Time-dependent induction of connected cells by overexpression of cdc5ΔN. (A) Strain KLY1083 expressing three copies of GAL1-EGFP-cdc5ΔN homogeneously induced a chained cell phenotype. Typical cell morphologies (DIC images) were shown after culturing these cells under the induction conditions for 7 h (left), 12 h (middle), or 24 h (right). These cells continue to increase cell body numbers without an apparent cell division. Bar: 5 μm. (B) Strain KLY1229 expressing three copies of the corresponding FAA triple mutant failed to induce this phenotype even after 24 h induction. Bar: 5 μm. (C) Quantification of connected cells as a function of time. Percentage of cells in chains was determined by dividing the sum of cells in chains by the total number of cells. cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229. (D) Strain KLY1083 grows without increasing cell numbers under the induction conditions. Cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were taken at the indicated time points upon transferring cultures into YEP-galactose. Cell number was determined by plating serial dilutions on YEP-glucose and counting the colony numbers. The number of cells at time 0 was 1.2 × 106 cells/ml with an OD600 of 0.05. The resulting cell number and OD600 at each time point were divided by those at time 0 to give relative cell number and OD600. (E) The FAA mutations do not influence the stability of EGFP-cdc5ΔN. EGFP-cdc5ΔN was detected by an anti–GFP antibody, whereas Cdc28 (loading control) was recognized by an anti–Cdc28 antibody. 1X, one copy of cdc5ΔN; 3X, three copies of cdc5ΔN.
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