Targeting of cardiac muscle titin fragments to the Z-bands and dense bodies of living muscle and non-muscle cells

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 seque nced, 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 2-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-b and titin, linked to GFP, were expressed in cultured cardiomyocytes to dete rmine 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. S imilar transfections of skeletal myotubes and non-muscle cells lead to the localization of these cardiac z-repeats in the Z-bands of the myofibrils an d 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 myo fibrils or stress fibers, respectively. The transfection experiments also i ndicated that small domains of a protein, 40 to 50 amino acids, can be stud ied for their localization properties in living cells if a suitable linker is placed between these small domains and the much larger 28 kDa GFP protei n. Cell,Motil.: Cytoskeleton 45.67-82, 2000. (C) 2000 Wiley-Liss, Inc.