Dynamic properties of Tub4 at SPBs. (A) Photobleaching of Tub4-yeGFP at the SPB in a nonsynchronized cell. The SPB of the cell shown on the bottom was bleached in late anaphase (yellow arrow) and then was monitored for 2 h over the next cell cycle. After separation of mother SPB (mSPB) and daughter SPB (dSPB; blue arrow), the relative fluorescence intensities (rel. fl. int.) of both the mother and daughter SPBs were measured over time. (B) Tub4-yeGFP FRAP experiments of cells arrested in G1 with α-factor, G1/S by SIC1 overexpression, metaphase by Cdc20 depletion, and anaphase by Tem1 depletion. The Tub4-yeGFP signal at SPBs was bleached with a laser pulse (time −1), and the recovery was followed over time. The mean relative fluorescence signal of nonbleached cells with SD error bars (top), the fitted mean FRAP recovery curves with SD error bars (middle), and examples of bleached cells (bottom) are shown. The mean of t_1/2, Y_max ± SEM, and p-values from the t test for Y_max by comparison with SIC1 overexpression are given at the bottom. Arrows indicate the recovery of Tub4-yeGFP signal over time. (C) As in B (pGal1-SIC1) but with pMet25-SIC1 cells to show that the growth conditions do not influence the outcome of the experiment. (D) TUB4-yeGFP pMet25-UPL-TEM1 cells were arrested in anaphase by Tem1 depletion. One of the SPBs was bleached with a laser pulse, and the relative fluorescence intensities of both SPBs in the cells were followed over time. Fitted mean FRAP recovery curves with SD error bars of bleached SPBs are shown in green; the mean of nonbleached SPBs of cells used for FRAP experiments with SD error bars are shown in black. n, number of analyzed SPBs. Bars: (A–C) 4 µm; (B and C, magnified SPB images) 1.41 µm².