(A) i, bundle expulsion mediated by cytosolic proteins; ii, newly polymerized actin filaments that are unable to incorporate into a highly curved actomyosin ring. (B) Time-lapse micrograph shows contraction of an actomyosin ring in a cell ghost after addition of LifeAct-GFP (concentration: ~4 ng/µl) and 0.5 mM ATP. Arrows: expulsion of actomyosin bundles. (C) Cell ghosts containing Rlc1p-GFP were treated with 0.5 mM ATP for 20 min or 40 min respectively, and stained with CF633-phalloidin to visualize F-actin structures. (D) Quantification of the number of expelled bundles, total bundle length, and average bundle length in each cell ghost after 40 min ATP treatment (n = 32 cell ghosts, 137 bundles). (E) Quantification of the sum actin intensity in cell ghosts (ring + bundles) or ring (ring) after treatment with ATP. To measure the total actin intensity in a cell ghost stained with CF633-phalloidin, a square to cover the actomyosin ring and the associated bundles as the region-of-interest was selected, and the sum intensity of the region-of-interest was measured. For the actin intensity of the ring in cell ghosts, a line along the ring circumference to cover the ring area was selected as the region-of-interest for sum intensity measurement. n = 23, 24 cell ghosts for 20 min, 40 min time point, respectively. ns: no significant difference. ***p<0.001. (F) Immunoblots of actin and Cdc8p from cell ghosts treated with ATP for 20 min and 40 min. S, Supernatant: soluble G-actin or short F-actin. P, Pellet: actomyosin rings and associated bundles. Four blots were used for quantifications. Scale bar: 5 µm. Error bars: s.d.