INTRODUCTION

In cytokinesis of animal cells, a contractile ring consisting mainly of actin bisects a cell at an appropriate position. It usually occurs along the equatorial plane of the cell and the position is strictly defined, insuring that chromosomes segregated by the mitotic spindle become equally contained in each daughter cell.

The molecular mechanism of determination of the division plane is one of central questions in cytokinesis. Many experiments have demonstrated that the placement of the division plane is connected with preceding mitosis, and mitotic apparatus specifies the position of the cleavage furrow at a critical period in the cell cycle, reviewed in (Mabuchi, 1986). A signal stimulating cell cortex to form the cleavage furrow is called “cleavage stimulus”. It may be transmitted from the cytoplasm to the cortex via the astral microtubules. The nature of the cleavage stimulus is unknown for over 30 years. The study on the positioning mechanism of division plane is thus important for understanding linkage between mitosis and cytokinesis and also various cell motilities based on actin filaments and/or microtubules.

Fission yeast, Schizosaccharomyces pombe, is attractive as a model system for investigating cytokinesis. Excellent molecular genetic techniques can be applied, and it is found that fission yeast has similar cytokinetic machinery to animal cells, forming a contractile ring at the cell equator (Kanbe et al., 1989). Moreover, it is also shown that an actin binding protein, profilin, is involved in cytokinesis in fission yeast (Balasubramanian et al., 1994) like in the organisms undergoing animal cell-type of cytokinesis (Edamatsu et al., 1992; Haugwitz et al., 1994). Several mutants concerning cytokinesis have been isolated, reviewed in (Fankhauser and Simanis, 1994), but no mutant defective in correct positioning of septum has been reported so far.

In this study, we have succeeded in isolating pos mutants (called for positioning of septum) displaying incorrect positioning of septum in fission yeast.

MATERIALS AND METHODS

Schizosaccharomyces pombe strains used were 972h⁻, JY6 (h⁺ leu1 his2), JY333 (h⁻ leu1 ade6-M216) and JY336 (h⁺ leu1 ade6M210). In isolation of pos mutants, a 972h⁻ strain was first mutagenized by N-methyl-N'-nitro-N-nitrosoguanidine (Uemura and Yanagida, 1984). After incubated at 25°C for several days for recovery, the cells were transferred to 36°C for 5 to 6 hr to be visually screened by a microscope (Olympus CK) equipped with a micromanipulator (Narishige MN-151). The mutants showing unequal septation with a high frequency were back-crossed to JY6 strain three times prior to analyses.

Standard genetic procedures for linkage analysis, tetrad analysis and analysis of pos⁺/pos⁻ diploids are carried out according to (Alfa et al., 1993). In pairwise crosses of the linkage analysis and construction of pos⁺/pos⁻ diploids, the mutants were first crossed to JY333 (h⁻ leu1 ade6-M216) or JY336 (h⁺ leu1 ade6-M210) to obtain stable diploids using intragenic complementation between the two ade6 allele and were then used in the analyses.

To examine the phenotype of the mutants, synchronous cultures were prepared on lactose gradients by the method of Mitchison (Mitchison and Vincent, 1965). The procedure for DAPI (4′,6′-diamidino-2-phenylindole, Sigma) and Calcofluor (fluorescent brightener 28, Sigma) staining, and immunofluorescence microscopy were followed as described in (Alfa et al., 1993). For microtubule staining, anti-alpha-tubulin monoclonal antibody (Sigma) and FITC-conjugated anti-mouse IgG polyclonal antibody (Bio Source International) were used, and rhodamine-conjugated phailoidin (Sigma) was used for actin staining.

RESULTS AND DISCUSSION

Mutants defective in correct positioning of septum must produce big and small daughter cells resulting from unequal septation and not be lethal. We therefore carried out the visual screening for isolation of the mutants rather than that based on lethality at restrictive temperature widely used for isolating cell cycle mutants such as cdc mutants.

First of all, a haploid 972h⁻ strain was mutagenized by N-methyl-N'-nitro-N-nitrosoguanidine. About one thouthand of cells displaying unequal septation were visually selected by a micromanipulator. Among the cells, 16 strains frequently displayed unequal septation at restrictive temperature (Fig. 1A). After back-crossed three times, all the strains showed 2:2 segregation of the temperature-sensitive phenotype in tetrad analysis, and thus the phenotype is caused by a single mutation. All pos⁺/pos⁻ diploids were normal phenotype, so the mutations were recessive. Random spore analysis in linkage analysis demonstrated that the mutants were classified into three linkage groups, pos1-pos3 (Table 1). The result were confirmed by pairwise crosses among the mutants. Although most of the mutations (14/16) were on the same locus, the reason is not known.

Fig. 1.

Unequal septation of pos mutants. (A) Phase contrast micrographs of pos mutants showing unequal septation, (a) wild-type, (b) pos1, (c) pos2, (d) pos3. Bar, 10 μm. (B) Percentages of cells showing unequal septation in all septated ceils. Synchronous cultures were incubated at 36°C. Samples were taken at one-hour intervals, stained with Calcofluor and analyzed by a fluorescence microscopy. Unequal septation is defined as one occurring at less than one third of the cell length. (▪) wild-type, (○) pos1, (□) pos2, (•) pos3

Table 1.

Linkage analysis of pos mutants

In examining the phenotype of pos mutants, the septum was distant from the normal equatorial position with a high frequency, 42% (pos1), 25% (pos2) and 45% (pos3) at 3 hr after temperature shift-up (Fig. 1B). The position is quite divergent from the cell equator to the pole.

We then examined mitosis and cytokinesis of pos mutants by an immunofluorescence microscopy. At restrictive temperature, pos1-pos3 were able to form normal mitotic spindles at the cell center like a wild-type cell, which separated chromosomes and elongated to complete nuclear division (Fig. 2). In cytokinesis, however, a contractile ring was located at the site independent of the mitotic spindle, so it was frequently distant from the cell equator (Fig. 3). These observations suggest that the positional information for cytokinesis is not accurately transmitted to cortex of the cell equator in the mutants.

Fig. 2.

Mitosis of pos mutants at restrictive temperature. Cells exponentially grown at 25°C were incubated at 36°C for 2.5 hr. Cells were stained by anti-alpha-tubulin antibody (A, C, E, G) and DAPI (B, D, F, H). (A, B) wild-type; (C, D) pos1, (E, F) pos2; (G, H) pos3. Bar, 10 μm.

Fig. 3.

Cytokinesis of pos mutants at restrictive temperature. Cells exponentially grown at 25°C were incubated at 36°C for 2.5 hr. Cells were stained by rhodamine-conjugated phailoidin (A, C, E, G) and DAPI/ Calcofluor (B, D, F, H). (A, B) wild-type; (C, D) pos1, (E, F) pos2; (G, H) pos3. Bar, 10 μm.

Moreover, it was found that the cells unequally septated often exhibited wrong orientation and/or distortion of septum at restrictive temperature. This irregular septation was also observed in equally septated cells. In the cells of irregular septation, the septum was greatly tilted from the transverse plane (Fig. 4A, c) or even longitudinal to the long axis of the cell (Fig. 4A, b), and/or wavy septum was also observed (Fig. 4A, d). As shown in Fig. 4B, 55-70% of septated cells formed such irregular septum at 3 hr after temperature shiftup. No significant difference is detected among three pos mutants. These results suggest the possibility that the septum positioning is intimately related to the correct orientation and organization of septum. In spite of such irregular septation, the mutants were able to form colony at restrictive temperature, although the growth rate was severely reduced.

Fig. 4.

Incorrect orientation and/or distortion of septum in pos mutants. Synchronous cultures were incubated at 36°C. Samples were taken at one-hour intervals, stained with DAPI and Calcofluor, and analyzed by a fluorescence microscopy. (A) Fluorescence micrographs of pos mutants with irregular septum, (a) wild-type, (b) pos1, (c) pos2, (d) pos3. Bar, 10 μm. (B) Percentages of cells with incorrect orientation or distortion of septum in all septated cells. Septum of incorrect orientation is defined as one tilted to the cell axis at more than half rectangle. (▪) wild-type, (○) pos1, (□) pos2, (• ) pos3.

In this study, we carried out visual screening for isolating pos mutants influencing septum positioning, because the mutants are not lethal at restrictive temperature unlike cdc mutants. Recently, a similar method was used for isolating the mutants displaying abnormal morphologies, spherical (orb1-5), T-shaped (tea1), or banana-shaped (ban1) (Snell and Nurse, 1994). Thus the visual screening can be widely applied for isolating various types of mutants.

The pos mutants exhibit quite different phenotypes from the other mutants influencing septum formation isolated so far. Those mutants, mainly isolated as cdc mutants, can not form septum (cdc7, cdc11, cdc14 and cdc15) (Nurse et al., 1976), deposit disorganized septum material (cdc3, cdc4, cdc8 and cdc12) (Streiblova et al., 1984), show multiple septum (cdc16) (Minet et al., 1979), or can not or can partially separate in the septation (sep1 and spl1) (Spiczki et al., 1993). In those mutants capable of forming septum, the septum positioning is not defective. Therefore, it is unlikely that the gene products are directly implicated in the positioning of septum.

In transition from mitosis to cytokinesis, many events occur concerning initiation of cytokinesis as well as positioning of the division plane. Recently, it is suggested that protein phosphorylation regulates the initiation events, using the inhibitor of myosin light chain kinase, ML-9 (Mabuchi and Takano-Ohmuro, 1990) and that of protein phosphatases, calyculin A (Tosuji et al., 1992). It is also suggested that a small G-protein, rho, is involved in the events (Kishi et al., 1993; Mabuchi et al., 1993). Furthermore, uncoordinated cytokinesis cuts an undivided nucleus in the cut mutants, thus cut gene products may play a role in the initiation process (Hirano et al., 1986; Samijima et al., 1993). Initiation of furrow formation occurs at the site defined by preceding positioning process, so it is likely to interact between the factors concerning positioning and the initiation processes. The investigation of pos gene products, combined with that of the factor for the initiation, will lead to elucidating coordination between mitosis and cytokinesis. We are now attempting to identify pos gene products using molecular genetic techniques.