Vimentin filaments are assembled from a soluble precursor in avian erythroid cells
The synthesis and assembly of vimentin was studied in erythroid cells from 10-d-old chicken embryos. After various periods of [35S]methionine incorporation, cells were lysed in a Triton X-100-containing buffer and separated into a soluble and an insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis shows that vimentin is almost exclusively present in the cytoskeletal fraction and that newly synthesized vimentin is rapidly incorporated into this fraction. However, after a short pulse-labeling period, a prominent labeled protein at the position of vimentin is present in the soluble fraction. By immunoautoradiography and immunoprecipitations with vimentin antibodies, this protein was identified as vimentin. The vimentin in the soluble fraction is not sedimented by high speed centrifugation, suggesting that it does not consist of short filaments. After different pulse-labeling periods, assembly of newly synthesized vimentin in the cytoskeletal fraction increases linearly, while the radioactivity in the soluble vimentin remains constant. During a 2-h pulse-chase period, the vimentin in the soluble fraction is chased into the cytoskeletal fraction, with a half-life of 7 min. These results suggest that in chicken embryo erythroid cells newly synthesized vimentin is rapidly assembled into filaments from a soluble precursor.
Additional Information© 1983 by The Rockefeller University Press. Received for publication 28 December 1982, and in revised form 3 March 1983. We thank Dr. Bruce Granger for the vimentin antisera and Dr. Randal T. Moon for providing us with the immunoprecipitation protocol. We also thank Dr. Lars Carlsson and Dr. Bruce Granger for many helpful suggestions during the course of this work. This work was supported by grants from the National Institutes of Health, the National Science Foundation, and the Muscular Dystrophy Association of America. Dr. I. Blikstad was also supported by a postdoctoral fellowship from the Swedish Natural Science Research Council. Dr. E. Lazarides is a recipient of a Research Career Development Award from the National Institutes of Health.
Published - BLIjcb83.pdf