Actin Polymerization in Vitro Proceeds in Three Steps

The in vitro polymerization of G-actin to form F-actin filaments can be monitored by viscometry, sedimentation, fluorescence spectroscopy, and fluorescence microscopy. When actin filaments become long enough to become entangled, the viscosity of the solution increases, which is measured as a decrease in its flow rate in a viscometer. The basis of the sedimentation assay is the ability of ultracentrifugation (100,000g for 30 minutes) to pellet F-actin but not G-actin. The third assay makes use of G-actin covalently labeled with a fluorescent dye; the fluorescence spectrum of the modified G-actin monomer changes when it is polymerized into F-actin. Finally, growth of the labeled filaments can be imaged with a fluorescence microscope. These assays are useful in kinetic studies of actin polymerization and during purification of actin-binding proteins, which cross-link or depolymerize actin filaments.

The in vitro polymerization of G-actin proceeds in three sequential phases (Figure 19-6a). The first nucleation phase is marked by a lag period in which G-actin aggregates into short, unstable oligomers. When the oligomer reaches a cer-

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