We have purified the transformation and shape change sensitive isoform of an actin associated polypeptide doublet previously described by us (Shapland, C., P. Lowings, and D. Lawson. 1988. J. Cell Biol. 107:153-161) and have shown that it is evolutionarily conserved as far back as yeast. The purified protein: (a) binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of approximately 7.5 x 10(5) M-1; and (b) causes actin filament gelation within 2 min. Although these activities are controlled by ionic strength (and may be mediated by positively charged amino acid residues) the molecule remains as a monomer irrespective of ionic conditions. EM reveals that the addition of this protein to actin filaments converts them from a loose, random distribution into a tangled, cross-linked meshwork within 1 min, and discrete tightly aggregated foci after 10 min. By use of an "add-back" cell permeabilization system we can rebind this molecule specifically to actin filaments in cells from which it has previously been removed. Since the protein is transformation sensitive and gels actin, we have named it transgelin.
By using a monoclonal antibody we have identified a new polypeptide doublet (C4h and C4l) of Mr approximately 21 kD and pI 8 and 7, respectively, that is associated with and (at the immunofluorescence level) uniformly distributed on actin filament bundles in rat, mouse, and other vertebrate species. C4 is absent in neurones, erythrocytes, and skeletal muscle but the epitope is evolutionarily conserved as it is present in invertebrates such as molluscs and crustaceans. C4h is not found in cells such as lymphocytes and oncogenically transformed mesenchymal cells where actin stress fiber bundles are reduced in number or absent. C4l, on the other hand, is always present. C4h expression can also be blocked by switching normal nontransformed mesenchymal cells from adherent to suspension culture. Reexpression of C4h occurs 24 h after these cells are returned to normal adherent culture conditions, but can be blocked by either actinomycin D or cycloheximide, suggesting that the expression of this epitope is regulated at the transcriptional level.