Figure 4. Half-bridge elongation is a discrete step in SPB duplication.
(A) SIM images from asynchronously growing cells (SLJ9741) containing GFP-Sfi1 (green) and Spc42-mCherry (red). A merged image showing the cell outline (dashes) was used together with spindle length to approximate the cell cycle position indicated. Bar, 2 µm. The SPB(s) are shown to the right of each cell. Arrowheads in the merged images point to the satellite. Bar, 200 nm. (B) GFP-SFI1 mps1-1 cells (SHJ3829) were grown to log phase at 24°C, shifted to restrictive temperature (37°C, 4 hr) and then prepared for immunoEM with nanogold secondary label. Shown are two representative cells with labeling at the SPB (left) and the distal tip of the elongated half-bridge (right). Some cells also contained labeling closer to the center of the elongated half-bridge (left). +: label representation. (C) Wild-type (JA372) and mps1-1 (JA368) cells containing GFP-Sfi1 and Spc42-mCherry were grown at 24°C or shifted to 37°C for 4 hr then analyzed by SIM. Because mps1-1 cells arrest in mitosis at the non-permissive temperature (Winey et al., 1991), only large budded cells were examined. The SPBs from early mitotic wild-type cells showed co-localization of GFP-Sfi1 and Spc42-mCherry (95 ± 4%, n = 90, at 24°C or 94 ± 1%, n = 117, at 37°C), with 87 ± 14% (24°C) and 80 ± 17% (37°C) displaying co-localization at both poles. 94 ± 2% (n = 104) of mitotic mps1-1 cells grown at 24°C showed the same localization as wild-type, with 86 ± 1% of cells exhibiting co-localization at both poles. At 37°C, 49 ± 7% of cells showed co-localization of GFP-Sfi1 and Spc42-mCherry, with the majority of cells displaying a single focus of each (40% of all mps1-1 cells at 37°C). 24 ± 7% and 19 ± 3% of SPBs (n = 85) contained a single focus of Spc42-mCherry or two Spc42-mCherry, respectively, with two GFP-Sfi1 foci. Error bars, SEM.