Z-bands, beyond both of which clear sarcomeres have been formed, indicates that connectin is not a preformed scaffold of myofibrils on which sarcomeric proteins accumulate.