Figure 1. Tropomyosin Cdc8 loads cooperatively onto actin filaments.
(A) Two-color TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488 labeled) with a range of concentrations of tropomyosin Cdc8 (Cy5-labeled). Scale bar, 5 µm. (B) Plot of the fraction of actin filament bound by Cdc8 (‘Cdc8 occupancy’) over free Cdc8 dimer concentration. Data were fit to a Hill function, revealing a Hill coefficient >1 (Hill=14.6), that indicates cooperativity. Error bars represent standard error of the mean; n = 2 reactions. (C) Schematic of Cdc8 loading onto an actin filament. Observed cooperativity of Cdc8 could be the result of end-to-end binding of tropomyosin molecules (‘End-to-end cooperativity’) and/or indirect interactions between tropomyosin molecules via changes in the actin filament (‘Indirect cooperativity’).
Figure 1—figure supplement 1. Characterization of Tropomyosin Cdc8 mutants L38C, I76C, and D142C in vitro.
(A) Characteristic tropomyosin coiled-coil heptad repeat organization. Residues with low sequence conservation localized at b-, c-, or f-sites on the outside of the coiled-coil were chosen for mutation to cysteine. (B–C) High-speed sedimentation assay of 1 μM of wild-type (WT) or mutant (L38C, I76C, or D142C) tropomyosin Cdc8 dimer binding to increasing (0–10 μM) concentrations of actin (B) and quantification of Cdc8 in pellet normalized to actin in pellet (C). (D) Two-color TIRFM of 1.5 μM Mg-ATP actin (15% Alexa 488-labeled) and 2.5 μM Cdc8 mutants L38C (left), I76C (middle), or D142C (right) (Cy5-labeled).
Figure 1—figure supplement 2. Characterization of Tropomyosin Cdc8 mutants L38C, I76C, and D142C in vivo.
(A) Morphology (DIC and DAPI/calcofluor) and actin organization (BODIPY-phallicidin) of cells with Cdc8 mutants replacing the endogenous cdc8 gene. Scale bars, 5 μm. (B–D) Quantification of the number of nuclei (B), abnormal septa (C), and the time of ring assembly (D) in cells expressing tropomyosin cdc8 mutants. Two-tailed t-tests for data sets with equal variance yielded p-values: WT vs. L38C: *p-value=1.80×10−10; WT vs. I76C: p-value=0.27, WT vs. D142C: p-value=0.57. n ≥ 10 cells for each condition.