Incorporation of microinjected mutant and wildtype recombinant tropomyosins into stress fibers in fibroblasts.
The structural requirements for assembly of tropomyosin into stress fibers were investigated by microinjecting wildtype and four mutant striated chicken muscle alpha-tropomyosins expressed in E. coli as fusion and nonfusion proteins into cultured rat embryo fibroblasts, followed by localization of tropomyosin using indirect immunofluorescence. The results show that the determinants for stress fiber incorporation in living cells correlate with the in vitro actin affinity of these tropomyosins. Wildtype recombinant protein incorporated into stress fibers both when the amino terminus was unacetylated and when it was blocked with an 80-residue fusion protein [Hitchcock-DeGregori, S.E., and Heald, R.W. (1987): J. Biol. Chem. 262:9730-9735]. The pattern of incorporation was indistinguishable from that of tropomyosin isolated from chicken pectoral muscle. The striated alpha-tropomyosin incorporated into stress fibers, even though this isoform is not found in nonmuscle cells. Three recombinant mutant tropomyosins in which one-half, two-thirds, or one actin binding site was deleted were tested [Hitchcock-DeGregori, S.E., and Varnell, T.A. (1990): J. Mol. Biol. 214:885-896]. Only the fusion protein with a full actin binding site deleted incorporated into stress fibers. However, the unacetylated, nonfusion proteins with one half and one actin binding site deleted incorporated into stress fibers, consistent with the ability of troponin to promote the actin binding in vitro. A fourth mutant, in which the conserved amino-terminal nine residues were deleted, did not incorporate into stress fibers, consistent with the complete loss of function of this mutant [Cho, Y.J., Liu, J., and Hitchcock-DeGregori, S.E. (1990): J. Biol. Chem. 265:538-545].
Ranucci
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Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Yamakita
Y
Y
Matsumura
F
F
Hitchcock-DeGregori
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SE
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002645:Chickens / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D008845:Microinjections; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism
D000199:Actins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin