Figure 3.

Live-Cell Imaging NScc1’s Dissociation from Smc3

(A and B) An array of 448 tetracycline operators (TetO) was integrated between the BMH1 and PDA1 genes on the long arm of chromosome V in haploid (C, K23761 and K23764) or diploid (A, K23388; B, K23183) cells expressing a version of Scc3 fused to the Tet repressor as well as low levels of a Tet repressor protein fused to mCherry (TetR-mCherry) to mark the location of operators. Exponentially growing cells (in YPD medium at 25°C) were placed on 2.5% agarose pads made of synthetic complete medium containing glucose. Live-cell imaging was performed under a spinning disk confocal system at 25°C. The recruitment of C-terminally GFP-tagged Scc1 (A) and C-terminally GFP-tagged Smc3 (B) to the TetO arrays through Scc3-TetR fusion protein is shown (arrows).

(C) The localization of N-terminally GFP-tagged Scc1 to TetO arrays in wild-type (upper panel) waplΔ cells (lower panels) is marked with arrows. We failed to detect GFP-NScc1 at the Tet operators in 20 or more late anaphase/telophase nuclei in Wpl1⁺ cells.