Study of cyclin proteolysis in anaphase-promoting complex (APC) mutant cells reveals the requirement for APC function in the final steps of the fission yeast septation initiation network

Abstract

Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombe mutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APC cut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cut mutants and the normal reorganization of septation initiation network components during anaphase.

FIG. 1

Septation in APC cut mutants proceeds through the SIN. The lid1-6 (A) and lid1-6 cdc11-119 (B) strains were grown in YE medium at 25°C to mid-log phase and shifted to 36°C for 4 h. Cells were collected, fixed with ethanol, and stained with aniline blue to visualize the cell wall and with DAPI to visualize DNA. (C) The lid1-6 sid2-GFP strain was grown at 25°C to mid-log phase, and cells in the G₂ phase were collected by centrifugal elutriation. The cells were then shifted to 36°C, and pictures were taken 220 min into the temperature shift. (D) Illustration of the cut phenotype scored in subsequent experiments.

FIG. 2

Cdc2p kinase activity cycles in APC cut mutants. Wild-type (A and B), lid1-6 (A and C), and cut9-665 (A and D) cells were grown to mid-log phase at 25°C, synchronized in early G₂ phase by centrifugal elutriation, filtered, and released into prewarmed medium at 36°C. (A) Cells were collected at 15-min (for wild type) or 20-min (for APC mutants) intervals and processed for DNA content by flow cytometry. (B to D) The septation index and percentage of cut cells (in APC mutants) were also determined for each sample. The definition of the cut cell phenotype is provided in the Materials and Methods and shown in Fig. 1D. Cell samples were processed for H1 kinase activity, which was visualized by autoradiography. The amount of Cdc2p in the assayed immunoprecipitates was determined by immunoblotting using PSTAIR monoclonal antibodies.

FIG. 3

Cdc2p phospho-Tyr15 levels continue to cycle in the APC cut mutants. Wild-type (A), lid1-6 (B), and cut9-665 (C) cells were grown to mid-log phase at 25°C in YE medium, synchronized in early G₂ by centrifugal elutriation, filtered, and then released into prewarmed medium at 36°C. The cells were collected at 15-min (for wild-type cells) or 20-min (for APC mutants) intervals, and the septation index and percentage of cut cells were determined in each sample. Protein lysates were also prepared from each sample. Cdc2p levels and the Cdc2p-Tyr15 phosphorylation state were determined by immunoblotting using PSTAIR monoclonal and phospho-Cdc2 (Tyr15) polyclonal antibodies, respectively. anti-PTyr, antiphosphotyrosine.

FIG. 4

Cdc2p kinase activity cycles in APC cut mutants in the absence of Rum1p. (A to C) rum1Δ (A), lid1-6 rum1Δ (B), and cut9-665 rum1Δ (C) cells were grown to mid-log phase at 25°C in YE medium, synchronized in early G₂ phase by centrifugal elutriation, filtered, and then released to 36°C. Samples were collected every 15 min (for the rum1Δ single mutant) or 20 min (for APC mutants) and analyzed for H1 kinase activity, Cdc2p levels by immunoblotting using PSTAIR antibodies, and DNA content by FACS. The septation index and the percentage of cut cells were also measured at each time point. Representative images of DAPI-stained lid1-6 rum1Δ and cut9-665 rum1Δ cells at 300 min are shown at the right-hand ends of panels B and C. (D) Histograms of DNA content for rum1Δ, lid1-6 rum1Δ, and cut9-665rum1Δ cells from the experiments in panels A to C. Cells from each time point were fixed in ethanol and stained with Sytox green, and DNA content was measured by FACS analysis.

FIG. 5

Cdc13p is degraded in APC cut mutants. (A to C) Wild-type (A), lid1-6 (B), and cut9-665 (C) cells were grown to mid-log phase at 25°C in YE medium, synchronized in early G₂ phase by centrifugal elutriation, and then shifted to 36°C. Cells were collected at 15-min (for wild-type cells) or 20-min (for APC mutants) intervals, and the septation index and percentage of cut cells were determined in each sample. Protein lysates were also prepared from each sample. Cdc2p and Cdc13p levels were determined by immunoblotting with PSTAIR monoclonal antibodies and affinity-purified anti-Cdc13p antibodies, respectively. (D) The DNA content of each sample was also determined by flow cytometry.

FIG. 6

Analysis of Cdc13p localization in APC cut mutants. (A and B) Wild-type (A) and lid1-6 (B) cells were grown to mid-log phase at 25°C in YE medium, synchronized in early G₂ phase by centrifugal elutriation, and then shifted to 36°C. Cells were collected at 15-min (for wild-type cells) or 20-min (for lid1-6 cells) intervals and processed for indirect immunofluorescence. The percentage of binucleate cells and septated cells was determined by DAPI staining. The percentage of cells containing spindles was determined by staining cells with the anti-tubulin TAT-1 monoclonal antibody, and the percentage of Cdc13p-positive cells was determined with affinity-purified anti-Cdc13p. (C) Representative fields of lid1-6 cells at the times indicated stained with affinity-purified anti-Cdc13p, TAT-1, or DAPI. Arrows indicate cells containing nuclear Cdc13p, and arrowheads indicate cells with Cdc13p at SPBs.

FIG. 7

APC cut mutants are hypomorphic with respect to Cdc13p degradation. (A to C) The lid1::ura4⁺ allele was covered by an integrated version of the lid1⁺ cDNA under control of the nmt1-T81 promoter (KGY2675). Cells were grown to mid-log phase in the absence of thiamine. Thiamine was then added to repress lid1⁺ expression (time = 0). (A) DAPI-stained cells 27 h later. (B) Samples were examined for Cdc13p and Cdc2p abundance by immunoblotting at the indicated number of hours following thiamine addition. (C) Samples were examined for microtubule structures with TAT-1 antibody and Cdc13p localization using affinity-purified anti-Cdc13p at the same time point as in panel A. The arrowhead indicates a cut cell in which Cdc13p is no longer detectable. (D) lid1-6 cells were grown to mid-log phase at 25°C, shifted to 36°C for 4 h, and stained with DAPI.

FIG. 8

High Cdc2p activity delays septation in an APC cut mutant. cdc7-HA cells with an integrated copy of cdc13ΔDB (Cdc13p lacking the destruction box) under the control of the nmt1 promoter (KGY2474) were grown at 32°C in the presence or absence of thiamine, and samples were collected 18 h later. (A) Overproduction of Cdc13ΔDBp. Cells were collected before induction (+T) or 18 h following induction (−T), and Cdc13p and Cdc13ΔDBp levels were detected by immunoblotting using anti-Cdc13p antibodies. Cdc2p, as detected by PSTAIR antibodies, was used as a loading control. (B) Parallel samples to those used in panel A were processed for H1 kinase activity. Cdc2p, as detected by PSTAIR antibodies, was used as a loading control. (C) Samples collected in parallel to those used in panels A and B were fixed and stained with TAT-1 antibodies and Alexa-conjugated goat anti-mouse secondary antibodies. The percentage of cells containing an anaphase spindle was determined by microscopic examination before (+T) and after (−T) induction of Cdc13ΔDBp. (D) The APC is active in cells overexpressing Cdc13ΔDBp. The level of Cut2p-myc was determined in cdc10-V50 cells (KGY1573) 0 or 4 h after the shift to 36°C and in cells in which Cdc13ΔDBp was overproduced for 0 or 18 h (KGY3493). The lower band represents a degradation product. (E to H) The nuc2-663 mutant (KGY352) or the nuc2-663 mutant just beginning to express Cdc13ΔDBp (KGY3115) (19 h following thiamine removal) was synchronized in G₂ at 25°C by centrifugal elutriation and shifted to 36°C. The percentage of cells with septa (E) and the percentage of elongated cells containing a single nucleus and no septum (F) were determined following the temperature shift. Photomicrographs of nuc2-663 (G) and nuc2-663-overproducing Cdc13ΔDBp (H) at the 280-min time point are shown.

FIG. 9

Cdc13ΔDBp overproduction delays the relocalization of SIN components. (A and B) Cdc7p remains on both SPBs under conditions of high Cdc2p kinase activity. cdc7-HA cells with an integrated copy of cdc13ΔDB under the control of the nmt1 promoter (KGY2474) were grown at 32°C in the presence (A) or absence (B) of thiamine, and samples were collected 18 h later. Cells were fixed in methanol and stained with anti-HA (12CA5) monoclonal antibodies followed by Alexa-conjugated goat anti-mouse secondary antibodies. Rows: a, cells in metaphase or early anaphase; b to d, cells in later stages of anaphase. (C and D) Cells producing endogenously tagged GFP-Sid1p and containing the integrated copy of cdc13ΔDB were grown in the presence (C) or absence (D) of thiamine for 18 h and fixed in methanol, and the localization of GFP-Sid1p and DNA was examined. (E) Cells producing endogenously tagged Sid2p-GFP and containing an integrated copy of cdc13ΔDB were grown in the presence or absence of thiamine for 18 h, and the localization of Sid2p-GFP was examined. Rows: a and b, cells in metaphase; c and d, cells in anaphase.

FIG. 10

Model of SIN component rearrangements during the cell cycle.