(A–B) Low-speed (10,000 x g) sedimentation assays of 5 µM Mg-ATP preassembled actin filaments, 500 nM Ain1, and increasing concentrations of Cdc8 (0–5 µM). (A) Coomassie blue-stained gel of pellets and supernatants from a representative experiment. (B) Quantification of actin extracted from the pellet as a function of Cdc8 concentration. Error bars = s.d, n = 2. (C-D) TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) in the presence of 500 nM Cdc8 (C, top), 400 nM Ain1 (C, middle) or both (C, bottom). Dotted lines indicate the bundled region. Scale bars, 5 μm. (D) Quantification of the percent of bundled F-actin with 500 nM Cdc8 alone, 400 nM Ain1 alone, or 400 nM Ain1 with 50 nM, 125 nM, 250 nM or 500 nM Cdc8. Black lines indicate averages and green circles indicate values from independent TIRFM experiments. n = 2 independent experiments for each condition. (E-G) Spot density TIRFM experiments of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) and 500 nM Ain1 (0.5% TMR-labeled) alone or with 1 μM unlabeled Cdc8. (E, top) Representative images of Alexa-488-labeled F-actin bundles. (E, bottom) Representative max projection of all Ain1 spots on corresponding F-actin bundles. Scale bars, 5 μm. (F) Kymographs of the indicated bundle from (E) over time. Scale bar, 5 μm. Time bar, 11 s. (G) Ain1 spot density (Ain1 events/µm/s). Two-tailed t-tests for data sets with unequal variance yielded p-value *p=0.026. Error bars = s.e. n = 2 independent experiments.