Nuc2p, a subunit of the anaphase-promoting complex, inhibits septation initiation network following cytokinesis in fission yeast

Abstract

In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)-associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain-containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)-related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability.

Figure 1. Hyperactivation of SIN Signaling in nuc2-663 Mutant

(A) nuc2-663 cells were stained with DAPI and aniline blue to visualize nuclei and septum material, respectively. The left panel shows nuc2-663 cells at permissive temperature. The right panel shows nuc2-663 cells at restrictive temperature. (B) Quantification of multiseptated cells in the nuc2-663 mutant. (C) Wild-type and nuc2-663 cells expressing GFP tagged versions of Cdc7p, Sid1p, and Sid2p were grown at 36 °C for 4 h and were visualized using fluorescence microscopy. Arrowheads indicate mitotic cells with Sid1p at both SPBs. Arrows indicate cells in which Sid2p-GFP signal persisted at the division site after completion of cytokinesis. Scale bar, 5 μm.

Figure 2. Ectopic Actomyosin Ring and Septum Formation in the nuc2-663 Mutant After Septation

(A) The diagram schematically illustrates the experimental design. (B) Quantification of cells with normal or excessive septa after 1–2 h release from metaphase block. At least 200 cells were counted in each category. (C) Examples of septum patterns in nuc2-663 nda3-KM311 cells. Four examples of cells with excessive septum material are shown. i and ii, multiseptated cells; iii, excessive deposition of septum material at division site; iv, cell with ectopic misoriented septum. (D) Visualization of actomyosin rings in nuc2-663 nda3-KM311 cells by immunofluorescence microscopy. Top, middle, and bottom panels show cells with actomyosin rings that were formed straight, close to, or misoriented with respect to the previous division site, respectively. Microtubules were stained with TAT-1 antibody, and the actomyosin ring was stained with anti-Cdc4p antibody. (E) Septum assembly in S-phase–arrested cells. Wild-type, nuc2-663, and cdc16-116 cells arrested in S phase by hydroxyurea treatment were either formaldehyde fixed for DAPI and aniline blue staining or methanol fixed for immunostaining with TAT-1. (F) Quantification of septated cells versus non-septated cells upon hydroxyurea treatment. At least 300 cells were counted for each category. Scale bar, 5 μm.

Figure 3. Actomyosin Rings Are Assembled, but Not Maintained, at the Division Site in Cells Overexpressing Nuc2p

(A) Quantification of the number(s) of nuclei/cell and the frequency of their appearance upon overexpression of Nuc2p. Cells expressing Uch2p-GFP as a nuclear marker were used in the experiment. (B) Visualization of F-actin. Cells overexpressing Nuc2p were fixed and stained with phalloidin and DAPI to visualize the F-actin cytoskeleton and nuclei, respectively. (C) Visualization of Cdc15p. Cells carrying Cdc15p-GFP were induced to overexpress Nuc2p and visualized by fluorescence microscopy. +T/−T indicates medium supplemented with or without thiamine. (D) Time-lapse fluorescence microscopy to image the dynamics of actomyosin ring assembly and constriction. Cells were grown in medium with or without thiamine and imaged by time-lapse microscopy. The nucleus and actomyosin ring were marked by Uch2p-GFP and Rlc1p-GFP, respectively. Scale bar, 5 μm.

Figure 4. Overexpression of Nuc2p Inhibits SIN Signaling

(A) nmt1-nuc2 cells expressing Sid4p-GFP, Cdc7p-GFP, and GFP-Sid1p were visualized by fluorescence microscopy in the presence (+T, top panel) or absence (−T, bottom panel) of thiamine. The actomyosin ring (visualized with Rlc1p-GFP) was used as the marker for cells undergoing cytokinesis. (B) Fluorescence signal of Sid2p is reduced in cells overproducing Nuc2p. nmt1-nuc2 cells expressing Sid2p-GFP were visualized using fluorescence microscopy upon growth on medium supplemented with or without thiamine. SPBs and actomyosin ring were marked by Sid4p-mRFP and Rlc1p-mRFP, respectively. (C) Quantification of the relative fluorescence intensities of Sid2p-GFP and Sid4p-mRFP in Nuc2p-uninduced and -induced cells. (D) Quantification of actomyosin ring containing cells with Cdc7p-GFP or GFP-Sid1p at the SPBs. At least 80 cells were counted for each category. (E) Cdc7p is not recruited to the SPBs in cells overproducing Nuc2p. Top panel, control cell grown on minimal medium supplemented with thiamine. Arrowhead indicates localization of Cdc7p-GFP at the SPB. Bottom panel, cell grown in medium without thiamine to overexpress Nuc2p. Rlc1p-GFP was used in these cells to label actomyosin ring. Cells were grown on agarose pad containing growth medium and were imaged by laser scanning microscopy. Scale bar, 5 μm.

Figure 5. Overexpression of Nuc2p Does Not Affect the Steady-State Levels of Cdc7p-3HA and Sid2p-13Myc, but Disrupts the Binding of Spg1p-GFP and Cdc7p-3HA

(A) Protein levels of Cdc7p-3HA and Sid2p-13Myc in nmt1-nuc2 cells grown in medium with or without thiamine. Tubulin serves as the loading control. (B) Lysates from cells expressing Cdc7p-3HA and Spg1p-GFP in the presence or absence of Nuc2p overexpression were immunoprecipitated with GFP antibodies and immunoblotted with antibodies against the HA epitope. Lysates from a strain without Spg1p-GFP was included as the control. Tubulin serves as the loading control. Asterisk indicates heavy chain of IgG.

Figure 6. Inactivation of Cdc16p Function Promotes Localization of Cdc7p to SPBs and Allows Septation in Cells Overexpressing Nuc2p

(A) Diagram schematically illustrates the experimental design. Nuc2p was induced for 24 h at 26 °C, and the culture was shifted to 36 °C to inactivate Cdc16p and examined after 3 h of incubation. (B) The localization of Cdc7p-GFP in cdc16-116 cells overexpressing Nuc2p was visualized by fluorescence microscopy at the permissive or restrictive temperatures. (C) Septum assembly is restored upon inactivation of Cdc16p in cells overexpressing Nuc2p. DAPI and aniline blue staining of formaldehyde fixed cells is shown. (D) Quantification of septated cells versus non-septated cells in nmt1-nuc2 and nmt1-nuc2 cdc16-116. Note that only cells with four nuclei were counted. Scale bar, 5 μm.

Figure 7. Nuc2p Acts Independently of Dma1p to Regulate Cytokinesis

(A) Overexpression of Nuc2p in wild-type or dma1Δ mutant. (B) Wild-type, nuc2-663, dma1Δ, and nuc2-663 dma1Δ strains were diluted serially and spotted on YES agar and incubated at 24 °C or 26 °C. (C) Septum patterns in nuc2-663 and nuc2-663 dma1Δ mutants grown at 28 °C. Cells were fixed and stained with DAPI and aniline blue. (D) Visualization of actomyosin rings and microtubules in nuc2-663 dma1Δ cells by immunofluorescence microscopy. Microtubules were stained with TAT-1 antibody and the actomyosin ring was stained with antibodies against Cdc4p. About 150 cells were counted in each category. Arrowhead indicates septum assembled in previous cell division.

Figure 8. Model Summarizing the Temporal Regulation of Cytokinesis in S. pombe