Monomer-polymer equilibria in the axon: direct measurement of tubulin and actin as polymer and monomer in axoplasm

The monomer-polymer equilibria for tubulin and actin were analyzed for the cytoskeleton of the squid giant axon. Two methods were evaluated for measuring the concentrations of monomer, soluble (equilibrium) polymer, and stable polymer in extruded axoplasm. One method, the Kinetic Equilibration Paradigm ( KEP ), employs the basic principles of diffusion to distinguish freely diffusible monomer from proteins that are present in the form of polymer. The other method is pharmacological and employs either taxol or phalloidin to stabilize the microtubules and microfilaments, respectively. The results of the two methods agree and demonstrate that 22-36% of the tubulin and 41-47% of the actin are monomeric. The in vivo concentration of monomeric actin and tubulin were two to three times higher than the concentration required to polymerize these proteins in vitro, suggesting that assembly of these proteins is regulated by additional mechanisms in the axon. A significant fraction of the polymerized actin and tubulin in the axoplasm was stable microtubules and microfilaments, which suggests that the dissociation reaction is blocked at both ends of these polymers. These results are discussed in relationship to the axonal transport of the cytoskeleton and with regard to the ability of axons to change their shape in response to environmental stimuli.