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?2, B and D). For samples containing 7.5 ��M SpPrf, the steady-state bundle density is established at 800 s, but it takes >5000?s for 95% of the actin in the sample to polymerize. During this period of ?4000 s, bundles are locked in?place relative to one another and de novo bundle assembly is prohibited. As polymerization continues, the thickness of?existing bundles increases ( Fig.?S2), presumably by recruiting new filaments or by the elongation of existing filaments. Thus, limiting the F-actin nucleation rate promotes the formation of a smaller number of thicker bundles and increases the amount of time required to reach a steady state. In the presence of saturating SpPrf concentrations, the fission yeast formin Cdc12 accelerates filament nucleation but does not significantly affect filament elongation rates (Table 1) (16). As Cdc12 is added to profilin-actin Vorinostat purchase samples, the nucleation rate has been shown to increase proportionally with Cdc12 concentration (20?and?38). To assess this effect on actin assembly dynamics, we first examined actin assembly via pyrene fluorescence by spontaneously assembling samples of 5 ��M G-actin, 15 ��M SpPrf, and varying concentrations of Cdc12. Increasing the concentration of Cdc12 from 1 to 250?nM dramatically accelerates actin polymerization ( Fig.?3A), decreasing the time to 95% F-actin assembly from 5655?s to 975 s, with the effect of Cdc12 saturating at around 100?nM ( Fig.?3B). These changes in actin assembly have pronounced selleck effects on the kinetics and steady-state architectures of networks formed with 1.2 ��M ��-actinin ( Fig.?3C, Movie S3). As the concentration of Cdc12 increases, the time at which filaments and bundles first appear decreases, from 245?s for 0?nM to GPX5