Transfection of chicken skeletal muscle alpha-actinin cDNA into nonmuscle and myogenic cells: dimerization is not essential for alpha-actinin to bind to microfilaments.
alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F-actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin-binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments.
Tokuue
Y
Y
Department of Biology, Chiba University, Japan.
Goto
S
S
Imamura
M
M
Obinata
T
T
Masaki
T
T
Endo
T
T
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000185:Actinin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002642:Chick Embryo; D002645:Chickens; D003062:Codon / Q000235:genetics; D005347:Fibroblasts / Q000502:physiology; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
D003062:Codon; D000185:Actinin; D012313:RNA