Sarcomeric-alpha-actinin defective in vinculin-binding causes Z-line expansion and nemaline-like body formation in cultured chick myotubes.
The Z-line in each striated muscle has a precisely defined width that corresponds to muscle fiber type, and it can enlarge several fold in nemaline myopathy. To explore the mechanism(s) underlying Z-line width and structure maintenance, a series of sarcomeric-alpha-actinin mutants tagged with myc-epitope was transfected into cultured chick myotubes. By double-staining transfected myotubes with myc and myofibrillar protein antibodies, we found that alpha-actinin mutants with deletion of the region from the beginning of the fourth spectrin repeat to the start of the EF-hands resulted in expansion of Z-line width, often displayed a doublet staining pattern, and resulted in formation of nemaline-like bodies in older myotubes under fluorescence microscope. Yeast-two hybridization analysis demonstrated that this region was involved in vinculin binding, and for vinculin to bind alpha-actinin, residues 1-116 and 258-323 were required. Hence, we have defined a critical region of s-alpha-actinin that affects the width and integrity of the Z-line. This region is at least involved in the interaction with vinculin.
Zhang
Zhi-Qian
ZQ
Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Clinical Research Laboratory, Beijing Cancer Hospital and Institute, Peking University School of Oncology, Beijing 100142, PR China. zlzqzhang@bjmu.edu.cn
Bish
Lawrence T
LT
Holtzer
Howard
H
Sweeney
H Lee
HL
D000185:Actinin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D030161:Avian Proteins / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D064211:Connectin; D017353:Gene Deletion; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017696:Myopathies, Nemaline / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000662:veterinary; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000378:metabolism; D012518:Sarcomeres / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013049:Spectrin / Q000737:chemistry; D016596:Vinculin / Q000378:metabolism*
D030161:Avian Proteins; D064211:Connectin; D009124:Muscle Proteins; C035689:VCL protein, Gallus gallus; D000185:Actinin; D016596:Vinculin; D013049:Spectrin; D011494:Protein Kinases