Expression, cytoskeletal utilization and dimer formation of tropomyosin derived from retroviral-mediated cDNA transfer. Metabolism of tropomyosin from transduced cDNA.
Expression of the tropomyosin-1 isoform was enhanced by cDNA transfer in non-transformed murine 3T3 fibroblasts and also in v-Ki-ras transformed fibroblasts in which native tropomyosin-1 expression had been reduced and tropomyosin-2 synthesis virtually eliminated by action of the oncogene. The level of synthesis of insert-derived tropomyosin-1 was similar in normal and transformed transductants (3-5 times normal levels). The high level of insert-derived tropomyosin-1 expression resulted in a considerable increase in tropomyosin-1 utilization in the cytoskeleton of transformed cells, but this expression still did not reach normal levels, suggesting an oncogene-related inhibition of tropomyosin utilization. A large proportion of newly synthesized native tropomyosin-1 in normal, unmodified fibroblasts appeared in homodimers which, upon prolonged incubation, were largely converted to the heterodimers. Excess tropomyosin-1 derived from the inserted cDNA also appeared largely as the homodimer in both normal and transformed cells. This homodimer was utilized effectively in the formation of cytoskeletal structures but was partially converted to heterodimer by chain exchange. Under steady-state conditions, approximately 33% of the cytoskeletal tropomyosin-1-containing dimers were homodimers, compared to approximately 10% in normal fibroblasts. The results show that the increased amount of tropomyosin-1 homodimer entering the cytoskeleton under conditions of tropomyosin-1 excess, results in an atypical microfilament composition. The effect of this excess of tropomyosin-1 homodimers on stability or function of microfilament fibers remains to be determined. The results also confirm that the mechanisms of rapid homodimer formation with conversion to heterodimers by chain exchange, known from in vitro studies, also occur in vivo.
Prasad
G L
GL
Cell and Molecular Physiology Section, National Cancer Institute, Bethesda, MD 20892.
Fuldner
R A
RA
Braverman
R
R
McDuffie
E
E
Cooper
H L
HL
D016475:3T3 Cells; D000818:Animals; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D011108:Polymers; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D014335:Tropomyosin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D011108:Polymers; D014335:Tropomyosin