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.