The majority of rings in cell ghosts undergo full contraction upon stabilization of actin filaments. (A, top) Rlc1-GFP rings in cell ghosts incubated with 0.5 mM ATP and 100 µM blebbistatin. (Bottom) Rlc1-mCherry rings in cell ghosts incubated with 0.5 mM AMP-PNP. (B) Rlc1-mCherry rings in cell ghosts were stained with GST-LifeAct-GFP and incubated with 0.5 mM AMP-PNP (four rings) or 0.5 mM ATP (11 rings). (C) Contraction of rings in cell ghosts in the presence of 20 µM jasp (56 rings); rings in cell ghosts that underwent full-ring contraction versus those that formed clusters were quantitated. Full, full-ring contraction. (D) The change of Rlc1-GFP ring perimeters over time in cell ghosts was quantitated (11 rings each sample). (E) Rlc1-mCherry rings in cell ghosts were incubated with ATP with or without 5 µM Pha for 40 min, and then stained with purified GST-LifeAct-GFP. (F) Pha treatment stabilizes actin filaments in rings in cell ghosts. Proteins were extracted from ATP-treated rings in cell ghosts with or without Pha, and immunoblots were probed with α-actin or Cdc8p. Asterisks, actin; S, supernatant; P, pellet. (G) Quantification of the intensity of bands in protein blots (four protein blots). (H) An ultracentrifugation assay of the actin proteins during contraction of rings in cell ghosts. G, globular actin; F, filamentous actin; S, supernatant; P, pellet. Quantification of the band intensity on the protein blots (two protein blots). Shown at top and bottom are blots exposed for different durations. The intensity of bands (G and F lanes) in protein blots was quantitated. (I) Inhibition of loss of actin filaments (flux out) by jasp or Pha from the rings in cell ghosts improves ring contraction efficiency in the absence of actin polymerization (flux in).