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First published online 28 September 2004
doi: 10.1242/jcs.01409

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The GIN4 family kinase, Cdr2p, acts independently of septins in fission yeast

Jennifer L. Morrell^*, Connie B. Nichols and Kathleen L. Gould

Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Medical Center North B-2309, 1161 21st Avenue, Nashville, TN 37232, USA

View larger version (50K): [in a new window]   Fig. 6. Requirements for Cdr2p localization. (A) Schematic diagram of cdr2 deletion constructs. The black box indicates the N-terminal kinase domain; the asterisk indicates the D133A mutation. Cells expressing each construct were tested for their localization pattern and their ability to complement the nitrogen deprivation defect of cdr2 null cells; see Fig. 7. (B-H) Wild-type cells (KGY246) expressing (B) GFP-Cdr2p or (C) GFP-Cdr2p(E177A); cdr2 cells (KGY520) expressing (D) GFP-Cdr2p(747-775) or (E) GFP-Cdr2p(1-252) under the control of the weak nmt81 promoter; cdr2 cells (KGY520) expressing (F) Cdr2pE177A, (G) Cdr2p(1-252), or (H) Cdr2p(332-775) under the control of the strong nmt1 promoter. All cells were grown in the absence of thiamine for 18 hours at 32°C. Images of live cells were captured in B-E. Cells shown in F-H were fixed in 10% formaldehyde solution and stained with calcofluor.

View larger version (49K): [in a new window]   Fig. 1. Cdr2p-GFP localizes to the medial region of cells. (A) Cdr2p-GFP was visualized in live cells (KGY681) grown in YE medium at 25°C. (B) Interphase and mitotic cells showing a broad or thin band of Cdr2p-GFP, respectively. In panels on the right, cells are rotated to illustrate cortical localization. (C) Cdr2p-GFP was visualized in live cells also expressing Sid4p-GFP (KGY961) as a spindle pole body marker to determine cell cycle stage. Cells representing different cell cycle stages are shown. Individual cells are outlined with gray lines. Indicated stages of cell cycle: a, interphase; b and c, metaphase; d and e, anaphase/telophase; f, next cell cycle.

View larger version (56K): [in a new window]   Fig. 2. Mid1p does not influence Cdr2p localization. (A) cdr2-CFP mid1-GFP (KGY4421) cells were fixed in ethanol; both Cdr2p-CFP and Mid1p-GFP were photographed separately and the images were then merged. Cells representing different localization patterns through the cell cycle are shown. a, interphase; b, late G2; c, early mitosis; d, late mitosis. (B) mid1-GFP cdr2 (KGY4079) cells were grown at 25°C and Mid1p-GFP was visualized in ethanol fixed cells. (C) cdr2-GFP mid1 (KGY4393) cells were grown at 25°C and Cdr2p-GFP was visualized in live cells.

View larger version (62K): [in a new window]   Fig. 3. Cdr2p is mislocalized following disruption of sterol-rich membrane domains. (A) cdr2-GFP (KGY681) cells were grown for 1 hour at 32°C in 5 µg/ml filipin, 10 µg/ml filipin or 0.01% DMSO as a solvent control. Cdr2p-GFP was visualized in live cells. Control cells grown in DMSO were treated with filipin for 1 minute and observed immediately. (B) cdr2-GFP cdc25-22 cells (KGY2773) were grown to mid-log phase at 25°C and shifted to 36°C for 3.5 hours. Cdr2p-GFP was visualized in live cells.

View larger version (104K): [in a new window]   Fig. 4. Septins and Cdr2p localize independently. (A) spn1-CFP cdr2-YFP (KGY719) cells were grown in YE medium at 25°C and both Spn1p-CFP (red channel) and Cdr2p-YFP (green channel) were photographed and then the images merged. Individual cells are outlined with gray lines. (B) cdr2-GFP spn4 (KGY718) cells were grown at 25°C and Cdr2p-GFP was visualized in live cells. (C) spn3-GFP cdr2 (KGY138) cells were grown at 25°C and Spn3p-GFP was visualized in live cells. Cells are also rotated in the Z-axis to show Spn3p ring organization (lower panel). (D) spn3-GFP (KGY3244) cells were transformed with pREP1-cdr2+ and grown in the absence of thiamine to induce expression for 20 hours at 32°C. Images of live cells were captured. Cells are also rotated in the Z-axis to show Spn3p ring organization (lower panel).

View larger version (132K): [in a new window]   Fig. 5. Cdr2p and septins are functionally independent. (A,B) spn4-deleted cells (KGY925) were transformed with pREP1 or pREP1cdr2+ and colonies that formed in the presence of thiamine were struck to plates with and without thiamine (A) or grown in liquid cultures in the absence of thiamine for 18 hours before DIC images were captured (B). (C) spn4-deleted cells (KGY4509) were grown to exponential phase in minimal medium and then washed into minimal medium lacking nitrogen. DIC images were captured of cells 48 hours later.

View larger version (71K): [in a new window]   Fig. 7. Structure/function analysis of Cdr2p. A cdr2::ura4+ leu1-32 strain (KGY1519) was transformed with (A) empty pREP1 vector, (B) pREP1cdr2+, (C) pREP1cdr2747-775, (D) pREP1cdr2730-775, (E) pREP1cdr2679-775, (F) pREP1cdr2332-775, (G) pREP1cdr2730-746 or (H) pREP1cdr2E177A. Transformants were grown to mid-log phase in selective medium. Cells were then collected, filtered, washed, and released into selective medium lacking nitrogen for 48 hours, at which time phase micrographs were taken. Bar, 10 µm.