Figure 7.

Newly induced Spc110p-YFP incorporates into both the new and the old SPB, consistent with exchange during G1/S. (A) Quantification of the amount of “extra” Spc110p at the SPB due to induction. SPC110-YFP, MET3 SPC110-YFP cells (TYY80-5A) were grown in SD-met+met and arrested with α-factor (see MATERIALS AND METHODS). Half of the culture was induced by removal of methionine, whereas the other half was instead washed with SD-met+met and incubated for 90 min further in the continued presence of α-factor. Both cultures were then washed with SD-met+met and released into the cell cycle in SD-met+met. The fluorescence intensity of YFP at the SPBs in induced and uninduced cells that had separated their SPBs was compared. N = 47 and 70 for uninduced and induced, respectively. Bars represent the means. (B) An SPC110-CFP, MET3 SPC110-YFP (TYY69-2A) culture was subject to a 90-min pulse of induction of Spc110p-YFP (see MATERIALS AND METHODS). Images in the CFP and YFP channels were captured at regular intervals. Time is indicated in min after release: 0–10 min (G1), cells are unbudded; 30 min (S phase), SPBs are duplicated but not yet separated; 40–60 min (G2), cells are budded and SPBs have separated; 70 min, anaphase. Bar, 5 μm. (C) YFP fluorescence intensity of both SPBs was measured (see MATERIALS AND METHODS) in cells 40, 50, and 60 min after release from α-factor arrest for the experiment shown in B. The average ratio of YFP fluorescence intensity at the old SPB/YFP fluorescence intensity at the new SPB, determined for each cell (N = 37–41), was 1.06 ± 0.25. The SPB closest to the bud neck was designated the old SPB. Time is in min after release from α-factor arrest. Bars represent means.