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.

Quantification of γ-TuSC proteins and receptors at SPBs. (A) Tub4-yeGFP, Spc97-yeGFP, yeGFP-Spc98, Spc72-yeGFP, and Spc110-yeGFP signal at SPBs of metaphase and anaphase cells. Bar, 2 µm. (B) γ-TuSC signal at the SPB in anaphase cells. Mean signals ± SEM are given. n, number of analyzed SPBs. (C) Bar diagram of γ-TuSC, Spc72, and Spc110 numbers at the SPB in meta- and anaphase cells with SEM error bars. The molecule numbers were calculated by comparison of the corresponding yeGFP signals to Cse4-yeGFP as a reference. Numbers of yeGFP-tagged proteins per single MT minus end are also given in the diagram (red, cMT and nMT; green, cMT; and blue, nMT). [Tub4]/[Spc97] is the ratio of Tub4 to Spc97. CI, confidence interval.

Analysis of detached cMTs by electron tomography in kar1-Δ15 cells. (A) 3D model of an α-factor–treated kar1-Δ15 cell with three single detached cMTs. Green lines indicate cMTs, purple indicates the nuclear envelope, and blue indicates the plasma membrane. Red dots indicate MT minus ends. Purple, white, and blue dots mark open MT plus ends with different conformations (flared, curled, and blunt). (A1–4) Electron tomograms of MTs and their end structures from the cell on the left. MTs were tracked using the slicer tool in the IMOD software package. (A1) Closed minus end of a single detached cMT (corresponds to the MT end labeled with A1 on the left. (A2) Open plus end of a single detached cMT (A2 on the left). (A3) Closed minus end of an nMT. (A4) Open plus end of an nMT. (A1 and A3) Red arrows denote closed MT minus ends. (A2 and A4) Purple arrows indicate open MT plus ends. (B) 3D model of another α-factor–treated kar1-Δ15 cell with two single detached cMTs. Color coding is as in A. (B1) Electron tomogram of a closed minus end of a single detached cMT (B1 on the left). (B2) Closed minus end of an nMT. (B3 and B4) Open plus ends of two nMTs. (B1–4) Color coding of arrows is as in A1–4. Bars: (A and B) 300 nm; (A1, A3, B1, and B2) 30 nm; (A2, A4, B3, and B4) 50 nm.

Levels of γ-tubulin, nuclear receptors, and SPB core proteins during MT nucleation. (A) MT renucleation assay of TUB4-yeGFP mCherry-TUB1 cells. Nocodazole-arrested cells were released from the cell cycle block by washing the cells to remove nocodazole. The removal of nocodazole triggered MT renucleation. The relative fluorescence intensities (rel. fl. int.) of Tub4-yeGFP were quantified during the time of MT regrowth. The graph on the left shows relative intensities of Tub4-yeGFP at SPBs. Note that the Tub4-yeGFP SPB signal intensity is not changing during the experiment. Deconvolved images of the same cell are shown on the right. One representative experiment is shown. mSPB, mother SPB; dSPB, daughter SPB. (B) MT renucleation assay of SPC110-yeGFP, Spc42-yeGFP, and TUB4-yeGFP cells. Experiments were performed as in A. One representative experiment for each cell type is shown. Note that cells in B do not contain mCherry-TUB1. (A and B) Bars, 5 µm.

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.

Influence of αβ-tubulin on γ-tubulin binding to the SPB. (A) Localization of β-tubulin at SPBs in TUB2 wild-type and tub2-403 cells with TUB4-yeGFP. Both cell types were synchronized with α-factor, subsequently incubated with nocodazole, fixed, and stained with anti-Tub2 antibodies. (top) Images of fixed cells. Signals of anti-Tub2, Tub4-GFP, and stained DNA (blue) are shown. (bottom) Quantification of absolute intensities of anti-Tub2 at the SPB in tub2-403 and TUB2 cells. n, number of analyzed SPBs; fl. int., fluorescence intensity; a.u., arbitrary unit. (B) FRAP experiments of Tub4-yeGFP of TUB2 and tub2-403 cells treated with nocodazole for 1 h after synchronization with α-factor. (top) Relative fluorescence intensity (rel. fl. int.) of nonbleached cells. (middle) Mean FRAP recovery curve with SD error bars. (bottom) Images of photobleached cells and mean of t_1/2 and Y_max ± SEM are shown. The t test (**, P = 0.0024) is for the t_1/2 of the experiment. Bars: (A and B) 5 µm; (B, magnified SPB images) 2.7 µm².

γ-TuSC molecules at free cMT minus ends. (A) kar1-Δ15 mCherry-TUB1 cells with the indicated yeGFP-tagged genes were incubated with α-factor. This triggered detachment of cMTs from the SPB (illustrated in the cartoon). The images in the middle show cells with detached cMTs (single planes from z stacks). Pictures on the right show enlargements of detached cMTs. Blue arrows indicate minus ends of detached MTs. (B, top) Plot profiles of representative detached cMTs. Red lines indicate the signals of tagged cMTs, and green lines indicate the signals of tagged γ-TuSC proteins or Spc72 on MT minus ends. The numbers in the graph indicate MTs with yeGFP signals at MT ends versus the total number of analyzed MTs. rel. int., relative intensity. (bottom) Images of the cMTs that were used to create plot profiles (single planes from z stacks). The long white arrows indicate the region used for the line scans. (C) Functionality of detached γ-TuSC in MT nucleation. α-factor–arrested and nocodazole-treated kar1-Δ15 cells were washed with medium containing α-factor to remove nocodazole (t = 0). Images were acquired every 5 min to monitor MT renucleation. The enlargement on the bottom shows the detached cMT nucleation site with Spc72-yeGFP (green) and nucleated MTs (red). Images are single planes from z stacks. (D) Distribution of the numbers of tagged γ-TuSC proteins and Spc72 per single MT minus end plotted against the number of analyzed MTs. Mean number of molecules ± SEM, number of analyzed MTs (n), and number of independent datasets are given in the graphs. [Tub4]/[Spc97] and [Tub4]/[Spc98] indicate the ratio of Tub4 to Spc97 and Tub4 to Spc98, respectively. CI, confidence interval. Bars: (A, B, and C) 2 µm; (A and C, enlargements) 0.5 µm.

Levels of γ-TuSC and receptors during the cell cycle. (A, left) The signal intensity of the indicated yeGFP-tagged γ-TuSC proteins was analyzed by time-lapse analysis (graph of mean relative intensity with SD error bars) n is the number of cells, and t = 0 min indicates the time of SPB separation. The time of SPB duplication is indicated from t = −20 to t = 0 min. During this time interval, the measured signal is derived from two SPBs. dSPB, daughter SPB; mSPB, mother SPB. (right) Images from time-lapse videos of representative cells expressing the gene fusion indicated in the figure. (B) As in A but with SPC72-yeGFP or SPC110-yeGFP. (A and B) rel. fl. int., relative fluorescence intensity. Bars, 2 µm.

Model for MT nucleation of γ-TuSCs at the SPB. (A) An extended ring of 17 γ-tubulin molecules is assembled as a nucleation platform. This template is stably associated with the SPB in a cell cycle–dependent manner and further stabilized by the nucleation of MTs. Tubulin dimers interacting more strongly with γ-tubulin than with the MT plus end are the basis for MT nucleation. (B) Model of how the excess of Tub4 and Spc98 could stabilize the γ-TuSC ring.