Bni4 and Kcc4 do not colocalize in unbudded cells. DIC and fluorescence images are shown. For merged images, Bni4-CFP and Kcc4-YFP are pseudocolored green and red, respectively. (A) Asymmetric localization of Bni4-CFP and Kcc4-YFP in small- and medium-budded cells of strain YLK189. (B) Spatial separation of Bni4-CFP and Kcc4-YFP in unbudded cells of strain YLK189. (C) Localization of Kcc4-CFP and Bni4-CFP to the inner and outer edges of the Cdc10-YFP septin ring, respectively. 1–3, strain YLK214; 4–6, strain YLK215. (D) Separation of Kcc4-YFP (arrowheads) and Bni4-CFP (arrows) during bud emergence. A cell of strain YLK189 was imaged before bud emergence (1 and 4) and 10 (2 and 5) and 20 (3 and 6) min later. (E) Model showing the location of Bni4 and Kcc4 during the transition of the septin ring into the septin collar upon bud emergence. Bni4 (blue) forms a larger ring that encircles the Kcc4 ring (yellow). At bud emergence, Kcc4 moves toward the growing bud, whereas Bni4 stays on the mother side of the neck. Bars, 1 μm.