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. 2009 Sep 7;186(5):739–753. doi: 10.1083/jcb.200902116

Figure 3.

Figure 3.

Etd1 helps coordinate spindle elongation with SIN activation. (A) mad2Δ cdc7-GFP cells were grown in YE at 25°C and then shifted to YE containing MBC. Cdc7-GFP was imaged by time-lapse microscopy in the presence of MBC. Changes in fluorescence of Cdc7-GFP were measured in arbitrary units (A.U.) for either of the two types of cells: those that had already completed anaphase at the time of MBC addition and displayed Cdc7-GFP signal at one SPB toward the cell tip (type 1) and those that entered mitosis after the addition of MBC and displayed Cdc7-GFP signal at one or two SPBs near the cell center (type 2). A total of 43 type 1 cells and 34 type 2 cells were imaged. One representative experiment for each type is shown. (B and C) mad2Δ cdc7-GFP rlc1-GFP cells (B) or mad2Δ nmt41-GFP-etd1 cdc7-GFP rlc1-GFP cells (C), which moderately overexpressed etd1+, were grown without thiamine and then imaged by time-lapse microscopy in the presence of MBC. (A–C) Time is indicated in minutes. (D) Targeting of Etd1 to the SPB induces ectopic activation of the SIN pathway. etd1Δ cdc7-mCherry or etd1Δ mCherry-atb2 cells carrying the pREP41-EGFP-Etd1-Ppc89261–783 plasmid were cultured at 36°C in EMM liquid medium plus thiamine and shifted to media without thiamine for an additional 24 h. Samples were taken at 0 (repressed) and 24 h after thiamine removal (induced), fixed, and visualized for DIC, DAPI, CW (top panels), and Cdc7-mCherry and mCherry-Atb2 fluorescence (bottom panels). The number of mononucleate cells (1n) with a septum and binucleate cells (2n) with more than one septum versus the total number of cells is shown for each condition.