Analysis of αβ-tubulin–γ-tubulin interactions. (A) Comparison of colchicine-binding site in β-tubulin with the sequence in yeast γ-tubulin. An alignment of calf brain β-tubulin with S. cerevisiae β-tubulin (TUB2) and γ-tubulin (TUB4) is shown. The sequence alignment was created with CLUSTALW 2.0.12 (Chenna et al., 2003). Asterisks indicate identical residues. Conserved residues are indicated by two dots and semiconserved residues by one dot. Residues, which interact with colchicine, are marked in red. (B) Analysis of binding of tubulin to KTs and SPBs. SPC42-yeGFP and NUF2-yeGFP cells were incubated with or without nocodazole. Cells were fixed and prepared for indirect immunofluorescence. TUB4 auxin degron cells (tub4Δ; Nishimura et al., 2009) with SPC42-yeGFP were analyzed after auxin addition and Tub4 depletion by indirect immunofluorescence with anti-Tub2 and anti-Tub4 antibodies. DNA was stained with DAPI. Bars, 5 µm. (C) GFP-TUB1 SPC42-eqFP611 cells were incubated with nocodazole for 1 h after synchronization with α-factor. GFP-Tub1 at the SPB was bleached with a laser pulse, and the recovery was followed over time. The fitted mean FRAP recovery curves with SD error bars are shown (top). An example of a bleached cell over time is shown (bottom). n, number of analyzed cells; rel. fl. int., relative fluorescence intensity. (D) FLIP experiment of GFP-TUB1 SPC42-eqFP cells. The cells were synchronized with α-factor and subsequently released into YPAD medium with nocodazole until cells had arrested with a large bud in metaphase. The daughter cells were consecutively bleached, and the intensities of GFP-Tub1 at SPBs were recorded. (top) Mean graphs of relative intensities at the SPB with SD error bars. (bottom) Image of a representative bleached cell over time. X indicates the bleached region in the daughter cell. Bars: (C) 4 µm; (C, magnified SPB images) 2.44 µm²; (D) 2 µm; (D, magnified SPB images) 2.06 µm².