Figure 5.
Figure 5. Tetrad fluorescence microscopy reveals that for3Δ cdc12 truncation double mutants are defective in contractile ring assembly and constriction. (A) Sections of tetrad plates from crosses between for3Δ and cdc12 truncations grown at 25°C. Double mutants from each tetrad (vertical) are boxed. (B) Differential interference contrast images of a tetratype tetrad from the for3Δ × Δ503-cdc12 cross (JW1665 × JW5124) 3 d after tetrad dissection. The entire colony of the double mutant is shown, while the wt and single mutants are focused at the edge of the colonies where the cells are in a single layer. (C) Same as B for for3Δ × Δ841-cdc12 cross (JW5281 × JW5283). (D) Time courses showing three Δ503-cdc12 for3Δ CRIB-3GFP mRFP-atb2 cells that undergo mitosis during the movies. Time 0 is the presence of a short spindle. Tetrads were dissected from a cross JW5281 × JW5282-2 and imaged on YE5S agar (see Materials and methods) at 23°C. CRIB-3GFP is a marker for cell polarization and mRFP-Atb2 is a marker for MTs and the spindle. (E and F) for3Δ Δ503-cdc12 cells are defective in contractile ring assembly and constriction. Tetrad fluorescence microscopy of a cross between for3Δ rlc1-tdTomato and Δ503-cdc12 rlc1-tdTomato. (E) Timing (mean ± SD) of ring assembly and constriction as shown in F from tetrad colonies. Ring constriction here is from the appearance of a compact ring to ring constriction to a dot (including the ring maturation time). *, P < 0.05; **, P < 0.005 compared with wt. (F) Time courses of four representative cells from the same tetratype tetrad. (G) For3 is crucial for the localization of Cdc12 N-terminal truncations. Tetrad fluorescence microscopy of crosses between Δ503-cdc12-3YFP or Δ841-cdc12-3YFP with for3Δ. The localizations to rings (arrowhead) and clumps (arrow) are marked. Dead cells with high autofluorescence in for3Δ are marked with asterisks. (H) Micrographs (left) of mRFP-Atb2 spindles and Δ503-Cdc12-3GFP localization to faint rings (arrowhead) or clumps (arrow). Graph (right) shows timing (mean ± SD) of Δ503-cdc12-3GFP localization to the medial region of the cell after the appearance of a spindle using a 5–10-min delay in tetrad fluorescence microscopy. *, P < 0.001. Bars: (D and F–H) 5 µm; (B and C) 10 µm.

Tetrad fluorescence microscopy reveals that for3Δ cdc12 truncation double mutants are defective in contractile ring assembly and constriction. (A) Sections of tetrad plates from crosses between for3Δ and cdc12 truncations grown at 25°C. Double mutants from each tetrad (vertical) are boxed. (B) Differential interference contrast images of a tetratype tetrad from the for3Δ × Δ503-cdc12 cross (JW1665 × JW5124) 3 d after tetrad dissection. The entire colony of the double mutant is shown, while the wt and single mutants are focused at the edge of the colonies where the cells are in a single layer. (C) Same as B for for3Δ × Δ841-cdc12 cross (JW5281 × JW5283). (D) Time courses showing three Δ503-cdc12 for3Δ CRIB-3GFP mRFP-atb2 cells that undergo mitosis during the movies. Time 0 is the presence of a short spindle. Tetrads were dissected from a cross JW5281 × JW5282-2 and imaged on YE5S agar (see Materials and methods) at 23°C. CRIB-3GFP is a marker for cell polarization and mRFP-Atb2 is a marker for MTs and the spindle. (E and F) for3Δ Δ503-cdc12 cells are defective in contractile ring assembly and constriction. Tetrad fluorescence microscopy of a cross between for3Δ rlc1-tdTomato and Δ503-cdc12 rlc1-tdTomato. (E) Timing (mean ± SD) of ring assembly and constriction as shown in F from tetrad colonies. Ring constriction here is from the appearance of a compact ring to ring constriction to a dot (including the ring maturation time). *, P < 0.05; **, P < 0.005 compared with wt. (F) Time courses of four representative cells from the same tetratype tetrad. (G) For3 is crucial for the localization of Cdc12 N-terminal truncations. Tetrad fluorescence microscopy of crosses between Δ503-cdc12-3YFP or Δ841-cdc12-3YFP with for3Δ. The localizations to rings (arrowhead) and clumps (arrow) are marked. Dead cells with high autofluorescence in for3Δ are marked with asterisks. (H) Micrographs (left) of mRFP-Atb2 spindles and Δ503-Cdc12-3GFP localization to faint rings (arrowhead) or clumps (arrow). Graph (right) shows timing (mean ± SD) of Δ503-cdc12-3GFP localization to the medial region of the cell after the appearance of a spindle using a 5–10-min delay in tetrad fluorescence microscopy. *, P < 0.001. Bars: (D and F–H) 5 µm; (B and C) 10 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal