The Scw1 RNA-Binding Domain Protein Regulates Septation and Cell-Wall Structure in Fission Yeast

Corresponding author: Paul G. Young, Rm. 2443, Biosciences Complex, Queen's University, Kingston, ON K7L-3N6, Canada., youngpg{at}biology.queensu.ca (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Loss of the nonessential RNA-binding domain protein, Scw1, increases resistance to cell-wall-degrading enzymes in fission yeast. Surprisingly, scw1 null mutations also suppress the lethality of mutations (cdc11-136, cdc7-24, cdc14-118, sid1-239, sid2-250, sid3-106, sid4-A1, and mob1-1) at all levels of the sid pathway. This pathway forms part of the septation initiation network (SIN), which regulates the onset of septum formation and ensures the proper coupling of mitosis to cytokinesis. In contrast, scw1^- mutations do not suppress ts alleles of the rng genes, cdc12 or cdc15. These mutations also prevent the formation of a septum and in addition block assembly and/or function of the contractile acto-myosin ring. sid mutants exhibit a hyper-sensitivity to cell-wall-degrading enzymes that is suppressed by loss of Scw1. Furthermore, scw1^--mediated rescue of sid mutants is abolished in the presence of calcofluor white, a compound that interferes with cell-wall synthesis. These data suggest that Scw1 acts in opposition to the SIN as a negative regulator of cell-wall/septum deposition. Unlike components of the SIN, Scw1 is predominantly a cytoplasmic protein and is not localized to the spindle pole body.

CYTOKINESIS requires a large network of genes that function in a highly coordinated manner (LE GOFF et al. 1999 ; BALASUBRAMANIAN et al. 2000 ). One of our best models of cytokinesis is the fission yeast Schizosaccharomyces pombe, in which the process of cytoplasmic division can be separated into four phases: (1) the selection of the site of division; (2) the assembly of an acto-myosin-based medial ring; (3) contraction of the ring concurrent with the deposition of a primary septum and two flanking secondary septa; and (4) degradation of the primary septum leading to the physical separation of two daughter cells.

Mutants specifically defective in each of these processes have been identified and constitute the pos, mid, and pom mutants defective in positioning of the division site (EDAMATSU and TOYOSHIMA 1996 ; SOHRMANN et al. 1996 ; BAHLER and PRINGLE 1998 ; BAHLER et al. 1998A ; PAOLETTI and CHANG 2000 ); the rng mutants defective in assembly of the medial acto-myosin ring (BALASUBRAMANIAN et al. 1992 , BALASUBRAMANIAN et al. 1994 , BALASUBRAMANIAN et al. 1998 ; MCCOLLUM et al. 1995 ; CHANG et al. 1996 ; BEZANILLA et al. 1997 ; CHANG et al. 1997 ; GOULD and SIMANIS 1997 ; KITAYAMA et al. 1997 ; MAY et al. 1997 ; MOTEGI et al. 1997 ; ENG et al. 1998 ; NAQVI et al. 1999 ; WONG et al. 2000 ); the sid (septation initiation defective) mutants defective in the initiation of septum deposition (NURSE and THURIAUX 1976 ; MARKS et al. 1992 ; SCHMIDT et al. 1997 ; BALASUBRAMANIAN et al. 1998 ; SPARKS et al. 1999 ; CHANG and GOULD 2000 ; GUERTIN et al. 2000 ; HOU et al. 2000 ; SALIMOVA et al. 2000 ); and the sep mutants defective in cell separation (SIPICZKI et al. 1993 ; GRALLERT et al. 1999 ; RIBAR et al. 1999 ; ZILAHI et al. 2000A , ZILAHI et al. 2000B ).

The sid class of mutants belongs to a regulatory module (referred to as the septation initiation network, or SIN) that localizes to the spindle pole body and functions in signaling the onset of septum formation once per cell cycle. This network consists of the products of the cdc16, byr4, cdc11, cdc7, cdc14, spg1/sid3, sid1, sid2, sid4, and mob1 genes (reviewed in SAWIN 2000 ; MCCOLLUM and GOULD 2001 ).

Sid4 and Cdc11 are the most upstream members of this network and are localized constitutively to the spindle pole body (SPB) throughout the cell cycle. Sid4 is required for the recruitment of all other tested SIN components (Cdc11, Byr4, Spg1, Cdc7, Sid1, Sid2, Mob1, and Cdc14) to this subcellular location, whereas Cdc11 is required for the recruitment of Spg1, Sid2, Cdc7, Mob1, and Byr4, but not Sid4. Sid4 and Cdc11 physically interact, suggesting that the Sid4-Cdc11 complex may act as a scaffold (CHANG and GOULD 2000 ; SAWIN 2000 ; KRAPP et al. 2001 ).

The Spg1 GTPase, which acts as an on/off switch for septum formation, is also constitutively localized to the SPB but is kept in an inactive guanosine diphosphate-bound state by the GTPase activating protein (GAP) activity of a complex formed between Byr4 and Cdc16 (FURGE et al. 1998 , FURGE et al. 1999 ). Upon mitotic entry Spg1 is activated at both SPBs and in this form is able to recruit the protein kinase Cdc7. Intriguingly, Spg1 becomes deactivated at one SPB during anaphase, leaving only the other SPB colocalized with Cdc7 (SCHMIDT et al. 1997 ; SOHRMANN et al. 1998 ). The Cdc7 kinase is then required, after cyclin proteolysis at the end of anaphase, for the recruitment of the Sid1-Cdc14 protein kinase complex to the SPB (GUERTIN et al. 2000 ). This in turn is required for the translocation of the Sid2-Mob1 protein kinase complex from the SPB to the acto-myosin ring where it is thought to phosphorylate targets that trigger ring constriction and septum deposition (SPARKS et al. 1999 ).

Here we describe the initial characterization of an RNA-binding domain protein, Scw1, whose loss results in increased resistance to cell-wall-degrading enzymes. scw1 null mutations are also able to suppress the lethality and septation defects associated with sid mutants. In this report we demonstrate that sid mutants themselves exhibit a decrease in resistance to cell-wall-degrading enzymes and, in addition, that loss of scw1 counteracts this effect. Furthermore, we show that scw1^--mediated suppression of sid mutants is abolished in the presence of the cell-wall synthesis inhibitor, calcofluor white. These data suggest that scw1 acts as a negative regulator of cell-wall/septum deposition acting in opposition to the SIN.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, and growth conditions:
All S. pombe strains used in this study (Table 1) were derived from strains 972 h^- or 975 h⁺. Cells were grown in yeast extract medium supplemented with adenine (YEA; ALFA et al. 1993 ) or in appropriately supplemented Edinburgh minimal medium (EMM) adjusted to pH 5.5 or 3.5 (SALEKI et al. 1997 ). Strains were streaked to YEA supplemented with 0.5 mg/ml calcofluor white M2R (Polysciences, No. 4359) where indicated in the text. All genetic crosses and general yeast techniques were performed using standard methods (MORENO et al. 1991 ).

Isolation and cloning of the scw1 gene:
The scw1-1 (strong cell wall) mutation was isolated in a screen analogous to SALEKI et al. 1997 aimed at identifying mutants blocked for cell cycle progression under conditions of lowered extracellular pH. The scw1 gene was cloned by functional complementation of the cell size and colony morphology phenotype displayed by a scw1-1 leu1-32 h⁺ strain when plated on EMM pH 3.5 at 36°. Cells were transformed with a HindIII partial genomic library in pWH5 (P. G. YOUNG and D. BEACH, unpublished data) and a single leu⁺ transformant displaying a wild-type colony morphology was isolated after visually screening 18,000 transformants. Plasmid co-loss experiments showed that the complementing activity was due to the presence of the plasmid (p5ARES) and integrative mapping showed that the plasmid had integrated at or near the scw1 locus (indicating that it contained the scw1⁺ gene and not a multicopy suppressor). Recovery of the plasmid, followed by sequencing from either end of the genomic insert, comparison to Sanger Centre sequence, subcloning, and plasmid complementation, identified the scw1⁺ open reading frame (ORF) as SPCC16C4.07.

Disruption of the scw1 gene:
The scw1 gene was disrupted using the one-step gene disruption method (ROTHSTEIN 1983 ). A 2.1-kb SalI-SphI fragment containing the scw1⁺ gene was cloned into the unique SalI and SphI sites of the pGEMT plasmid (Promega, Madison, WI). The HindIII-NcoI fragment encompassing the translational start and 85% of the scw1 open reading frame (including the highly conserved RNP1 and RNP2 subdomains required for RNA binding) was then replaced with a 1.8-kb ura4⁺ fragment PCR amplified to incorporate HindIII and NcoI sites at its termini (forward: 5'-ggg gga agc tta gct aca aat ccc act gg-3'; reverse: 5'-ggg ggc cat ggg ctt gtg ata ttg acg aaa ctt ttt gac-3'). The plasmid was then digested with SalI and SphI releasing the disruption construct, which was purified and used to transform a ura4-D18 h^-/ura4-D18 h⁺ diploid strain using the LiAc-DMSO method (BAHLER et al. 1998B ). Stable ura⁺ transformants were sporulated and the progeny examined by tetrad analysis. One clone displaying 2:2 segregation of wild type (associated with uracil auxotrophy) to a mutant phenotype indistinguishable from the original scw1-1 mutant (associated with uracil prototrophy) was isolated and analyzed by PCR (forward: 5'-gta ttc tag gtt gtt gcc ctt cta att-3'; reverse: 5'-gta tcc taa aaa ttc ttg tat gta agc-3') to confirm the disruption (data not shown). The disruption of the scw1 gene was also confirmed genetically by constructing a scw1-1/scw1::ura4⁺ diploid (which was observed to display a scw1^- phenotype; data not shown).

Overexpression of the scw1 gene:
The scw1 gene was PCR amplified (forward: 5'-gga att cca tat gtt tgt ggg atc acc gag c-3'; reverse: 5'-acg cgt cga cct att tgc cat aca tta gat tat tac ccc-3') using the Expand high-fidelity PCR system (Roche, Indianapolis) and cloned into the unique NdeI and SalI sites of the pREP1, pREP41, or pREP81 vectors (BASI et al. 1993 ; MAUNDRELL 1993 ) using standard molecular techniques (AUSUBEL et al. 1995 ). Plasmids were transformed into both scw1-1 leu1-32 and leu1-32 strains and leu⁺ transformants examined on EMM media in the presence or absence of 10 µM thiamine added after autoclaving.

Fluorescence microscopy:
For 4',6-diamidino-2-phenylindole [DAPI; Sigma (St. Louis) D1388] and calcofluor staining (Polysciences, No. 4359) cells were grown to mid-log phase, collected by centrifugation (3000 rpm, 5 min), washed, and then fixed by adding one-tenth volume 37% formaldehyde to cells resuspended in 1x PEM (100 mM PIPES, 1 mM EGTA, and 1 mM MgSO₄). Cells were incubated with rotation for 20 min, collected by centrifugation, and then washed and resuspended in 1x PEM. One microliter of cell suspension was then mixed on a microscope slide, with 1 µl of DAPI at a concentration of 1 µg/ml and 1 µl of calcofluor at a concentration of 2 µg/ml. Images were acquired using a Leitz DMRB fluorescence microscope (Leica Microsystems) and a high-performance cooled CCD camera (Cooke Sensicam) operated by Slidebook image analysis software (Intelligent Image Innovations). Methyl blue (Sigma M6900) staining was performed according to the protocol of KIPPERT and LLOYD 1995 . Cells were collected (3000 rpm, 5 min) and washed with 10 mM Tris pH 7.6 buffer. Methyl blue was then added to the cell suspension at a concentration of 0.5 mg/ml. Images were acquired as described above. Alternatively, cells were fixed (by adding 2 volumes of ice-cold methanol to 1 volume of culture) before being washed with 10 mM Tris pH 7.6 buffer.

Scw1-GFP fusion:
The scw1 gene was PCR amplified (forward: 5'-gga att cca tat gtt tgt ggg atc acc gag c-3'; reverse: 5'-acg cgt cga cga ttt gcc ata cat tag att att acc cca ac-3') using the Expand high-fidelity PCR system (Roche) and cloned into the unique NdeI and SalI sites of the pREP41-GFP(S65T) plasmid (TARICANI et al. 2002 ) to create a C-terminal Scw1-green fluorescent protein (GFP) fusion. The construct was transformed into a scw1-1 leu1-32 strain and was able to complement the strong cell-wall, colony morphology, cell length at septation, and increased septation index phenotypes of the mutant in the absence of thiamine (data not shown). The plasmid was integrated by homologous recombination and a stable wild-type leu⁺ clone whose GFP expression was independent of thiamine was chosen for analysis (this selected for clones in which the GFP-tagged version was under control of its native promoter and the mutant version was under control of the nmt41 promoter; see DECOTTIGNIES et al. 2001 ). Integrative mapping confirmed that the plasmid had integrated at or near the scw1 locus. Localization was monitored in EMM in the presence of 10 µm thiamine. Images were acquired as described above.

Cell-wall digestion:
Cell-wall digestion experiments were performed as described in LEVIN and BISHOP 1990 . Cells were grown to mid-log phase in EMM at 30°, collected by centrifugation (3000 rpm, 5 min), washed, lightly sonicated (to prevent cell clumping), and then resuspended to an optical density of 1.0 in 50 mM sodium phosphate/50 mM sodium citrate buffer pH 5.6 containing 0.2–0.5 mg/ml Novozym 234 (-glucanase; Interspex Products) or in TE buffer pH 7.5 (10 mM Tris-HCl, 1 mM EDTA) containing 0.5 mg/ml Zymolyase 20T (ß-glucanase; ICN Biomedicals). Digestion buffers contained ß-mercaptoethanol at 0.01%. The cell suspension was then incubated with constant shaking at 36° and cell lysis was monitored by measuring the optical density at 600 nm at the time points indicated in the text. In experiments using temperature-sensitive (ts) SIN mutants, cultures were grown in YEA at 25° and then transferred to 36° for 4 hr prior to suspension in the digestion buffer.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Phenotypic characterization of the scw1-1 mutant:
The recessive scw1-1 mutation was isolated in a screen for mutants with pH-sensitive cell cycle defects (see MATERIALS AND METHODS). The mutation conferred increased cell size at division as well as an unusual, disorganized colony morphology. The phenotype was exacerbated by increased temperatures and by lowered external pHs (Fig 1A, Table 2). Logarithmically growing populations of scw1-1 mutants also displayed an abnormally high septation index, as well as a significant proportion of multiseptate cells (Fig 1B). Unlike other multiseptate mutants that display incorrectly placed septa both spatially and temporally (LE GOFF et al. 1999 ), the multiseptate cells in the scw1-1 mutant invariably contained a single nucleus per compartment and had thickened septa that were symmetrically placed perpendicular to the long axis of the cell (Fig 1C).

View larger version (92K): In this window In a new window Download PPT slide   Figure 1. Characterization of scw1-1 and scw1::ura4+ mutants. (A) Colony morphology. The indicated strains were freshly streaked to EMM pH 5.5 or EMM pH 3.5 plates and incubated for 24 hr at 25° or 36°, respectively. Unintegrated multicopy plasmids or integrated (int.) plasmids carried by the strains are shown in parentheses following the listed genotype. Bar, 100 µm. (B) scw1- mutants exhibit an increased septation index as well as cells with multiple septa. Cells of the indicated genotype were grown to mid-log phase in EMM pH 3.5 at 36° (top) or in EMM pH 5.5 at 25° (bottom). The solid portion of the bars indicates the percentage of cells containing a single septum while the open portion of the bars indicates the percentage of cells with multiple septa. Error bars indicate standard deviations. (C) Wild-type and scw1-1 cells were grown to mid-log phase in EMM pH 3.5 at 36° before septate or multiseptate cells were stained with calcofluor (cell wall and septa) and DAPI (DNA). Bar, 10 µm.

Taken together, these data suggest that the fundamental defect in the scw1-1 mutant was not in the control of cell cycle progression, but rather stemmed from an inability to degrade septal material in a timely manner (i.e., cell separation). Since S. pombe grows only by extension in length at its tips this would lead to the appearance of abnormally long, or in more extreme instances (where separation was delayed past the next mitosis), to multiseptate cells.

Septal material is similar in composition to the cell wall (ISHIGURO 1998 ). Also, degradation of the primary septum is triggered by a localized rupture of the cell wall at the point where the cell wall and the primary septum meet (SIPICZKI and BOZSIK 2000 ). We therefore examined the ability of wild-type and scw1-1 mutants to resist the action of cell-wall-degrading enzymes. Intriguingly, the cell wall of the scw1-1 strain showed a dramatically increased resistance to both - and ß-glucanases, supporting the hypothesis that the fundamental defect caused by this mutation is in altered cell-wall structure (Fig 2).

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. scw1- mutants exhibit increased resistance to cell-wall-degrading enzymes. Strains were incubated in the presence of either 0.5 mg/ml Novozym 234 (top) or 0.5 mg/ml Zymolyase 20T (bottom). Cell lysis was monitored by measuring the optical density at 600 nm. Error bars indicate standard deviations. () Wild type; () scw1-1; () scw1::ura4+; (•) wild type transformed with pREP41-scw1+; () scw1::ura4+ transformed with pREP81-scw1+.

Cloning and disruption of the scw1 gene:
The scw1 gene was cloned by functional complementation and shown to correspond to an open reading frame designated SPCC16C4.07 by the S. pombe sequencing project (see MATERIALS AND METHODS; Fig 3A). scw1 encodes a polypeptide of 561 amino acids and contains an RNA-binding domain in its C terminus (Fig 3B). RNA-binding domains (also referred to as RNA recognition motifs or ribonucleoprotein domains) are a common eukaryotic motif found in proteins with roles in the maturation, transport, stability, localization, and translation of mRNAs (NAGAI et al. 1995 ; VARANI and NAGAI 1998 ).

View larger version (52K): In this window In a new window Download PPT slide   Figure 3. scw1 encodes an RNA-binding domain protein with homology to S. pombe Mde7 and S. cerevisiae Whi3 and Whi4. (A) Schematic showing subcloning of the original rescuing genomic fragment and disruption of the scw1 gene (SPCC16C4.07). (+) a restriction fragment is able to complement a scw1-1 mutant; (-) no complementation. Arrows indicate a predicted ORF. H, S, Sp, and N indicate HindIII, SalI, SphI, and NcoI restriction sites, respectively. (B) Predicted amino acid sequence of the protein encoded by scw1. The RNA-binding domain is indicated by boldface type. The highly conserved RNP2 and RNP1 subdomains are indicated by a single or double underline, respectively. (C) ClustalW alignment of the RNA-binding domains of S. pombe Scw1 and Mde7, as well as S. cerevisiae Whi3 and Whi4. White type on black background indicates an identity. Boldface type on a shaded background indicates a strongly conserved group (score 0.5 using Gonnet PAM250 matrix) whereas regular type on a shaded background indicates a weakly conserved group (a positive score <0.5 using Gonnet PAM 250 matrix).

scw1 is part of a conserved family of four genes, which includes fission yeast mde7, as well as the budding yeast WHI3 and WHI4 genes (see DISCUSSION). The four proteins are most highly similar in the RNA-binding domain, but do show weaker similarity elsewhere in the proteins (Fig 3C, Table 3). Caenorhabditis elegans Mec8 also shows significant similarity to Scw1, mainly in the RNA-binding domain. In humans, Scw1 shows highest similarity to poly-pyrimidine tract binding protein. Strains carrying a disruption of the scw1 ORF (see MATERIALS AND METHODS; Fig 3A) behaved very similarly to scw1-1 point mutants under all conditions tested (Fig 1A and Fig B; Fig 2; Table 2).

Overexpression of the scw1 gene:
Overexpression of scw1 from the strong thiamine repressible nmt1 promoter (BASI et al. 1993 ; MAUNDRELL 1993 ) was lethal and could not be rescued by the inclusion of 1.2 M sorbitol to the growth media (data not shown). However, examination of cells grown for 24 hr under nonrepressing conditions revealed that 11% of the population exhibited cell-wall or septation defects (viability at this point was 21%). Of the defective class 28% exhibited incompletely formed septa (Fig 4E), 41% displayed incompletely formed septa opposite cell-wall ruptures (Fig 4F and Fig G), and 31% displayed cell-wall ruptures and cell lysis with little or no septal material (Fig 4H). Cells with completely formed septa were also observed and accounted for 5% of the total number of cells examined (Fig 4D).

View larger version (15K): In this window In a new window Download PPT slide   Figure 4. Strong overexpression of scw1 results in a proportion of cells displaying cell-wall and/or septation defects. Unfixed wild-type cells carrying either the empty pREP1 vector (a and b) or the pREP1-scw1+ vector (c–h) were examined with the cell-wall marker methyl blue after 24 hr growth in nonrepressing conditions. Arrows indicate cell-wall ruptures. Bar, 10 µm.

Logarithmically growing cells expressing scw1 from the intermediate strength nmt41 promoter were viable, but expression did result in a slower rate of growth (60% that of wild type). Overexpression did not, however, result in septation defects or in a weakened cell wall relative to wild type (Fig 2; data not shown). Since the small percentage of cells exhibiting cell-wall/septation defects could not account for the large drop in viability seen upon strong overexpression, and since moderate overexpression resulted in a slow-growth phenotype in the absence of cell-wall/septation defects, we suggest that scw1 overexpression may have a dominant negative effect due to a general interference with mRNA metabolism. Expression of scw1 from the nmt41 promoter, as well as expression from the weakest nmt81 promoter, could suppress the colony morphology, cell length at septation, and strengthened cell-wall phenotypes of both the scw1-1 point mutant and scw1::ura4⁺ disruption (Fig 1 and Fig 2; Table 2; data not shown).

scw1 null mutations suppress defects in the septation initiation network:
Since scw1 loss-of-function mutants displayed defects in septation/cell separation, and since strong overexpression resulted in a proportion of cells displaying cell-wall and/or septation defects, we searched for genetic interactions between the scw1::ura4⁺ disruption and available septation mutants. These mutants included cdc11-136, cdc7-24, cdc14-118, sid1-239, sid2-250, sid3-106, sid4-A1, and mob1-1. These mutants fall into the class of sid mutants, which arrest with an elongated morphology, no septa, and multiple nuclei and which eventually lyse (MARKS et al. 1992 ; BALASUBRAMANIAN et al. 1998 ; CHANG and GOULD 2000 ; GUERTIN et al. 2000 ; HOU et al. 2000 ). Remarkably, the scw1::ura4⁺ disruption was able to suppress the lethality and septation defects associated with each of these mutations at the restrictive conditions (Fig 5A; data not shown). Importantly, the double mutants displayed an elongated, multiseptate phenotype similar to that seen in the scw1::ura4⁺ single mutant.

View larger version (89K): In this window In a new window Download PPT slide   Figure 5. scw1 null mutations suppress the lethality and septation defects associated with sid mutants. (A) Cells of the indicated genotype were freshly streaked to YEA plates and incubated for 24 hr at 36°. As a control scw1::ura4+ cdc7-24 double mutants transformed with the pREP81-scw1+ plasmid were streaked to EMM in the presence (EMM + T) or absence (EMM - T) of 10 µM thiamine (to repress and induce, respectively, scw1+ transcription). Bar, 75 µm. (B) Cells of the indicated genotype (from a typical tetratype tetrad derived from a cross of scw1::ura4+ mob1-1 and scw1::ura4+ sid2-250 strains) were examined 3 days after tetrad dissection at a temperature of 25°. Bar, 100 µm. (C) Cells of the indicated genotype (from a typical tetratype tetrad derived from a cross of scw1::ura4+ sid2-250 and scw1::ura4+ cdc16-116 strains) were freshly streaked to YEA plates and incubated for 24 hr at 36°. Bar, 100 µm. (D) Cells of the indicated genotype were grown to mid-log phase in YEA at 25°, shifted to 36° for 5 hr, and then stained with DAPI and calcofluor. Bar, 10 µm.

The strength of suppression (in terms of growth) varied, depending on which sid mutant was tested, but did not show any relationship to the location of the gene within the sid pathway. The most complete suppression was seen in scw1::ura4⁺ cdc11-136 and scw1::ura4⁺ sid1-239 double mutants, which were indistinguishable from scw1::ura4⁺ single mutants. The weakest suppression was observed in the scw1::ura4⁺ mob1-1 double mutant, which grew relatively poorly and displayed cells that were both wider and more elongated than wild type. All other scw1::ura4⁺ sid double mutants displayed intermediate levels of growth between these two extremes (Fig 5A, data not shown).

Importantly, the scw1::ura4⁺ disruption was unable to rescue the phenotypes associated with the cdc12-112 (Fig 5A), or the cdc15-140 mutations (data not shown). These mutants also arrest with an elongated morphology and no septa, but are unable to correctly form the acto-myosin ring (MARKS et al. 1992 ; FANKHAUSER et al. 1995 ; CHANG et al. 1997 ; BALASUBRAMANIAN et al. 1998 ). These data strongly suggest that loss of scw1 acts at the level of deposition of septal material.

To determine whether the scw1::ura4⁺ disruption could suppress multiple defects in the SIN we mated scw1::ura4⁺ mob1-1 and scw1::ura4⁺ sid2-250 strains. Out of 20 analyzed tetrads, 14 displayed a three-to-one segregation of colony- to non-colony-forming ability (at 25°), three tetrads displayed a two-to-two segregation of this phenotype, and the three remaining tetrads displayed a four-to-zero segregation. Microscopic inspection of the non-colony-forming spores revealed that they had germinated and formed cdc^- microcolonies. Genetic analysis of the viable spores in a tetratype tetrad allowed us to infer that the genotype of the cdc^- microcolonies was in fact scw1::ura4⁺ mob1-1 sid2-250, as expected (Fig 5B). Thus the presence of the scw1::ura4⁺ disruption cannot entirely bypass the requirement of the SIN.

We next constructed scw1::ura4⁺ cdc16-116 double mutants. At 36° cdc16-116 strains display a terminal phenotype opposite to that of sid mutants, arresting with a predominantly multiseptate phenotype (MINET et al. 1979 ; MARKS et al. 1992 ; FANKHAUSER et al. 1993 ). We found that scw1::ura4⁺ cdc16-116 double mutants were indistinguishable from the cdc16-116 single mutant under restrictive conditions and furthermore that the scw1::ura4⁺ disruption did not lower the restrictive temperature of this ts cdc16 allele (data not shown).

Interestingly, since the sid2-250 mutation is epistatic to cdc16-116 (TANAKA et al. 2001 ), we were also able to determine whether the scw1::ura4⁺-mediated suppression of the sid phenotype required Cdc16 activity. We found that scw1::ura4⁺ sid2-250 cdc16-116 triple mutants were viable under restrictive conditions (Fig 5C). Since suppression occurs in the absence of a fully functional Cdc16, this suggests that scw1 does not normally operate through modulation of Spg1 GAP activity.

We next examined scw1::ura4⁺ sid double mutants with DAPI and calcofluor 5 hr after shift to restrictive conditions. Although able to form septa, it was clear that scw1::ura4⁺ sid double mutants displayed defects in the coupling of mitosis to cytokinesis. scw1::ura4⁺ cdc7-24, scw1::ura4⁺ mob1-1, scw1::ura4⁺ sid2-250, scw1::ura4⁺ sid3-106, scw1::ura4⁺ sid4-A1, and scw1::ura4⁺ cdc14-118 double mutants all exhibited multiseptate cells with anucleate or multinucleate compartments. scw1::ura4⁺ cdc11-136 and scw1::ura4⁺ sid1-239 double mutants, which were the most strongly suppressed in terms of growth (see above), exhibited these defects to a much lesser extent and appeared similar to scw1::ura4⁺ single mutants alone (Fig 5D, Table 4). These data demonstrate that the proper coupling of mitosis to cytokinesis can be defective in scw1^- sid double mutants even if the lethality and septum formation defects are suppressed.

The SIN is a regulator of cell-wall integrity:
Since scw1^- mutants displayed increased resistance to cell-wall-degrading enzymes we examined the ability of sid mutants to resist degradation of their cell walls by the -glucanase Novozym 234. We discovered that after a 4-hr incubation under restrictive conditions cdc7-24 cells exhibited a marked decrease in resistance to the enzyme relative to wild type while scw1::ura4⁺ cdc7-24 cells exhibited similar levels of resistance to those seen in scw1::ura4⁺ single mutants. Furthermore, hyper-activation of the pathway caused by loss of cdc16 led to Novozym 234 resistance even greater than that seen in scw1::ura4⁺ cells (Fig 6A). These data demonstrate that the SIN module has dosage-dependent effects on cell-wall integrity.

View larger version (61K): In this window In a new window Download PPT slide   Figure 6. SIN mutants display altered resistance to cell-wall-degrading enzymes. (A and B) Cell lysis was monitored by measuring the optical density at 600 nm. Error bars indicate standard deviations. () Wild type; () scw1::ura4+; () cdc7-24; (•) scw1::ura4+ cdc7-24; () cdc16-116; () scw1::ura4+ cdc16-116. (A) Strains were incubated as described in MATERIALS AND METHODS in the presence of 0.2 mg/ml Novozym 234 (top) or 0.5 mg/ml Novozym 234 (bottom). (B) Strains were incubated as described in MATERIALS AND METHODS in the presence of 0.5 mg/ml Zymolyase 20T. (C) Cells of the indicated genotype were grown to mid-log phase in YEA at 25°, shifted to 36° for 5 hr, fixed with methanol, and then stained with methyl blue. Arrowhead indicates cell-wall rupture. Bar, 10 µm. (D) Cells of the indicated genotype were streaked to YEA plates supplemented with 0.5 mg/ml calcofluor white and incubated for 36 hr at 36°. Bar, 100 µm.

As expected, when experiments were performed using the ß-glucanase Zymolyase 20T, we found similar interactions between the scw1::ura4⁺ disruption and the cdc7-24 allele (with the exception that scw1::ura4⁺ cdc7-24 cells displayed intermediate levels of resistance between scw1::ura4⁺ and cdc7-24 single mutants). cdc16-116 mutants, on the other hand, displayed a profile similar to wild type (with the exception in this case being that the mutant consistently displayed altered kinetics, i.e., increased sensitivity at earlier time points and increased resistance at later time points). In addition we found that cdc16-116 scw1::ura4⁺ strains displayed increased resistance over strains carrying the cdc16-116 mutation alone (Fig 6B). The differential response of the cdc16-116 mutant to two different cell-wall-degrading enzymes suggests that the changes in cell-wall structure associated with hyper-activation of the SIN involve more than simple increases in the deposition of cell-wall material. Rather it may involve more complex changes in cell-wall architecture. Similar results to those obtained with the cdc7-24 mutant were observed with the other sid mutants used in this study (data not shown).

Since methyl blue has recently been reported to be an excellent cell-wall marker (KIPPERT and LLOYD 1995 ; FEOKTISTOVA et al. 2001 ), we examined sid mutants with this dye. Interestingly, when cdc7-24 cells at the restrictive conditions were fixed with methanol and stained, we noted that 20% of the cells had lysed. Visible ruptures in the cell wall (generally in the medial regions of the cell) and the spilling of cytoplasmic contents were also observed. This phenomenon was not noted in wild-type, scw1::ura4⁺, or scw1::ura4⁺ cdc7-24 cells (Fig 6C). This phenomenon was also not noted in unfixed cdc7-24 cells or in cdc7-24 cells fixed with formaldehyde (data not shown). Therefore, although the phenomenon is artifactual, most likely due to the harsher nature of methanol fixation, it does reveal an inherent weakness in the cell wall of cdc7-24 cells. Similar results were obtained with the other sid mutants used in this study (data not shown). Otherwise, results obtained using methyl blue were similar to those obtained using the more widely used cell-wall marker, calcofluor white (data not shown).

Taking all data together, our results suggested that the stronger cell wall conferred by the scw1::ura4⁺ disruption played a key role in the observed suppression of sid mutations. If this were true, then one would expect conditions that act against cell-wall integrity to negatively affect this rescue. We thus examined the growth of wild-type, scw1::ura4⁺, cdc7-24, and scw1::ura4⁺ cdc7-24 cells at 36° in the presence of 0.5 mg/ml calcofluor white. This compound (in addition to acting as a cell-wall marker) inhibits the formation of glucan and chitin microfibrils and thus interferes with cell-wall synthesis (HAIGLER et al. 1980 ; RONCERO et al. 1988 ; RAM et al. 1994 ; LUSSIER et al. 1997 ). Although growth in media containing calcofluor did not affect the multiseptate phenotype of the scw1::ura4⁺ mutant (data not shown), scw1::ura4⁺ cdc7-24 double mutants did behave as cdc7-24 cells and displayed a lethal cdc^- phenotype (Fig 6D). Similar interactions were observed among the scw1::ura4⁺ disruption and other sid mutants in media containing calcofluor (data not shown). These data strongly suggest that the scw1::ura4⁺-mediated rescue of sid mutants is achieved through altered cell-wall synthesis and/or structure.

SIN-mediated effects on cell-wall integrity are influenced by cell cycle position:
The effects of cdc7 overexpression on septation are reduced in cells arrested at the G₂/M transition (FANKHAUSER and SIMANIS 1994 ). Furthermore, following mitotic entry, translocation of the Sid1/Cdc14 complex to the SPB is promoted by the inactivation of Cdc2 through cyclin proteolysis (GUERTIN et al. 2000 ). If activation of the SIN affects cell-wall structure, then changes in cell-wall integrity mediated by cdc7 overexpression should be influenced by cell cycle position. We thus examined the effects of increased Cdc7 levels on cell-wall integrity in populations arrested at different points of the cell cycle. Wild type, as well as cdc10-129 and cdc2-33 mutants (which arrest predominantly at the G₁/S and G₂/M transitions, respectively, at 36°), were transformed with the pREP3-cdc7 plasmid (which places cdc7 under control of the nmt1 promoter).

Consistent with our previous results, hyper-activation of the SIN in exponentially growing cells resulted in increased resistance to Novozym 234. A similar effect was seen in cells arrested at the G₁/S transition. Intriguingly, however, the increased resistance was partially abrogated in cells arrested predominantly in G₂ (Fig 7). When analogous experiments were performed using Zymolyase 20T we noted that exponentially growing and G₁-arrested cells displayed decreased resistance, but that G₂ arrest partially abrogated this effect (Fig 7). These data show that the effects of SIN hyper-activation on cell-wall integrity are modulated by cell cycle position.

View larger version (20K): In this window In a new window Download PPT slide   Figure 7. Altered resistance to cell-wall-degrading enzymes mediated by overexpression of Cdc7 is partially abrogated in G2-arrested cells. (A) Strains carrying the pREP3-cdc7 plasmid were grown to mid-log phase at 25° in EMM containing 10 µM thiamine to repress cdc7 transcription. Cells were subsequently centrifuged, washed, and used to reseed EMM cultures containing no thiamine (open symbols) or 10 µM thiamine (solid symbols). Cultures were grown to early-log phase for a further 13 hr at 25° before being shifted to 36° for 4 hr. Strains were then incubated as described in MATERIALS AND METHODS in the presence of either 0.5 mg/ml Novozym 234 (top) or 0.5 mg/ml Zymolyase 20T (bottom). Cell lysis was monitored by measuring the optical density at 600 nm. Error bars indicate standard deviations. ( and ) Wild type; ( and ) cdc2-33; ( and ) cdc10-129. (B) Cells treated as in A and then stained with methyl blue 4 hr after the shift to 36°. (Top) Plus thiamine; (bottom) minus thiamine; (left) wild type; (middle) cdc10-129; (right) cdc2-33. Bar, 30 µm.

Zymolyase 20T resistance assays were somewhat surprising since cdc7 loss-of-function mutants displayed a similar resistance to that demonstrated by cells overexpressing cdc7. We interpret these results to indicate that gross disturbances of Cdc7 function (either by loss or by strong overexpression) cause drastic changes in cell-wall structure, which manifest as a decrease in Zymolyase 20T resistance. This is to say that although the loss and overexpression of Cdc7 have opposing effects, the net result is a poorly structured cell wall vulnerable to attack by Zymolyase 20T.

Subcellular localization of Scw1:
Since members of the SIN localize to the spindle pole body we created a C-terminal Scw1-GFP fusion to determine its subcellular localization (see MATERIALS AND METHODS). Unlike members of the SIN, Scw1 was predominantly a cytoplasmic protein (Fig 8). Localization was not altered as a function of cell cycle position by changes in temperature or by external pH conditions (data not shown).

View larger version (50K): In this window In a new window Download PPT slide   Figure 8. Scw1 is localized predominantly to the cytoplasm. Scw1-GFP-expressing cells were grown in EMM pH 5.5 to mid-log phase and images taken using either Nomarski optics (left) or fluorescence microscopy (right). Bar, 10 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The cell wall of S. pombe is composed mainly of polymers of 1,3-ß-glucan, 1,3--glucan, and -galactomannan. It serves a wide variety of functions including protection from environmental stresses, cell adhesion during conjugation and mating, and the maintenance of cellular morphology (ISHIGURO 1998 ). Many different proteins, acting as regulators of a wide variety of pathways, have been shown to affect cell-wall structure. These include the rho1 and rho2 GTP-binding proteins, the pmk1/spm1 mitogen-activated protein kinase, the pab1 protein phosphatase 2A regulatory B subunit, and the pck2 protein kinase C homolog, among many others (ISHIGURO 1998 ).

In this report we show that loss of the scw1 RNA-binding domain protein also affects the cell wall as evidenced by increased resistance to both - and ß-glucanases. The mechanism by which loss of scw1 causes these differences is unknown, but on the basis of scw1 encoding an RNA-binding domain protein, the differences most likely result from an indirect means related to the regulation of mRNAs. RNA-binding domain proteins have been shown to affect many distinct cellular pathways through their effects on mRNA maturation, transport, stability, localization, and translation (see NAGAI et al. 1995 and VARANI and NAGAI 1998 for reviews).

The scw1 RNA-binding domain protein isolated in this study is part of a family of four genes that include S. pombe mde7, as well as S. cerevisiae WHI3 and WHI4. mde7 has not been characterized in detail, but is known to be one of nine meiosis-dependent transcripts under the control of the Mei4 forkhead transcription factor (ABE and SHIMODA 2000 ). In their study, sequence analysis revealed the presence of a conserved cis-element GTAAACAAACA(A/T)A (found in either forward or reverse orientation) starting within 26 and 282 bp upstream of the translational start in each of the nine mde genes. We examined the scw1 promoter for this sequence and were able to find only a single copy of GTAAACA in an intergenic region 735 bp upstream of the translational start. Furthermore, no defects in mating or spore formation were observed in scw1^- mutants (data not shown).

WHI3 was isolated as a dosage-dependent regulator of cell size in the budding yeast Saccharomyces cerevisiae (NASH et al. 2001 ). Whi3 acts as a negative regulator of Cln3 (a G₁ cyclin) through specifically binding CLN3 mRNA and localizing the transcripts to discrete cytoplasmic foci (GARI et al. 2001 ). WHI4, on the other hand, was isolated on the basis of its sequence similarity to WHI3. whi3 null mutants maintain a cell size 78% that of wild type, whereas whi4 null mutants do not have any cell size or other observable phenotype. whi3whi4 double mutants are viable, but display an additive interaction (being 64% the size of wild-type cells; NASH et al. 2001 ). No decreased cell size phenotypes were observed for scw1^- mutants at pHs ranging from 3.5 to 5.5 or at temperatures ranging from 25° to 35°. Taken together these data suggest that (although the RNA-binding domain is conserved) these proteins have evolved unique functions in the more recent evolutionary past. In this respect it is interesting to note that in contrast to the highly conserved RNA-binding domain, flanking sequences accounting for 80% or more of the proteins show much greater levels of divergence (Table 3).

In addition to the characterization of a previously unknown protein, these results are more significant in terms of highlighting the role of cell-wall structure in the onset of septation in S. pombe. We are able to show here that both scw1, and the SIN module itself, act as regulators of cell-wall integrity. We further demonstrate that loss-of-function mutations in scw1, which counteract the cell-wall changes associated with reduced SIN activity, also allow the formation of septa. Lastly, we show that conditions that negatively regulate cell-wall integrity also abolish the ability of the scw1::ura4⁺ disruption to suppress sid mutations. These data suggest that scw1 normally acts in opposition to the SIN as a negative regulator of septum/cell-wall deposition and/or structure. Alternatively, loss of scw1 may bring about changes in cell-wall architecture that are more permissive for SIN-induced septum formation.

Interestingly, scw1 is one of several recently identified genes whose loss suppresses defects in the SIN. The first, zfs1, encodes a zinc-finger-containing protein, which, when deleted, reduces the restrictive temperatures of ts alleles of cdc7, cdc11, cdc14, spg1, sid1, sid2, and sid4 (mob1 was not tested; BELTRAMINELLI et al. 1999 ). par1 and par2, on the other hand, encode B' regulatory subunits of protein phosphatase 2A (PP2A). Cells deleted for both par1 and par2 are able to suppress defects in a loss-of-function allele of spg1, but are unable to rescue sid mutants downstream of spg1. Interestingly, par1 par2 double mutants (like scw1^- mutants) display a multiseptate phenotype (JIANG and HALLBERG 2001 ).

Whether or not zfs1, par1, or par2 act as regulators of cell-wall structure has not been tested explicitly, but loss-of-function mutants of the pab1 B subunit of PP2A have been shown to display reduced resistance to Zymolyase 100T, demonstrating that the cell wall is sensitive to PP2A activity (KINOSHITA et al. 1996 ). Furthermore, overexpression of pab1 can rescue the spg1-B8 ts allele (LE GOFF et al. 2001 ). These results are made even more intriguing by the fact that B and B' subunits most likely compete for binding to the core PP2A enzyme (as has been suggested in S. cerevisiae; SHU et al. 1997 ; EVANS and HEMMINGS 2000 ). Thus both loss of par1 and par2 or overexpression of pab1 would be expected to generate PP2A holoenzymes with predominantly B subunits.

The mechanism(s) by which these genes affect the SIN is unknown, but par1 and par2 have been proposed to act as negative regulators either at the level of cdc7 or at a point upstream, whereas zfs1 has been proposed to act as a negative regulator of a SIN component, an inhibitor of a SIN substrate, or in a parallel pathway whose loss is capable of compensating for reduced SIN signaling (BELTRAMINELLI et al. 1999 ; JIANG and HALLBERG 2001 ; LEGOFF et al. 2001 ).

On the basis of genetic analysis scw1 is clearly acting at the level of septum deposition since scw1^- mutations are able to suppress ts mutants at all levels of the SIN pathway and since scw1^- mutations have no ability to suppress either the cdc12-112 or the cdc15-140 mutations (which cause defects in acto-myosin ring formation and/or function; FANKHAUSER et al. 1995 ; BALASUBRAMANIAN et al. 1998 ). Genetic analysis also suggests that Scw1 is not acting through modulation of Cdc16/Byr4 since unlike cdc16-116 sid2-250 double mutants, which display a sid2-250 terminal phenotype, scw1::ura4⁺ cdc16-116 sid2-250 triple mutants are viable at restrictive temperatures. Also, fluorescence-activated cell sorter analysis demonstrated that wild-type and scw1::ura4⁺ cells synchronized by nitrogen starvation and released into rich media at 36° progressed into the cell cycle with similar kinetics (our unpublished observations). It is thus unlikely that suppression of the sid phenotype is due to an indirect mechanism such as a scw1::ura4⁺-mediated increase in the duration of G₁ (which could have increased the time available to form a septum).

The ability of the scw1::ura4⁺ disruption to suppress the mob1-1 and sid2-250 mutations singly, but not in combination, also demonstrates that the scw1::ura4⁺ disruption does not simply bypass the requirement of the SIN. Instead, scw1 most likely acts as a negative regulator of the network either at a point upstream of Sid2/Mob1 or possibly in a parallel pathway, which also plays a role in modulating cell-wall structure and counteracts the effects of the SIN. Interestingly, a role for cell-wall structure in regulating septation has been suggested by the cloning and characterization of the cps1 gene. cps1 encodes a 1,3-ß-glucan synthase subunit whose loss results in an inability to initiate septum formation, but does not affect normal growth (LIU et al. 1999 ).

It is interesting to note that clear functional homologs of many SIN members have not been found in more complex, multicellular eukaryotes to date (BARDIN and AMON 2001 ). Given that the module functions as a regulator of septum formation, this is not surprising since most eukaryotes do not have an analogous structure to the fungal septum or cell wall. In this respect it is interesting to note that scw1 also has close homologs in budding yeast, but not in humans. Genes homologous to members of the SIN, however, are present in S. cerevisiae. These homologs form a regulatory module referred to as the mitotic exit network or MEN (reviewed in BARDIN and AMON 2001 ; MCCOLLUM and GOULD 2001 ).

Unlike members of the SIN, loss-of-function mutants of the MEN display a late mitotic arrest. However, a cell lysis phenotype has been observed in the MEN mutant cdc15-lyt1, as well as in mutant alleles of DBF2 and TEM1 (homologs of S. pombe cdc7, sid2, and spg1, respectively; JIMENEZ et al. 1998 ). Furthermore, 28–38% of cdc15-lyt1/cdc15-lyt1 cells are present in the form of budded chains without septa under prolonged incubation at the restrictive conditions. Each compartment of the chain contains a nucleus due to leakage past the late mitotic block (JIMENEZ et al. 1998 ). This phenotype (i.e., growth in the absence of septum formation) could be considered analogous to the sid phenotype observed in S. pombe SIN mutants. Thus, despite the fact that S. pombe sid mutants do not display a late mitotic arrest, other phenotypes, including growth in the absence of septum formation and cell-wall integrity defects, are applicable to at least some MEN mutants.

	FOOTNOTES

¹ Present address: Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Toronto, ON M5G-2C1, Canada.

	ACKNOWLEDGMENTS

We thank D. McCollum, M. K. Balasubramanian, and V. Simanis for strains and/or plasmids, as well as L. Taricani for constructing the pREP41-GFP (S65T) plasmid. This research was supported by the Natural Science and Engineering Council of Canada through grants to P.G.Y. J.K. was supported by the Bauman Foundation and by an Ontario Graduate Scholarship.

Manuscript received March 4, 2002; Accepted for publication June 6, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ABE, H. and C. SHIMODA, 2000  Autoregulated expression of Schizosaccharomyces pombe meiosis-specific transcription factor Mei4 and a genome-wide search for its target genes. Genetics 154:1497-1508.[Abstract/Free Full Text]

ALFA, C., P. FANTES, J. HYAMS, M. MCLEOD and E. WARBRICK, 1993 Experiments with Fission Yeast: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

AUSUBEL, F., R. BRENT, R. E. KINGSTON, D. D. MOORE, J. G. SEIDMAN et al., 1995 Short Protocols in Molecular Biology, Ed. 3. John Wiley & Sons, New York.

BAHLER, J. and J. R. PRINGLE, 1998  Pom1p, a fission yeast protein kinase that provides positional information for both polarised growth and cytokinesis. Genes Dev. 12:1356-1370.[Abstract/Free Full Text]

BAHLER, J., A. B. STEEVER, S. WHEATLEY, Y. L. WANG, and J. R. PRINGLE et al., 1998a  Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. J. Cell Biol. 143:1603-1616.[Abstract/Free Full Text]

BAHLER, J., J. WU, M. S. LONGTINE, N. G. SHAH, and A. MCKENZIE, III et al., 1998b  Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943-951.[Medline]

BALASUBRAMANIAN, M. K., D. M. HELFMAN, and S. M. HEMMINGSEN, 1992  A new tropomyosin essential for cytokinesis in the fission yeast S. pombe.. Nature 360:84-87.[Medline]

BALASUBRAMANIAN, M. K., B. R. HIRANI, J. D. BURKE, and K. L. GOULD, 1994  The Schizosaccharomyces pombe cdc3 gene encodes a profilin essential for cytokinesis. J. Cell Biol. 125:1289-1301.[Abstract/Free Full Text]

BALASUBRAMANIAN, M. K., D. MCCOLLUM, L. CHANG, K. C. WONG, and N. I. NAQVI et al., 1998  Isolation and characterization of new fission yeast cytokinesis mutants. Genetics 149:1265-1275.[Abstract/Free Full Text]

BALASUBRAMANIAN, M. K., D. MCCOLLUM, and U. SURANA, 2000  Tying the knot: linking cytokinesis to the nuclear cycle. J. Cell Sci. 113:1503-1513.[Abstract]

BARDIN, A. J. and A. AMON, 2001  MEN and SIN: What's the difference? Nat. Rev. Mol. Cell Biol. 2:815-826.[Medline]

BASI, G., E. SCHMID, and K. MAUNDRELL, 1993  TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription start point or thiamine repressibility. Gene 123:131-136.[Medline]

BELTRAMINELLI, N., M. MURONE, and V. SIMANIS, 1999  The S. pombe zfs1 gene is required to prevent septation if mitotic progression is inhibited. J. Cell Sci. 112:3103-3114.[Abstract]

BEZANILLA, M., S. L. FORSBURG, and T. D. POLLARD, 1997  Identification of a second myosin II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis. Mol. Biol. Cell 8:2693-2705.[Abstract/Free Full Text]

CHANG, F., A. WOOLARD, and P. NURSE, 1996  Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring. J. Cell Sci. 109:131-142.[Abstract]

CHANG, F., D. DRUBIN, and P. NURSE, 1997  cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J. Cell Biol. 137:169-182.[Abstract/Free Full Text]

CHANG, L. and K. L. GOULD, 2000  Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast. Proc. Natl. Acad. Sci. USA 97:5249-5254.[Abstract/Free Full Text]

DECOTTIGNIES, A., P. ZARZOV, and P. NURSE, 2001  In vivo localization of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis. J. Cell Sci. 114:2627-2640.

EDAMATSU, M. and Y. Y. TOYOSHIMA, 1996  Isolation and characterization of pos mutants defective in correct positioning of septum in Schizosaccharomyces pombe.. Zool. Sci. 13:235-239.[Medline]

ENG, K., N. I. NAQVI, K. C. WONG, and M. K. BALASUBRAMANIAN, 1998  Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body. Curr. Biol. 8:611-621.[Medline]

EVANS, D. R. and B. A. HEMMINGS, 2000  Mutation of the C-terminal leucine residue of PP2Ac inhibits PR55/B subunit binding and confers supersensitivity to microtubule destabilization in Saccharomyces cerevisiae.. Mol. Gen. Genet. 264:425-432.[Medline]

FANKHAUSER, C. and V. SIMANIS, 1994  The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast. EMBO J. 13:3011-3019.[Medline]

FANKHAUSER, C., J. MARKS, A. REYMOND, and V. SIMANIS, 1993  The S. pombe cdc16 gene is required both for maintenance of p34^cdc2 kinase activity and regulation of septum formation: A link between mitosis and cytokinesis? EMBO J. 12:2697-2704.[Medline]

FANKHAUSER, C., A. REYMOND, L. CERUTTI, S. UTZIG, and K. HOFMANN et al., 1995  The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis. Cell 82:435-444.[Medline]

FEOKTISTOVA, A., P. MAGNELLI, C. ABEIJON, P. PEREZ, and R. L. LESTER et al., 2001  Coordination between fission yeast glucan formation and growth requires a sphingolipase activity. Genetics 158:1397-1411.[Abstract/Free Full Text]

FURGE, K. A., K. WONG, J. ARMSTRONG, M. K. BALASUBRAMANIAN, and C. F. ALBRIGHT, 1998  Byr4 and Cdc16 form a two-component GTPase-activating protein for the Spg1 GTPase that controls septation in fission yeast. Curr. Biol. 8:947-954.[Medline]

FURGE, K. A., Q. CHENG, M. JWA, S. SHIN, and K. C. SONG et al., 1999  Regions of Byr4, a regulator of septation in fission yeast, that bind spg1 or cdc16 and form a two-component GTPase-activating protein with cdc16. J. Biol. Chem. 274:11339-11343.[Abstract/Free Full Text]

GARI, E., T. VOLPE, H. WANG, C. GALLEGO, and B. FUTCHER et al., 2001  Whi3 binds the mRNA of the G1 cyclin CLN3 to modulate cell fate in budding yeast. Genes Dev. 15:2803-2808.[Abstract/Free Full Text]

GOULD, K. L. and V. SIMANIS, 1997  The control of septum formation in fission yeast. Genes Dev. 11:2939-2951.[Free Full Text]

GRALLERT, A., B. GRALLERT, E. ZILAHI, Z. SZILAGYI, and M. SIPICZKI, 1999  Eleven novel sep genes of Schizosaccharomyces pombe required for efficient cell separation and sexual differentiation. Yeast 15:669-686.[Medline]

GUERTIN, D. A., L. CHANG, F. IRSHAD, K. L. GOULD, and D. MCCOLLUM, 2000  The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast. EMBO J. 19:1803-1815.[Medline]

HAIGLER, G. H., R. M. BROWN, and M. BENZIMAN, 1980  Calcofluor white ST alters the in vivo assembly of cellulose microfibrils. Science 210:903-906.[Abstract/Free Full Text]

HOU, M., J. SALEK, and D. MCCOLLUM, 2000  Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast. Curr. Biol. 10:619-622.[Medline]

ISHIGURO, J., 1998  Genetic control of fission yeast cell wall synthesis: the genes involved in wall biogenesis and their interactions in Schizosaccharomyces pombe.. Genes Genet. Syst. 73:181-191.