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First published online 29 November 2005
doi: 10.1242/jcs.02687

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Ace2p contributes to fission yeast septin ring assembly by regulating mid2⁺ expression

Howard Hughes Medical Institute, and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

View larger version (47K): [in a new window]   Fig. 1. Ace2p regulates mid2 RNA and protein levels and functions downstream of Sep1p. (A) cdc25-22 ace2 (KGY3236) cells or (C) cdc25-22 mid2-myc13 ace2 (KGY4727) cells were arrested in G2 by shifting to 36°C for 4 hours. Cultures were released to 25°C and cell pellets were collected every 15 minutes for northern blot or immunoblot analysis, respectively. RNA collected from the cdc25-22 ace2 pellets was used for northern blot analysis alongside RNA from wild-type cells using the his3+ transcript as a loading control. (B) Protein lysates prepared from mid2-myc13 ace2 (KGY4518) and mid2-myc13 (KGY2432) strains were run on a 4-12% Bis-Tris gel and immunoblotted with 9E10 antibody to determine Mid2p levels and to determine the levels of Cdc2p that served as a loading control. (C) Protein lysates were prepared from the cdc25-22 mid2-myc13 ace2 (KGY4727) pellets and resolved by SDS-PAGE. Immunoblotting was performed as in (B) along with anti-Cdc13p (determined by blotting with GJG56 anti-Cdc13 rabbit serum) as a marker for cell-cycle progression. (D) The nmt1sep1-HA3 mid2-myc13 (KGY4235) strain was grown under conditions that repressed (+T) or derepressed (–T) sep1+ expression at 32°C for 20 hours. Protein lysates were prepared and resolved alongside wild-type (KGY246) lysates on a 4-12% Bis-Tris gel. Mid2p-Myc13 levels were determined by immunoblotting with the 9E10 antibody and Cdc2p (loading control) levels were detected with anti-PSTAIRE. (E) The mid2-myc13 (KGY2432) strain containing the pREP3X-ace2 plasmid (pKG3141) was grown and immunoblotted as in (D). (F) sep1 mid2-HA3 (KGY3420) and mid2-HA3 (KGY3131) cells containing either empty pREP3X vector (1) or pREP3X-ace2 vector (2) were grown in the absence of thiamine at 32°C for 20 hours to express ace2+. Protein lysates were prepared and immunoblotted as in (D). (G) A mid2-myc13 strain lacking ace2+ and with sep1 under control of the nmt1 promoter (KGY4740) was grown and immunoblotted alongside mid2-myc13 lysate as in (D).

View larger version (83K): [in a new window]   Fig. 2. Ace2p affects cell morphology and septin ring organization. (A) Wild-type (KGY246) cells containing the pREP3X-ace2 (pKG3141) plasmid were incubated at 25°C for 22 hours in the absence of thiamine to induce production of Ace2p. Cells were fixed in ethanol and stained with DAPI to visualize nuclei. (B) mid2-GFP (KGY2419) cells alone (left panels) or containing the pREP3X-ace2 (pKG3141) plasmid (right panels) were grown and visualized as in (A). (C) spn3-GFP (KGY3337) cells alone (top panel) or containing the pREP3X-ace2 (pKG3141) plasmid (bottom panel) were grown at 25°C for 22 hours in the absence of thiamine to induce Ace2p production. Cells fixed in 70% ethanol were visualized. (D) The spn3-GFP (KGY3337) and spn3-GFP ace2 (KGY4774) strains were grown at 25°C. Confocal images of live cells were obtained. Z-series optical sections were taken at 0.5 µM spacing. Images were rotated on the Z-axis to visualize the septin rings. Bars, 5 µm. Arrows indicate split ring structures that form after septation.

View larger version (85K): [in a new window]   Fig. 3. Mid2p functions in parallel with other Ace2p targets. (A) The spn3-GFP agn1 (KGY5091), spn3-gfp adg1 (KGY5093), spn3-GFP adg2 (KGY5094), and spn3-gfp adg3 (KGY5095) strains were grown at 32°C and visualized as in (Fig. 2D). (B) mid2 (KGY3135), eng1 (KGY5032), mid2 eng1 (KGY5467) and mid2 eng1 agn1 (KGY5505) cells were grown at 32°C and stained with DAPI to visualize DNA and Methyl Blue to visualize septa. (C) Quantification of the number of septa in cells from (B) in addition to wild-type (KGY246), agn1 (KGY5033), and eng1 agn1 (KGY5034) cells. In each case, 200 cells were examined. (D) spn3-gfp ace2 (KGY4774) containing the pREP41-mid2+ plasmid (pKG2208) was grown at 32°C for 18 hours in media lacking thiamine (–T). Confocal images of live cells were obtained. Z-series optical sections were taken at 0.5 µM spacing. Images were rotated on the Z-axis to visualize the septin rings. Arrows indicate split ring structures that form after septation. Bars, 5 µm.

View larger version (30K): [in a new window]   Fig. 4. Cell-cycle regulation of Ace2p. (A) cdc25-22 ace2-myc13 (KGY4472) cells were shifted to 36°C for 4 hours to block cells in G2. Cell pellets were collected every 15 minutes after shift down to 25°C and were fixed in ethanol or frozen. The septation index (SI) was determined to monitor progression through the cell cycle. The ethanol-fixed samples were stained with DAPI to visualize the nuclei and allow microscopic calculation of the percentage of binucleates (BN). Protein lysates were prepared and resolved by SDS-PAGE. Ace2p-Myc13 levels were determined by immunoblotting with 9E10 antibody. In these and other experiments with multi-septated cells, the fluctuation of Cdc13p abundance (determined as in Fig. 1C) was used to assess mitotic progression and Cdc2p abundance (determined by blotting with anti-PSTAIRE) served as a loading control. (B) Denatured protein lysates prepared from wild-type (KGY246), ace2-myc13 (KGY1124) and ace2-myc13 sep1 (KGY1141) strains were resolved on a 4-12% Bis-Tris gel and immunoblotted with antibodies to myc (9E10) (top panel) or to Cdc2p (PSTAIRE) (lower panel). (C) The nmt1sep1-HA ace2-myc13 (KGY4735) strain was grown to mid-log phase in the absence or presence of thiamine (–T/+T) at 32°C for 20 hours. Protein lysates were prepared, resolved on a 4-12% Bis-Tris gel, and immunoblotted as in (B) or with the 12CA5 antibody. (D) The nmt1sep1-HA3 (KGY4221) strain was grown as in (C). Cells were fixed in ethanol, stained with Methyl Blue and DAPI and imaged. (E) The ace2-myc13 mts3-1 (KGY4536) and ace2-HA3 mts3-1 (KGY5314) strains were grown at 25°C and then shifted to 36°C for 4 hours to block proteasome function and arrest cells in mitosis. Ace2p-Myc13 was immunoprecipitated with the 9E10 antibody and Ace2p-HA3 was immunoprecipitated with the 12CA5 antibody, followed by incubation in the presence or absence of -phosphatase. (F) The mid2-myc13 mts3-1 (KGY1976; lane 1), mts3-1 (KGY1135; lane 2), ace2-myc13 mts3-1 (KGY5231) strains containing the pREP1-His6-Ubiquitin plasmid (pKG1284; lane 4) and the ace2-myc13 mts3-1 (KGY4536; lane 3) strain without vector were grown at 25°C for 22 hours in the absence of thiamine in order to induce His-Ub production. This was followed by a 4-hour shift to 36°C. Substrates modified with ubiquitin were isolated and detected by immunoblotting with anti-Myc antibodies.

View larger version (42K): [in a new window]   Fig. 5. Ace2p localization and regulation. (A) The ace2-GFP (KGY4385) strain was grown at 25°C and confocal images of live cells were obtained. (B) The ace2-GFP sid4-GFP (KGY1311) strain was fixed in ethanol and the spindle pole body marker Sid4p was used to determine cell-cycle stage. 100 cells at each cell-cycle stage were examined to determine the percentage of cells expressing Ace2p-GFP. Representative images of different stages are shown. (C) Ace2p-HA3 was immunoprecipitated from native protein lysates prepared from wild-type (KGY246; lane 1), ace2-HA3 (KGY1123; lane 2), ace2-HA3 fkh2 (KGY5388; lane 3) and ace2-HA3 mbx1 (KGY5394; lane 4) strains with the 12CA5 antibody. Immunoprecipitates were resolved as in Fig. 4B as well as an aliquot of protein lysate set aside prior to immunoprecipitation. Immunoblotting was performed using antibodies against HA (12CA5; upper panel) or against Cdc2p (PSTAIRE; lower panel) to demonstrate that immunopreciptation was performed on equal amounts of protein within the lysates. (D) ace2-gfp fkh2 (KGY5361) (left panel) or ace2-gfp mbx1 (KGY5393) (right panel) cells were grown in at 32°C. Confocal images of live cells were obtained. Bars, 5 µm.

View larger version (29K): [in a new window]   Fig. 6. The SIN is not required for timely expression of ace2+ or mid2+. (A) cdc7-24 (KGY2061) cells were synchronized at the G2-M boundary by centrifugal elutriation. The synchronous population was shifted to 36°C. Cell pellets were collected every 20 minutes beginning 30 minutes after the shift. RNA recovered from the pellets was used for northern blot analysis of mid2+, ace2+ and his3+ (loading control). An aliquot of cells was also collected at each time point, fixed in ethanol and stained with DAPI in order to microscopically visualize the cell nuclei and monitor progression through the cell cycle. (B) mid2-GFP cdc16-116 (KGY741) cells were synchronized by centrifugal elutriation and treated as in (A). The percentage of binucleate cells (BN) was determined by DAPI staining. Septation index was monitored throughout the time course.

View larger version (13K): [in a new window]   Fig. 7. Model for transcriptional control of cell separation. In separate waves of transcription, Sep1p and Ace2p promote expression of genes critical for cell separation. Ace2p controls expression of four sets of genes. The first group includes genes encoding the glucanases Agn1p and Eng1p; the second group, consisting of cfh4+, adg1+, adg2+ and adg3+, encodes proteins with as-yet-unknown function. Thirdly, Ace2p controls the expression of mid2+, whose protein product is responsible for proper septin ring organization, an event necessary for proper cell separation.

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