Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green f luorescent protein (GFP) linked to the Z-band protein, ␣-actinin. The expression of this f luorescent protein provided an in vivo label for structures containing ␣-actinin. The GFP-␣-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of ␣-actinin, termed Z-bodies. The punctate Zbodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils-i.e., the precursors of mature myofibrils.The formation of a myofibril involves the precise ordering of multiple subunits into a linear array of sarcomeres. One of the challenges in muscle research is to delineate the sequence of steps occurring in a cell during the assembly of the thick and thin filaments and Z-bands to form sarcomeres and myofibrils. We have recently proposed a model for the assembly of myofibrils based on antibody staining of chicken cardiomyocytes fixed at different times after spreading in culture (1). This model postulates that premyofibrils, characterized by banded patterns of ␣-actinin-rich Z-bodies and nonmuscle myosin IIB, form at the edges of spreading cardiomyocytes and develop into mature myofibrils (Fig. 1). During the transition from premyofibril to myofibril, it is postulated that there is an exchange of nonmuscle myosin IIB filaments for muscle myosin II filaments and a growth and fusion of Z-bodies into Z-bands (1, 2). The Z-bodies appear initially as discrete aggregates of ␣-actinin along the premyofibrils. As the myofibrils increase in width, the Z-bands appear to be composed of laterally aligned Z-bodies and finally continuous bands of ␣-actinin (see Fig. 1). A second model of myofibrillogenesis proposes that the first fibrils that form at the periphery of spreading cardiomyocytes are temporary scaffolds along which myofibrils assemble (3). Finally, there is a third hypothesis that spatially separate complexes of actin filaments and Z-bands, I-Z-I brushes, and groups of myosin thick filaments assemble independently of one another and become spliced together by titin filaments and then inserted at the en...

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ABSTRACT. Originally, zeugmatin was identified as a 600-800 kD muscle specific protein in Z-bands of cardiac and skeletal muscles by Maher et ah (1985). In this presentation we review our work on myofibrillogenesis and present evidence that zeugmatin is actually part of the Z-Band region of titin and that this region of titin plays an important role in the assembly of the Z-bands and myofibrils. Rhee et al. (1994) reported that during myofibrillogenesis, zeugmatin antibody localization is detected in fully formed Z-bands in the mature myofibrils, in the Z-bodies of the nascent myofibrils, but not in the Z-bodies of the premyofibrils. These observations lead to the suggestion that zeugmatin might be responsible for the fusion of the Z-bodies to form the solid Zbands of the mature myofibrils (Rhee et al. 1994). As part of a study to test aspects of this model of myofibrillogenesis, we isolated a 1.8 kb CDNAfrom a chicken cardiac expression library using an anti-zeugmatin antibody (Turnacioglu et al. , 1996). We found this chicken CDNAto be 60%identical at the amino acid level to a segment of the Z-band region of human cardiac titin (connectin) sequenced by Labeit and Kolmerer (1995). This homology along with Western blot analysis with purified titin, suggested that zeugmatin is in fact part of the N-terminal region of chicken titin. Whenexpressed in non-muscle cells, Zl.l product colocalized with the alpha-actinin in stress fiber dense bodies and focal adhesions. Cultures of non-muscle cells, skeletal myotubes and cardiomyocytes were also transfected with a fusion construct (Z1.1GFP) consisting of the Zl.l kb CDNAlinked to the CDNAfor green fluorescent protein (GFP). The Zl.l kb CDNAencodes only 362 of the approximately 2,000 amino acids which comprise the Z-band region of titin; neverthelss, the Z1.1GFPfusion protein targets in vivo to the alpha-actinin rich Z-bands of contracting myofibrils. A dominant negative phenotype was observed in living cells expressing highlevels of this Z1.1GFPfusion protein with inhibition of myofibrillogenesis as well as the disassembly of preexisting myofibrils in these cells. These data indicate that the Z-band region of titin (connectin) plays an important role in organizing and maintaining the structure of the myofibril.

Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Zl.lGFP fusion protein targets the a-actininrich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Zl.lGFP fusion protein localizes to the a-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Zl.lGFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac Agtl 1 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Zl.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin. The Z-bands of striated muscles anchor the thin fila-sin filaments (titin). Nebulin, a 775-kDa protein with ments of the sarcomere and are also sites of insertion its C-terminal end embedded in the Z-band, binds for the two largest muscle proteins: nebulin and titin. along the entire 1-.m length of the thin filament (Labeit and Kolmerer, 1995b). Titin, also known as conThese proteins exist as single polypeptides that bind nectin, therest polypeptide known a monectin, iS the largest polypeptide known with a mo-

A 6.5-kb N-terminal region of embryonic chick cardiac titin, including the region previously reported as part of the protein zeugmatin, has been sequenced, further demonstrating that zeugmatin is part of the N-terminal region of titin, and not a separate Z-band protein. This Z-band region of cardiac titin, from both 7-and 19-day embryos as well as from adult animals, was found to contain six different small motifs, termed z-repeats [Gautel et al., 1996: J. Cell Sci. 109:2747-2754, of approximately 45 amino acids each sandwiched between flanking regions containing Ig domains. Fragments of Z-band titin, linked to GFP, were expressed in cultured cardiomyocytes to determine which regions were responsible for Z-band targeting. Transfections of primary cultures of embryonic chick cardiomyocytes demonstrated that the z-repeats play the major role in targeting titin fragments to the Z-band. Similar transfections of skeletal myotubes and non-muscle cells lead to the localization of these cardiac z-repeats in the Z-bands of the myofibrils and the dense bodies of the stress fibers. Over-expression of these z-repeat constructs in either muscle or non-muscle cells lead to the loss of the myofibrils or stress fibers, respectively. The transfection experiments also indicated that small domains of a protein, 40 to 50 amino acids, can be studied for their localization properties in living cells if a suitable linker is placed between these small domains and the much larger 28 kDa GFP protein.