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First published online 22 March 2005
doi: 10.1242/jcs.02286

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Interplay between septin organization, cell cycle and cell shape in yeast

Amy S. Gladfelter^*, Lukasz Kozubowski, Trevin R. Zyla and Daniel J. Lew

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA

View larger version (61K): [in a new window]   Fig. 1. Septins contribute to shaping the wide necks in cdc42V36T,K94E mutants and the base of the bud in wild-type cells. (A) Strains DLY3067 (GAL1p-CDC42) and DLY4412 (cdc12-6 GAL1p-CDC42) were transformed with pMOSB55 (CDC42) or pMOSB57 (cdc42V36T,K94E). Cells were photographed after growth to exponential phase at 24°C, the permissive temperature for cdc12-6 mutants, on dextrose-containing medium. (B) The same strains were grown to stationary phase, stained with FITC-ConA to visualize cell wall mannan, washed, and inoculated into fresh medium at 37°C, the restrictive temperature for cdc12-6 mutants. Morphology of new buds (unstained by FITC-ConA) was documented 3 hours later. Bar, 10 µm. (C) Strains DLY4035 (GAL1p-SWE1) and DLY7452 (cdc12-6 GAL1p-SWE1) were grown to stationary phase in raffinose-containing medium at 24°C, and inoculated into galactose-containing medium (to induce Swe1p expression) at 37°C (to inactivate septins). Cell morphology was documented 5 hours later. Bar, 5 µm.

View larger version (39K): [in a new window]   Fig. 2. Septins, but not Bnr1p, contribute to shaping the base of the bud. Strains DLY4035 (GAL1p-SWE1), DLY7452 (cdc12-6 GAL1p-SWE1) and DLY6136 (bnr1 GAL1p-SWE1) were grown to stationary phase in raffinose-containing medium at 24°C, stained with FITC-ConA, washed, and inoculated into galactose-containing medium (to induce Swe1p expression) at 37°C (to inactivate septins). Bud morphology was documented 5 hours later by DIC microscopy, and scored for new buds (unlabeled with FITC-ConA). (A) Image showing the measurements of bud length, neck diameter and bud diameter at 1 µm and 2 µm from the neck. (B) Plots of neck diameter versus bud length for wild-type (DLY4035; CDC12) and septin mutant (DLY7452, cdc12-6) buds. Each circle represents one cell (n=200). (C) Quantitation of neck diameter and bud diameter at 1 µm and 2 µm from the neck. Values are mean ± standard deviation (n=130 for the wild-type, n=184 for the cdc12-6 and n=125 for the bnr1 strains) Asterisk indicates that the bud diameter difference between cdc12-6 and wild-type cells was statistically significant (P<0.001).

View larger version (35K): [in a new window]   Fig. 3. Swe1p-dependent G2 delay promotes assembly of ectopic septin rings. (A,B) Strains DLY4037 (cdc42V36T,K94E), DLY1028 (swe1) and DLY4136 (cdc42V36T,K94E swe1) were grown to exponential phase in YEPD at 30°C and processed to visualize septin distribution. The proportion of budded cdc42 or cdc42 swe1 cells displaying ectopic septins in the bud (top graph: mislocalized septins) or defective (faint, patchy, or absent) septins at the neck (bottom graph: misorganized septins) were scored. Representative cells (B) show that septins at the neck are still defective in cdc42V36T,K94E cells that lack Swe1p. (C) Strains RSY136 (GAL1p-SWE1), DLY4141 (cdc42V36T,K94E GAL1p-SWE1), and DLY4838 (cdc12-6 GAL1p-SWE1) were grown to exponential phase in sucrose-containing medium at 24°C, induced to overexpress Swe1p by addition of galactose (2% final concentration), and processed to visualize septin distribution or cell morphology after a further 4 hours at 24°C. Bar, 10 µm.

View larger version (88K): [in a new window]   Fig. 4. Arrest with high Clb/Cdc28p activity maintains neck-localized septins. (A) Strains DLY7326 (GAL1p-CLB1152) and DLY7327 (cdc42V36T,K94E GAL1p-CLB1152) were grown to exponential phase in sucrose-containing medium at 29°C, induced to overexpress Clb1p152 by addition of galactose (2% final concentration), and processed to visualize septin distribution after a further 4 hours. (B) Strains DLY5 (WT) and DLY5080 (cdc42V36T,K94E) were grown to exponential phase in YEPD at 29°C and nocodazole was added to 15 µg/ml to arrest cells in mitosis. After 3 hours of arrest cells were fixed and processed to visualize septin distribution. Bar, 10 µm.

View larger version (52K): [in a new window]   Fig. 5. Septins disassembled from the neck assemble into new rings at the bud tip during G2 but not M phase. (A) Strain JMY1141 (cdc12-6) was grown to exponential phase in YEPD at 24°C, shifted to 37°C for 30 min, and then shifted back down to 24°C. Samples were taken before the shift (24°C), 30 minutes after shift-up (), and at various times after shift-down (). Arrow indicates original neck. For each sample following shift-down, we quantitated the percentage of cells with septins assembled at the tip or within the bud (B) or as rings in unbudded cells or at the neck (C). These categories were separated in the interests of clarity, and the remaining cells (25%, not graphed) showed no septin staining. (D) Cells were arrested in mitosis (75% large budded cells) by treatment with 15 µg/ml nocodazole for 2 hours at 24°C and then subjected to the same shift-up/shift-down regimen as above. Bar, 10 µm.

View larger version (50K): [in a new window]   Fig. 6. Septin organization in cln1 cln2 cells. Top panels: strains DLY5103 (WT) and DLY5102 (cln1 cln2) were grown to exponential phase in YEPD at 30°C, and the cells were fixed and processed to visualize septins. Bottom panels: strains DLY5106 (GAL1p-SWE1) and DLY5104 (cln1 cln2 GAL1p-SWE1) were grown to exponential phase in YEPsucrose at 30°C, induced to express Swe1p by addition of galactose (2% for 5 hours), and the cells were fixed and processed to visualize septins. Bar, 10 µm.

View larger version (31K): [in a new window]   Fig. 7. Proposed genesis of the cdc42V36T,K94E phenotype. See text for details.

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