Figure 5.

Localization of GFP-Chs4 and Bni4-GFP by time-lapse imaging. (A) In the BNI4 cells (KT1972 cotransformed with pAR26 and p366), GFP-Chs4 accumulates at the site of the presumptive bud (0 min, top arrow) and it disappears soon after bud emergence. GFP-Chs4 reappears at the neck before cytokinesis (100 arrowhead). (B) In the bni4^V-A/F-A cells (KT1972 cotransformed with pAR26 and pAR17), GFP-Chs4 does not accumulate at the site of bud emergence. However, it appears at the bud neck before cytokinesis (0 and 46 min, arrowheads). (C) Localization of Bni4-GFP by time-lapse imaging (YLK80). Bni4-GFP appears at the future bud emergence site ∼15 min before budding (15 min, arrow) and stays at the bud neck at relatively high levels for 60–80% of the cell cycle (100 and 175 min, arrowheads). Close to cytokinesis, Bni4 forms a double ring that is hardly detectable (175 min, arrow). (D) Relative fluorescence of GFP-Chs4 (solid line) and Bni4-GFP (dotted line) was plotted as a function of the progress through the cell cycle (percentage of the cell cycle completed) by using the first appearance of GFP at the incipient bud site as the start point and the appearance of GFP at the incipient bud site in the mother cells in the next generation as the endpoint. Shown are representative data from three cells of each strain for which complete cell cycle data were obtained. A video supplement to Figure 5, A–C, was prepared from images taken at 5-min intervals. Bars (A and C), 5 μm.