SIN-inhibitory phosphatase complex promotes Cdc11p dephosphorylation and propagates SIN asymmetry in fission yeast

Abstract

Background: Cytokinesis in many eukaryotes involves the function of an actomyosin-based contractile ring. In fission yeast, actomyosin ring maturation and stability require a conserved signaling pathway termed the SIN (septation initiation network). The SIN consists of a GTPase (Spg1p) and three protein kinases, all of which localize to the mitotic spindle pole bodies (SPBs). Two of the SIN kinases, Cdc7p and Sid1p, localize asymmetrically to the newly duplicated SPB in late anaphase. How this asymmetry is achieved is not understood, although it is known that their symmetric localization impairs cytokinesis.

Results: Here we characterize a new Forkhead-domain-associated protein, Csc1p, and identify SIN-inhibitory PP2A complex (SIP), which is crucial for the establishment of SIN asymmetry. Csc1p localizes to both SPBs early in mitosis, is lost from the SPB that accumulates Cdc7p, and instead accumulates at the SPB lacking Cdc7p. Csc1p is required for the dephosphorylation of the SIN scaffolding protein Cdc11p and is thereby required for the recruitment of Byr4p, a component of the GTPase-activating subunit for Spg1p, to the SPB.

Conclusions: Because Cdc7p does not bind to GDP-Spg1p, we propose that the SIP-mediated Cdc11p dephosphorylation and the resulting recruitment of Byr4p are among the earliest steps in the establishment of SIN asymmetry.

Figure 1. Csc1p, a new Forkhead-associated domain containing protein localizes to both SPBs and then transiently to one SPB during early mitosis

A. Schematic representation of Csc1p with the FHA domain at the N-terminus. The phosphopeptide binding domain is highlighted between amino acids 30 and 80. B. Csc1p localizes to both SPBs in cells with short mitotic spindles. csc1-GFP mCherry-atb2 cells were grown at 24°C till mid-log phase, fixed with methanol and the localization of Csc1p imaged. C. Csc1p localization depends on entry into mitosis and the spindle pole body component Ppc89p, but does not require microtubules and SIN activity. cdc25-22 cells and sid4-SA1 cells expressing Csc1p-GFP and mCherry-Atb2p were grown at 24°C, shifted to the semi-permissive temperature of 33°C for 5 hours and imaged for the two fluorescent proteins. nda3-KM311 cells expressing Csc1p-GFP and mCherry-Atb2p were grown at 30°C, shifted to the non-permissive temperature of 19°C and imaged for the two fluorescent proteins. nmt81-ppc89 cells expressing Csc1p-GFP and mCherry-Atb2p were grown in EMM medium containing thiamine for 36 hours. The cells were then fixed with methanol imaged and for the two fluorescent proteins.(n=50×3) D. Csc1p shows a transient asymmetric localization to one SPB. cdc25-22 cells expressing Csc1p-GFP and mCherry-Atb2p were arrested at G2 by incubation at 33°C for 3 hours. The cells were then released at 24°C and grown for 1 hour. Cells were fixed with methanol and the localization of Csc1p and microtubules imaged. The percentage of cells with asymmetric distribution of Csc1p is shown at the bottom of the figure (n=26). E–G. Csc1p and Cdc7p segregate to opposite SPBs. E. cdc25-22 cells expressing Csc1p-GFP and Cdc7p-mCherry were grown at the permissive temperature and fixed with methanol. The fluorescence patterns of Csc1p and Cdc7p were scored. Cells that showed signals of both Csc1p and Cdc7p were imaged to analyze the localization relationship of the two proteins at SPB(s). The percentage of cells showing symmetric distribution (top cell) and asymmetric distribution (bottom two cells) of Cdc7p and Csc1p are indicated (n=50×3). F. Csc1p-GFP asymmetry is observed in wild-type cells. Wild-type cells expressing Csc1p-GFP and mCherry-Atb2p were grown at 24°C till mid-log phase, fixed with methanol and scored for the symmetric or asymmetric distribution of Csc1p in cells with short spindles (n=50×3). G. cdc25-22 cells expressing Csc1p-GFP and Cdc7p-mCherry were grown at the permissive temperature and imaged by time-lapse microscopy. Elapsed time (in minutes) after start of the imaging is indicated above the panels. The arrows indicate positions when asymmetry of Cdc7p and Csc1p are clearly apparent.

Figure 2. Csc1p promotes asymmetric localization of SIN-kinases Cdc7p and Sid1p

A. csc1Δ generates uninucleate cells with a division septum and binucleate cells with more than one septum. csc1Δ cells were grown at 30°C, fixed, and stained with DAPI (for DNA) and aniline blue (for cell wall). B. Loss of Csc1p function can cause inappropriate septation in interphase. Wild type (WT) and csc1Δ cells were grown at 24°C or 30°C, treated with 12 mM HU for 6 hrs to arrest the cells at S-phase. Interphase cells with a division septum were quantitated (n=500×3). Cells with septum deposition were counted for WT as well as csc1Δ. C–D. Cdc7p and Sid1p localize symmetrically in csc1Δ cells. Wild type cells expressing Cdc7p OR Sid1p-GFP and mCherry Atb2p were fixed with methanol and the localization of the fluorescent proteins investigated. The fraction of cells displaying Cdc7p or Sid1p in an asymmetric configuration or the fraction of csc1Δ cells displaying a symmetric distribution of Cdc7p or Sid1p are indicated in the figure. E. Simultaneous compromise of Csc1p and Cdc16p function leads to growth defects. csc1Δ, cdc16-116 and csc1Δ cdc16-116 cultures were serially diluted 5 fold for 4 times and spotted on YES-agar medium and grown at 24°C and 30°C. The two parental strains were capable of colony formation at 24°C and 30°C. The csc1Δ cdc16-116 double mutant was capable of colony formation at 24°C, but not at 30°C. F. Simultaneous compromise of Csc1p and Cdc16p generates cells with multiple septa. csc1Δ, cdc16-116 and csc1Δ cdc16-116 mutants were grown at 24°C till early log phase, shifted to 30°C for 3 hours, fixed and stained with DAPI and aniline blue. G. Graphical representation of the percentage of hyperseptated cells in strains described F (n=500×3). H. Rescue of cdc16-116 growth defect with Csc1p overexpression (v-pREP41-GST and csc1-pREP41-csc1-GST). I. Csc1p-GFP cells do not display synthetic lethality with cdc16-116. csc1-GFP, cdc16-116 and csc1-GFP cdc16-116 cultures were serially diluted 5 fold and spotted on YES-agar plates and incubated at 24°C and 30°C.

Figure 3. Identification of a multiprotein complex (SIP) required for generation of Cdc7p asymmetry

A. Csc2p, Csc3p, and Paa1p bind Csc1p-Myc₁₃. GST, Csc3-GST, Csc2-GST and Paa1-GST were isolated from yeast cells expressing Csc1p and one of these proteins using glutathione linked agarose beads. The isolated proteins were immunoblotted with an antibody against the Myc-epitope. The top panel shows the total lysates probed with the anti-Myc antibody, whereas the bottom panel shows an immunoblot of the proteins isolated from glutathione-agarose beads and probed with the anti-Myc antibody. B. Csc2p, Csc3p, and Paa1p localize to early mitotic SPBs. Cells individually mCherry-Atb2p and one of the following fusion proteins, Csc2p-GFP, Csc3p-GFP and Paa1p-GFP were grown at 24°C and the localization patterns of the fluorescent proteins investigated. Shown are 3 examples in each case, of cells with short mitotic spindles. C. The SPB component Ppc89p is required for Csc3p localization to SPB. Spores from a ppc89::ura4⁺/ppc89⁺ strain expressing Csc3p-GFP were germinated in minimal media in the presence or absence of uracil. The cells were then observed for the localization of Csc3p during early mitosis. The proportion of spores germinated in medium containing uracil and exhibiting and exhibiting a short spindle and Csc3p on SPBs is indicated in the figure. In addition, the proportion of spores germinated in the absence of uracil and exhibiting a short spindle and Csc3p on the SPB is indicated in the figure. D. csc2Δ, csc3Δ, csc4Δ, and paa1-L566P display synthetic lethal interactions with cdc16-116. Serial dilutions of csc2Δ cdc16-116, csc3Δ cdc16-116, csc4Δ cdc16-116 and paa1-L566P cdc16-116 and the corresponding single mutants were spotted on YES-agar medium and incubated at 24°C or at 30°C. E. Csc2p, Csc3p, Csc4p, Paa1p, and Ppa3p are required for the generation / maintenance of Cdc7p asymmetry. csc2Δ, csc3Δ, csc4Δ, and ppa3Δ cells expressing Cdc7p-GFP and mCherry-Atb2p were grown at 24°C, fixed with methanol and imaged for Cdc7p localization during late anaphase. paa1-L566P cells expressing Cdc7p-GFP and mCherry-Atb2p were grown at 24°C and then shifted to 36°C and the localization of Cdc7p during late anaphase investigated. The proportion of cells exhibiting symmetric localization of Cdc7p in the various mutants and the proportion of wild-type cells showing asymmetric distribution of Cdc7p are indicated. F. Cdc7p asymmetry is not compromised in the absence of Ppa1p and Ppa2p. Wild-type, ppa1Δ, and ppa2Δ cells expressing mCherry-Cdc7p were grown at 24°C and the localization of Cdc7p during late anaphase investigated. G. Combined loss of Ppa2p and Ppa3p leads to a modest increase in uninucleate cells with division septum. ppa1Δ, ppa2Δ, ppa3Δ, ppa1Δ ppa3Δ, and ppa2Δ ppa3Δ cells were cultured in the presence of 12 mM hydroxyurea and the proportion of uninucleate cells with a division septum estimated (n=500×3).

Figure 4. SIP complex interacts physically with the SIN scaffold protein Cdc11p and promotes dephosphorylation of Cdc11p

A. Restoration of colony formation ability of cdc11-136 upon deletion of csc1 or csc4 genes. Overnight cultures of csc1Δ, csc4Δ, cdc11-136, csc1Δ cdc11-136, and csc4Δ cdc11-136 grown at 24°C were serially diluted 5 fold for 5 times and spotted on YES-agar plates and incubated at 36°C. B. cdc11-136 csc1Δ and cdc11-136 csc4Δ cells assemble normal-appearing septa. csc1Δ, csc4Δ, cdc11-136, csc1Δ cdc11-136, and csc4Δ cdc11-136 cells were grown at 24°C and shifted to 36°C. The cells were fixed and stained with aniline blue (cell wall) and DAPI (DNA). C. The percentage of multinucleate cells in all strains described in B. were estimated and plotted to gain a measure of the extent of suppression of cdc11-136 by loss of function of csc1 or csc4 (n=500×3). D. Cdc11p extracted from csc1Δ cells migrates slower than Cdc11p from wild type cells. cdc11-myc₁₃ and cdc11-myc₁₃ csc1Δ were arrested at S-phase using 12 mM hydroxyurea for four hours. The cells were released by washing three times with YES and samples were collected at indicated times. Cell extracts were resolved on 8% SDS-PAGE and immunoblot analysis was carried out using an anti-Myc antibody. E. SIP promotes dephosphorylation of Cdc11p. nda3-KM311 cdc11-myc₁₃ and nda3-KM311 cdc11-myc₁₃ csc1Δ cells were arrested at prometaphase by shift to 18°C for 4 hours and released to 32°C. The phosphorylation state of Cdc11p was then analysed after 50 min of release by immunoblot analysis using anti-Myc antibody. Cell lysates were prepared and immunoblotted with antibodies against the Myc-epitope (left panel). To establish that the slower migration observed in the csc1Δ cells was a result of increased phosphorylation of Cdc11p, lysates were treated with Lambda phosphatase (right panel).

Figure 5. Modulation of SIP affects the localization of SIN and its regulators

A, B. Byr4p localization to the mitotic SPB depends on SIP. A. Wild-type and csc1Δ cells expressing Cdc7p-3xHA were stained using antibodies against the HA-epitope and against Byr4p. After staining, cells were mounted in DAPI and images acquired (n=50×3). B. csc3Δ, ppa3Δ cells grown at 24°C and paa1-L566P cells grown at 36°C were stained with antibodies against tubulin and Byr4p, mounted in DAPI medium and images acquired. C. Overexpression of Csc1p leads to a failure in cytokinesis. Csc1p was overexpressed by growing nmt1-csc1-GFP cells in minimal medium lacking thiamine for 16 hours at 24°C, fixed and stained with aniline blue (cell wall) and DAPI (DNA). The percentage of multinucleated cells was then determined (n=500×3). D. Overproduction of Csc1p leads to recruitment of Byr4p to both SPBs in mitotic cells. nmt1-csc1-GFP cells were grown and fixed as in (C) above. The cells were then stained using antibodies against Byr4p and tubulin TAT-1 antibody. Arrows indicate an increased accumulation of GFP-Csc1p (n=50×3). E. Quantitation of the percentage of anaphase SPB pairs with Byr4p on one or both the SPBs (n=50×3). F. Cdc7p and Sid1p are displaced from mitotic SPBs upon Csc1p overexpression. nmt1-csc1-GFP expressing Cdc7p-mCherry were grown in EMM medium lacking thiamine for 16 hours at 24°C, fixed with methanol and imaged. To visualize Sid1p, nmt1-csc1-GFP cells expressing Sid1p-Myc₁₃ were stained with antibodies against the Myc-epitope.