Targeting of nebulin fragments to the cardiac sarcomere.
Nebulin, a vertebrate skeletal muscle actin binding protein, plays an important role in thin filament architecture. Recently, a number of reports have indicated evidence for nebulin expression in vertebrate hearts. To investigate the ability of nebulin to interact with cardiac myofilaments, we have expressed nebulin cDNA fragments tagged with green fluorescent protein (GFP) in chicken cardiomyocytes and PtK2 cells. Nebulin fragments from both the superrepeats and single repeats were expressed minus and plus the nebulin linker. Nebulin fragment incorporation was monitored by fluorescent microscopy and compared with the distribution of actin, alpha-actinin and titin. Expression of nebulin N-terminal superrepeats displayed a punctate cytoplasmic distribution in PtK2 cells and cardiomyocytes. Addition of the nebulin linker to the superrepeats resulted in association of the punctate staining with the myofibrils. Nebulin C-terminal superrepeats plus and minus the linker localized with stress fibers of PtK2 cells and associated with the cardiac myofilaments at the level of the Z-line. Expression of the single repeats plus and minus the nebulin linker region resulted in both a Z-line distribution and an A-band distribution. These data suggest that N-terminal superrepeat nebulin modules are incapable of supporting interactions with the cardiac myofilaments; whereas the C-terminal nebulin modules can. The expression of the N-terminal or C-terminal superrepeats did not alter the distribution of actin, alpha-actinin or titin in either atrial or ventricular cultures.
Panaviene
Zivile
Z
Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 S. Limestone, Lexington, KY 40536, USA.
Deng
Xiaodi A
XA
Esham
Michael
M
Moncman
Carole L
CL
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D060588:LIM Domain Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D012518:Sarcomeres / Q000378:metabolism*; D020079:Terminal Repeat Sequences; D014162:Transfection
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D060588:LIM Domain Proteins; D009124:Muscle Proteins; C486890:NEBL protein, human; C035620:nebulin