Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores

Abstract

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filaments. The rapidly growing ('barbed') end has higher association and dissociation rate constants than the slowly growing ('pointed') end. The value of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 μM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.