EFFECTS OF CYTOCHALASIN, PHALLOIDIN, AND PH ON THE ELONGATION OF ACTIN-FILAMENTS

We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1-mu-M CB and 0.02-mu-M CD for ATP-actin and at 0.1-mu-M CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2-mu-M) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem, 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin. Phalloidin reduces the dissociation rate constants at both ends to near zero and also reduces the association rate constant at the barbed end by about 50%. This confirms and extends the observations of Coluccio and Tilney [Coluccio, L. M., & Tilney, L. G. (1984) J. Cell Biol. 99, 529-535] and provides convincing evidence that phalloidin affects both subunit binding and dissociation. Over the pH range of 6.6-8.3, the pH has very little effect on the association rate constant at the barbed end, but the dissociation rate constant is larger at alkaline pH. At the pointed end, the association rate constant decreases slightly at alkaline pH. Together, these effects account for the higher critical concentration and slower rates of polymerization at alkaline pHs.