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 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.
Ayoob
J C
JC
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104-6804, USA.
Turnacioglu
K K
KK
Mittal
B
B
Sanger
J M
JM
Sanger
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D064211:Connectin; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D006321:Heart / Q000196:embryology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011784:Quail; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D064211:Connectin; D008164:Luminescent Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012313:RNA; D011494:Protein Kinases