TROPOMYOSIN SUBUNITS: NORMAL AND TRANSFORMED CELLS

  • Warren, Robert H., (PI)

Project: Research project

Description

The purpose of this project is to determine the role played by tropomyosin
in the organization of the actin cytoskeleton in normal and transformed rat
kidney cells in culture. The normal cells contain five isoforms of subunit
tropmyosin: two of 30 kilodalton apparent molecular weight (as determined
on two-dimensional polyacrylamide gels, one of 35\kilodalton, one of
37\kilodalton, and one of 40\kilodalton. Transformed cells contain the
30\kilodalton isoforms but have only trace amounts of the 40\kilodalton
isoform and are lacking the 35 and 37\kilodalton isoforms. Experiments are
being conducted on the effects of microinjecting complete mixtures of
isoforms of tropomyosin purified from normal cells into transformed cells
in order to determine if the transformed cells can acquire the ability to
organize stress fibers. Using a rabbit polyclonal antibody that binds to
the 35, 37, and 40\kilodalton isoforms but not to the 30\kilodalton
isoforms, we have found them to be localized in stress fibers in fully
spread cells. In freshly plated cells, we have found that the antibody
stains the ruffles at the peripheries of the cells, in contrast to previous
reports that ruffles lacked tropomyosin. The same antibody also stains
ruffles at the edges of transformed cells, which never contain stress
fibers. Injection of an affinity-purified derivative of this antibody into
normal cells disrupts stress fibers in the cells and alters cell shape.
Another antibody has been raised in rabbits to a 30\kilodalton protein from
transformed cells that appears to be an isoform of tropomyosin. This
antibody stains a reticular network just beneath the plasmalemma of normal
and transformed cells but does not stain stress fibers in normal cells.
The possibility that this is a membrane-associated isoform of tropomyosin
is being investigated. Monoclonal antibodies to individual tropomyosin
isoforms are now being raised. Actin binding studies indicate that the
30\kilodalton tropomyosin isoforms bind less tightly to actin than the
other three isoforms. (L)
StatusFinished
Effective start/end date8/1/821/31/89

Funding

  • National Institutes of Health
  • National Institutes of Health

Fingerprint

Tropomyosin
Protein Isoforms
Stress Fibers
Antibodies
Actins
Coloring Agents
Rabbits
Cell Shape
Actin Cytoskeleton

ASJC

  • Medicine(all)