SPB duplication and spindle function. (A) Photomicrograph of wild type SPC42:GFP cells from time course in C, 110 minutes after alpha-factor release. Arrowhead shows rebudded cell that has already undergone a second SPB duplication. (B) Photomicrograph of rad23Δdsk2Δddi1ΔSPC42:GFP cells from time course in C, 110 minutes after alpha factor release. Arrow shows cell with G2-like short inter-SPB distance. (C) SPB duplication kinetics. Wild type (WT) and rad23Δdsk2Δddi1Δ (3Δ) cells were arrested with alpha factor and released into rich medium at 37°C. Samples were taken every 10 minutes and SPBs in each cell were counted. (D) Photomicrographs showing SPBs (SPC42:GFP) in wild type, rad23Δdsk2Δ and rad23Δdsk2Δddi1Δ cells at 90 minutes after release from G1 arrest at the restrictive temperature. (E) Photomicrographs showing DIC, SPC42:GFP signals, and DAPI staining of DNA in rad23Δdsk2Δddi1Δ cells at 180 minutes after release from G1 arrest at the restrictive temperature of 37°C. Left two panels show G2-like cells (52% of the large budded cells) with a single nucleus and SPBs typically separated by less than 4 μm. Right two panels show anaphase cells with divided nuclei (44% of the large budded cells) and SPBs typically separated by > 4 μm. The remaining large budded cells had stretched single nuclei. (F) Timing of bud emergence and spindle assembly after release from alpha-factor induced G1 arrest in wild type, rad23Δdsk2Δ, and rad23Δdsk2Δddi1Δ cells. Photomicrograph shows rad23Δdsk2Δddi1ΔTUB1:GFP cells at 37°C with both G2 and anaphase spindles. (G) Nocodazole release experiment in rad23Δdsk2Δddi1ΔSPC42:GFP cells. Cells were arrested with alpha factor, released into medium containing nocodazole and grown at 30°C for 2 hours, then washed and released into medium containing alpha factor to prevent re-budding upon progression to G1. Samples were taken every 10 minutes and SPBs with short G2-like inter-SPB distance (1–4 μm, inset micrograph) were counted (upper panel). This protocol was repeated in rad23Δdsk2Δddi1Δ TUB1:GFP cells (lower panel). Samples were taken every 10 minutes and short G2 spindles (inset micrograph) were counted.