Genetic Interaction Between Calcineurin and Type 2 Myosin and Their Involvement in the Regulation of Cytokinesis and Chloride Ion Homeostasis in Fission Yeast

Corresponding author: Takayoshi Kuno, Department of Genome Sciences, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan., tkuno{at}kobe-u.ac.jp (E-mail)

Communicating editor: M. D. ROSE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Calcineurin plays a critical role in Ca²⁺ signaling in various cell types. In fission yeast, calcineurin is required for cytokinesis and chloride ion homeostasis. However, most of its physiological functions remain obscure. A genetic screen was performed to identify genes that share an essential function with calcineurin. We screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and to a high concentration of chloride ion and isolated a mutant, cis2-1/myp2-c2, which contains a novel allele of the myp2⁺/myo3⁺ gene that encodes a type 2 myosin heavy chain. The myp2-c2 mutant showed morphological defects similar to those associated with a calcineurin deletion mutant, such as multiseptated and branched cells. Consistently, myp2-null cells were hypersensitive to chloride ion and showed the multiseptated phenotype in the presence of immunosuppressants or at high chloride concentrations. Overexpression of constitutively active calcineurin suppressed the chloride ion-sensitive growth defect and cytokinesis abnormality of the myp2-c2 mutant and myp2-null cells. Interestingly, the essential myosin light chain mutant cdc4-8 failed to grow and could not form a normal contractile ring in the presence of immunosuppressants. Furthermore, calcineurin-null cells exhibited aberrant contractile rings, suggesting impaired contraction of the rings. These results indicate that calcineurin is involved in the regulation of cytokinesis and that chloride ion homeostasis is mediated by type 2 myosin.

CALCINEURIN, a Ca²⁺- and calmodulin-dependent protein phosphatase, is conserved from yeast to humans (CYERT et al. 1991 ; Y. LIU et al. 1991 ; YOSHIDA et al. 1994 ) and is a molecular target for immunosuppressive drugs, such as Cyclosporin A (CsA) and FK506 (J. LIU et al. 1991a). These drugs induce their biological effects by forming an initial complex with cytosolic proteins termed immunophilins (cyclophilin and FKBP12). These drug-immunophilin complexes then bind to and inhibit calcineurin. Calcineurin is widely distributed in mammalian tissues and may have additional functions other than its well-characterized role in lymphocytes. The inhibition of these functions may contribute to the side effects of these drugs. A better understanding of the biological roles of calcineurin in different cell types may lead to the development of improved strategies for immunosuppression.

We are studying the calcineurin signal transduction pathway in the fission yeast Schizosaccharomyces (S) pombe because this system is amenable to genetics and has many advantages in terms of relevance to higher systems. In addition, S. pombe is an excellent model organism in which to study cell division, since it shows the general features of higher eukaryotic cell division. S. pombe has a single gene encoding the catalytic subunit of calcineurin, ppb1⁺, which is essential for cytokinesis (YOSHIDA et al. 1994 ). We have previously shown that ppb1⁺ plays an essential role in maintaining chloride ion homeostasis and acts antagonistically with the Pmk1 mitogen-activated protein kinase pathway (SUGIURA et al. 1998 , SUGIURA et al. 1999 ). To identify new components that share an essential function with calcineurin, we screened for mutations that confer sensitivity to the calcineurin inhibitor FK506 and to a high concentration of chloride ion. We have isolated mutants and have identified five complementation groups (cis1-5 for chloride ion- and immunosuppressant-sensitive) to be described elsewhere in detail.

Here we report the isolation of a mutant, cis2-1/myp2-c2, a novel allele of the myp2⁺/myo3⁺ gene that encodes a type 2 myosin heavy chain. Functional analysis of myp2-c2 and cdc4-8, a conditional mutant of the essential myosin light chain, revealed that calcineurin and type 2 myosin play overlapping functions in cytokinesis and chloride ion homeostasis in fission yeast. Results also indicate that calcineurin exerts its regulatory roles at multiple sites of action in these cellular events.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, genetic and molecular biology techniques, and nomenclature:
Fission yeast strains used in this study are listed in Table 1. The complete medium, YPD, and the minimal medium, Edinburgh minimal medium (EMM), have been described previously (TODA et al. 1996 ). FK506 was provided by Fujisawa Pharmaceutical (Osaka, Japan). Standard methods for S. pombe genetics were followed according to MORENO et al. 1991 . Gene disruptions are denoted by lowercase letters representing the disrupted gene followed by two colons and the wild-type gene marker used for disruption (for example, myp2::ura4⁺). Gene disruptions are abbreviated by the gene preceded by (for example, myp2). Proteins are denoted by roman letters and only the first letter is capitalized (for example, Myp2).

Isolation of the cis2-1/myp2-c2 mutant:
The cis2-1/myp2-c2 mutant was isolated in a screen of cells that had been mutagenized with nitrosoguanidine. Cells of strain HM123 were mutagenized with 300 µM nitrosoguanidine (Sigma, St. Louis) for 60 min (10% survival) as described by MORENO et al. 1991 . Mutants were spread onto YPD plates to give 1000 cells/plate and incubated at 27° for 4 days. The plates were then replica plated to plates containing 0.5 µg/ml FK506 or 0.15 M MgCl₂. Mutants that showed both MgCl₂ sensitivity and FK506 sensitivity were selected. The original mutants isolated were backcrossed three times to wild-type strain HM123 and HM528.

To clone the mutated gene, the cis2-1/myp2-c2 mutant (KP333) was grown at 27° and transformed with an S. pombe genomic DNA library constructed in the vector pDB248 (MORENO et al. 1991 ). Leu⁺ transformants were replica plated to plates containing 0.15 M MgCl_2, and plasmid DNA was recovered from transformants that showed plasmid-dependent rescue. These plasmids had identical or overlapping inserts as judged from restriction digests, and all complemented both the chloride ion sensitivity and immunosuppressant sensitivity of the cis2-1/myp2-c2 mutant.

Deletion of the myp2⁺ gene:
A one-step gene disruption by homologous recombination (ROTHSTEIN 1983 ) was performed. The myp2::ura4⁺ disruption was constructed as follows. The BglII fragment containing the myp2⁺ gene was subcloned into the BglII site of pGEM-13Zf(+) (Promega). Then a BamHI fragment containing the ura4⁺ gene was inserted into the BamHI site of the previous construct. The fragment containing the disrupted myp2⁺ gene was transformed into diploid cells. Stable integrants were selected on medium lacking uracil, and disruption of the gene was checked by genomic Southern hybridization (data not shown).

Microscopic analysis:
For microscopic observation, cells were washed with phosphate-buffered saline (PBS; pH 7.0). Cells were stained with Hoechst 33342 and Calcofluor dissolved in PBS to visualize the DNA or septum, respectively. For F-actin staining, cells were fixed in 3% formaldehyde in PBS for 30 min (BALASUBRAMANIAN et al. 1997 ), and 1 µl of 100 µg/ml rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR) was added to 50 µl of fixed cell suspension. After 30 min at room temperature the excess phalloidin was washed away with PBS. To visualize components of the medial ring in living cells, wild-type, myp2-c2, myp2, ppb1, or cdc4-8 mutant cells were transformed with a plasmid encoding green fluorescent protein (GFP)-Cdc4 (BALASUBRAMANIAN et al. 1997 ), GFP-Myo2, or GFP-Myp2 (MOTEGI et al. 2000 ). The cells were examined by differential interference contrast (DIC) and fluorescent microscopy using an Axioskop microscope (Carl Zeiss, Thornwood, NY). Photographs were taken with a SPOT2 digital camera (Diagnostic Instruments). Images were processed with the CorelDRAW (Corel Corporation).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of the cis2-1/myp2-c2 mutant:
A genetic screen was performed to identify genes that share an essential overlapping function with calcineurin. For this purpose, we developed a genetic screen using the immunosuppressive drug FK506 for mutants that depend on calcineurin for growth. During the screening we observed that several immunosuppressant-sensitive mutants were also hypersensitive to chloride ion. As described previously, the chloride ion hypersensitivity is one of the most notable phenotypes of the calcineurin gene deletion (ppb1; SUGIURA et al. 1998 ). To find the mechanism for the regulation of chloride ion homeostasis through calcineurin action, fission yeast mutants that showed both immunosuppressant sensitivity and chloride ion hypersensitivity were selected. We have identified five complementation groups (cis1-5) to be described elsewhere in detail. One of the mutants, cis2-1 mutant, grew as well as the wild type at 27°. But the cis2-1 mutant could not grow in the presence of FK506 or CsA or in the presence of 0.15 M MgCl₂ or 0.4 M KCl, while the wild type grew well under these conditions (Fig 1A). Similar hypersensitivity was seen in YPD containing 0.3 M NaCl, or 0.15 M CaCl₂, and the sensitivity was due to the chloride ion rather than to the high osmolarity, as 1.2 M sorbitol did not affect the growth of cis2-1 mutant cells (data not shown). Cis2 is thus thought to be required for the regulation of chloride ion homeostasis. Previous reports showed that calcineurin is an in vivo target of FK506 in fission yeast, and inhibition of calcineurin activity by the addition of FK506 to the culture media resulted in phenotypes identical to those of calcineurin deletion (YOSHIDA et al. 1994 ; SUGIURA et al. 1999 ). It could be anticipated, then, that the cis2-1 mutant requires functional calcineurin activity for vegetative growth. Tetrad analysis was performed to examine the synthetic lethality with the ppb1 mutant. As expected, no double mutant was obtained, indicating that cis2-1 mutation and calcineurin deletion was synthetically lethal (data not shown).

View larger version (216K): In this window In a new window Download PPT slide   Figure 1. Immunosuppressant and chloride ion sensitivity of the cis2-1 mutant cells. (A) Wild-type (WT) and cis2-1 mutant cells were streaked onto each plate containing YPD, YPD plus 0.5 µg/ml FK506, YPD plus 50 µg/ml CsA, YPD plus 0.15 M MgCl2, or YPD plus 0.4 M KCl and incubated at 27° for 4 days. (B) Defective cytokinesis in the cis2-1 mutant. Wild-type (WT) and cis2-1 mutant cells grown to mid-log phase at 27° in liquid YPD medium were shifted to the medium containing 0.15 M MgCl2 or 0.5 µg/ml FK506 for 8 hr, stained with Calcofluor, and examined by DIC and fluorescent microscopy. Arrowhead indicates abnormally thick or misshapen septum. Bar, 10 µm.

The cis2-1/myp2-c2 mutant showed cytokinesis defects that were exacerbated by FK506 or MgCl₂:
We examined the morphological phenotypic changes in the cis2-1 mutant upon FK506 or MgCl₂ treatment in the cis2-1 mutant to search for the physiological function related to Cis2 (Fig 1B). Cells grown to mid-log phase at 27° in liquid YPD medium were subjected to a shift to the medium containing FK506 or MgCl₂. Even in the absence of FK506 or MgCl₂, 30–50% of the cis2-1 mutant cells had a division septum as compared with the 5–15% seen in a wild-type population (Fig 1B). Upon a shift to the medium containing 0.15 M MgCl₂, the cis2-1 mutant cells were enlarged, the frequency of septated cells significantly increased (up to 70–90%), and multiseptated or branched cells were frequent. Abnormally thick or misshapen septum was also frequently observed (Fig 1B, arrowheads). These results suggest the important role of Cis2 function in cell volume control and cytokinesis especially under the salt-stress condition. FK506 caused similar, but more severe, cytokinesis defects as those seen in 0.15 M MgCl₂, and nearly 100% of cells became septated (Fig 1B). In addition, branched cells and cells with two or more septa were very frequent, and some of the septa were abnormally positioned or misshapen (Fig 1B, arrowheads). Thus, it is suggested that calcineurin and Cis2 play overlapping functions for cytokinesis and cell polarity control.

cis2-1/myp2-c2 is a novel allele of the myp2⁺/myo3⁺ gene that encodes a type 2 myosin heavy chain:
The plasmids recovered from transformants that showed plasmid-dependent rescue had identical or overlapping inserts, and all complemented both the chloride ion sensitivity and immunosuppressant sensitivity of the cis2-1 mutant (Fig 2A). A BLAST search of protein sequence databases revealed that these plasmids contained a myp2⁺/myo3⁺ gene encoding a 2104-amino-acid S. pombe type 2 myosin heavy chain (BEZANILLA et al. 1997 ; MOTEGI et al. 1997 ), which is a homolog of S. pombe Myo2 (57%; KITAYAMA et al. 1997 ), Saccharomyces cerevisiae Myo1 (49%; WATTS et al. 1987 ), and human myosin heavy chain (47%; TOOTHAKER et al. 1991 ). Two groups have independently cloned and deleted the myp2⁺/myo3⁺ gene and found it to be nonessential (BEZANILLA et al. 1997 ; MOTEGI et al. 1997 ). Consistent with our present study, the possibility that Myp2 might interact with the salt-response pathway has been noted by Pollard's group (BEZANILLA et al. 1997 ; BEZANILLA and POLLARD 2000 ).

View larger version (67K): In this window In a new window Download PPT slide   Figure 2. Cloning and characterization of the cis2+/myp2+ gene. (A) Cloning of the cis2+ gene. Wild-type (WT) and cis2-1/myp2-c2 mutant cells transformed with a control vector, the vector containing the myp2+ gene, or the vector containing the myo2+ gene were streaked onto each plate containing YPD, YPD plus 0.15 M MgCl2, or YPD plus 0.5 µg/ml FK506 and incubated for 4 days at 27°. (B) The effects of FK506 and MgCl2 on the growth of the cis2-1/myp2-c2 mutant and myp2 cells. Wild-type (WT), myp2-c2 mutant cells, or myp2 cells were streaked onto each YPD plate containing 0.5 µg/ml FK506, 0.15 M MgCl2, or 0.3 M MgCl2 and incubated at 27° for 4 days.

To investigate the relationship between the cloned myp2⁺ gene and cis2-1 mutant, integration mapping was performed as follows. The entire myp2⁺ gene was subcloned into the pUC-derived plasmid containing S. cerevisiae LEU2 gene and integrated by homologous recombination into the genome of the wild-type strain HM123. The integrant was mated with the cis2-1 mutant. The resulting diploid was sporulated and tetrads were dissected. A total of 24 tetrads were dissected. In all cases, only parental ditype tetrads were found, indicating allelism between the myp2⁺ gene and cis2-1 mutation. Furthermore, random spore analysis of a cross between the cis2-1 mutant and the myp2 deletion confirmed the allelism (data not shown). Accordingly, we renamed the cis2-1 mutant as myp2-c2 mutant.

When expressed in multicopy, the myo2⁺ gene, which encodes another S. pombe type 2 myosin heavy chain, partially but clearly complemented the chloride ion-sensitive growth defect of the myp2-c2 mutant. Interestingly, however, it only weakly suppressed the FK506-sensitive growth defects (Fig 2A). These results suggest that the protein phosphatase activity of calcineurin is required for the proper function of the myo2⁺ gene product.

Disruption of myp2⁺ gene:
To further analyze Myp2 function, the myp2⁺ gene was knocked out in a diploid by homologous recombination using the ura4⁺ marker gene (ROTHSTEIN 1983 ). Southern blotting of the genomic DNA of one such transformant confirmed that the wild-type gene had been replaced by the derivative containing the ura4⁺ insertion (data not shown). Tetrad analysis revealed that the myp2⁺ gene is nonessential, in agreement with previous studies (BEZANILLA et al. 1997 ; MOTEGI et al. 1997 ). Interestingly, myp2 cells showed the chloride ion-sensitive growth defect that is similar to but less severe than that of the myp2-c2 mutant. They grew in the presence of 0.15 M MgCl₂, but could not grow in 0.3 M MgCl₂ (Fig 2B). As shown in Fig 2B, myp2 cells could grow in the presence of FK506. Consistently, calcineurin deletion was not synthetically lethal with myp2 (data not shown). However, microscopic observation revealed that most of the myp2 cells were also enlarged and multiseptated, similar to the myp2-c2 mutant, especially when the medium contained FK506 or a high concentration of chloride ion (data not shown). These findings suggest that Myp2 is implicated in the establishment of normal cell volume and cytokinesis.

Since the myp2-c2 mutation appears to be more extreme than that of myp2, it is suggested that the mutation is dominant and that an excess of wild-type protein from the multicopy plasmid may titrate out the dominant mutation. In agreement with this hypothesis, complementation in heterozygous myp2-c2/myp2⁺ diploid cells was incomplete, suggesting that the myp2-c2 mutation is semidominant. Consistently, when the myp2⁺ gene was knocked out in the myp2-c2 mutant, the cis2-1 phenotype was destroyed and the mutant came to show only the myp2 phenotype (data not shown).

Expression of constitutively active calcineurin (ppb1C) suppressed the chloride-sensitive growth defects of the myp2-c2 mutant and myp2 deletion:
To better understand the relationship between Myp2 function and calcineurin signaling, the myp2-c2 mutant and myp2 cells were transformed with a control vector, a vector containing a full-length ppb1⁺ calcineurin gene, or a constitutively active truncated calcineurin gene (ppb1C; SUGIURA et al. 1998 ). They were streaked onto each plate containing YPD plus 0.5 µg/ml FK506, YPD plus 0.15 M MgCl₂, or YPD plus 0.3 M MgCl₂ (Fig 3A). The myp2-c2 mutant cells transformed with a control multicopy vector or with the vector containing the ppb1⁺ gene could not grow in the presence of FK506 or 0.15 M MgCl₂, whereas myp2-c2 mutant cells transformed with a vector containing the myp2⁺ gene grew in the presence of FK506 or MgCl₂. Strikingly, myp2-c2 mutant cells transformed with the vector containing the ppb1C grew in the presence of 0.15 M MgCl₂, similar to the mutant cells transformed with the vector harboring the myp2⁺ gene. However, they could not grow in the presence of FK506 (Fig 3A).

View larger version (216K): In this window In a new window Download PPT slide   Figure 3. Expression of constitutively active calcineurin suppressed the chloride-sensitive defects of myp2-c2 mutant cells and myp2 cells. (A) Suppression of the growth defects. myp2-c2 or myp2 cells transformed with a control vector, the vector containing the myp2+ gene, the vector containing the full-length ppb1+ gene, or the vector containing the constitutively active truncated calcineurin gene (ppb1C) were streaked onto each YPD plate containing 0.15 M MgCl2, 0.3 M MgCl2, or 0.5 µg/ml FK506 and incubated for 4 days at 27°. (B) Suppression of the morphological defects. myp2-c2 mutant cells transformed with a control vector, the vector containing the myp2+ gene, or the vector containing ppb1C were grown to mid-log phase at 27° in liquid EMM, shifted to the medium containing 0.15 M MgCl2 or 0.5 µg/ml FK506 for 8 hr, and stained with Hoechst 33342 and Calcofluor. Bar, 10 µm. (C) The septation index of myp2-c2 mutant. Overnight cultures of the myp2-c2 mutant transformed with a control vector, the vector containing the myp2+ gene, ppb1+ gene, or ppb1C were subjected to a shift to the fresh EMM containing 0.1 M MgCl2 or 0.5 µg/ml FK506 for 8 hr and stained with Calcofluor. A septum of 100 cells was counted and a mean of septum/cell was calculated. (D) The septation index of myp2 cells. Overnight cultures of myp2 cells transformed with a control vector, the vector containing the myp2+ gene, ppb1+ gene, or ppb1C were subjected to a shift to the fresh EMM containing 0.2 M MgCl2 or 0.5 µg/ml FK506 for 8 hr and stained with Calcofluor. A septum of 100 cells was counted and a mean of septum/cell was calculated.

Consistently, myp2 cells transformed with a control vector or with the vector containing a full-length ppb1⁺ gene could not grow in the presence of 0.3 M MgCl₂. However, myp2 cells transformed with the vector containing ppb1C grew, similar to the cells harboring multicopy myp2⁺ gene (Fig 3A).

Constitutively active calcineurin also suppressed the cytokinesis defects of myp2-c2 and myp2 cells:
Then we examined the morphological changes of myp2-c2 mutants upon multicopy expression of ppb1C (Fig 3B and Fig C). Cells transformed with various constructs described above were grown to mid-log phase at 27° in liquid EMM and were subjected to a shift to the medium containing MgCl₂ or 0.5 µg/ml FK506 for 8 hr (Fig 3B and Fig C). As expected, nearly 100% of the myp2-c2 mutant cells transformed with a control vector showed abnormal morphology after the addition of FK506 or 0.15 M MgCl₂, whereas the myp2-c2 mutant cells transformed with a vector containing the myp2⁺ gene were morphologically indiscernible from the wild type (Fig 3B). Most notably, the mutant cells harboring the vector containing the ppb1C were also morphologically indiscernible from the wild type in the absence or presence of 0.15 M MgCl₂ (Fig 3B). Again, the suppression by ppb1C overexpression was observed only with the chloride ion-induced defects and it had no effect on the FK506-induced defects. On the other hand, overexpression of the full-length Ppb1 calcineurin had no effect on the chloride ion-induced cytokinesis defects of myp2-c2 mutant cells. These results suggest that the protein phosphatase activity of calcineurin is important for the suppression of the cytokinesis defects.

As described above, myp2 deletion also resulted in a significant increase in the frequency of septated cells upon treatment with FK506 or MgCl₂ (Fig 3D). As in the case of the myp2-c2 mutant, ppb1C could suppress these cytokinesis defects of myp2 cells caused by 0.2 M MgCl₂, but was ineffective on the defects caused by FK506 (Fig 3D).

Together, these results suggest that Myp2 is implicated in chloride ion homeostasis and cytokinesis and performs overlapping functions with Ppb1 calcineurin in these cellular events.

Subcellular localization of GFP-Cdc4 in myp2-c2 and myp2 cells:
Unlike other unicellular organisms, S. pombe has two structurally distinct type 2 myosin heavy chains, Myo2 and Myp2/Myo3, which are required under different conditions (BEZANILLA et al. 1997 , BEZANILLA et al. 2000 ; KITAYAMA et al. 1997 ; MAY et al. 1997 ; MOTEGI et al. 1997 ). These two heavy chains assemble into contractile rings at distinct times during mitosis (BEZANILLA et al. 2000 ). As the myosin light chain Cdc4 is a well-known contractile ring component complexed with Myo2, subcellular localization of GFP-tagged Cdc4 was examined in the myp2-c2 mutant and myp2 deletion (Fig 4). We also examined the effects of MgCl₂ and FK506 on the GFP-Cdc4 distribution. myp2-c2 and myp2 cells expressing GFP-Cdc4 were grown at 27° and then shifted to the medium containing MgCl₂ (0.1 M for myp2-c2 and 0.2 M for myp2 deletion) or 0.5 µg/ml FK506 for 8 hr. The cells were examined by DIC and fluorescence microscopy (Fig 4). GFP-Cdc4 localized in a medial ring in wild-type cells undergoing mitosis and cytokinesis, and a shift to the medium containing MgCl₂ or 0.5 µg/ml FK506 had no effect on the GFP-Cdc4 localization (data not shown). In the myp2-c2 or myp2 cells, GFP-Cdc4 was observed in the contractile ring-like structure even when these cells showed the cytokinesis defects upon a shift to the medium containing MgCl₂ or 0.5 µg/ml FK506. These findings suggest that Myp2 was not essential for contractile ring formation. However, many of these ring-like structures were aberrantly localized to the outside of the medial region or associated with thickened and misshapen septa (Fig 4, arrowheads). These results suggest that proper coordination between medial ring formation and septum formation is lost. They are also consistent with the hypothesis that Myp2 is required to improve the efficiency of cytokinesis and links septum formation to contractile ring contraction (BEZANILLA et al. 2000 ).

View larger version (105K): In this window In a new window Download PPT slide   Figure 4. Subcellular localization of GFP-Cdc4 in the myp2-c2 mutant and the myp2 deletion. The cells expressing GFP-Cdc4 were grown to mid-log phase at 27° in liquid EMM and were subjected to a shift to the medium containing 0.1 M MgCl2 (myp2-c2 mutant), 0.2 M MgCl2 (myp2 deletion), or 0.5 µg/ml FK506 for 8 hr. The cells were examined by DIC and fluorescent microscopy. Arrowhead indicates the contractile ring-like structure, which was aberrantly localized to the outside of the medial region or associated with thickened and misshapen septum. Bar, 10 µm.

cdc4-8 mutant cells were sensitive to immunosuppressants:
As a next step to study the functional relationship between calcineurin and type 2 myosin, we examined the effects of calcineurin inhibitor FK506 on the cdc4-8 (G107S) mutant. As described above, the cdc4⁺ gene encodes an essential myosin light chain complexed with Myo2 (MCCOLLUM et al. 1999 ; NAQVI et al. 1999 ). Our present study (Fig 2A) suggested that the protein phosphatase activity of calcineurin is required for the proper function of the myo2⁺ gene product. Thus, we expected that the temperature-sensitive cdc4-8 mutant (MCCOLLUM et al. 1995 ) might also be sensitive to immunosuppressants. Consistent with our expectation, cdc4-8 mutant cells could not grow in the presence of FK506 or CsA (Fig 5A). In addition, the cdc4-8 mutant showed synthetic lethality with calcineurin deletion (data not shown). These results again suggest that calcineurin and type 2 myosin play an overlapping function in cytokinesis. Interestingly, cdc4-8 mutant cells were not sensitive to chloride ion and grew in the presence of 0.3 M MgCl₂ (Fig 5A).

View larger version (174K): In this window In a new window Download PPT slide   Figure 5. cdc4-8 mutant cells were immunosuppressant sensitive. (A) Immunosuppressant-sensitive growth defect of cdc4-8 mutant cells. Wild-type (WT) and cdc4-8 mutant cells were streaked onto each plate containing YPD, YPD plus 0.5 µg/ml FK506, YPD plus 50 µg/ml CsA, or YPD plus 0.3 M MgCl2 and incubated at 27° for 4 days. (B) Defective contractile ring formation of cdc4-8 mutant. Overnight cultures of cdc4-8 cells were subjected to a temperature shift (33°) or a shift to the fresh medium containing 0.5 µg/ml FK506 for 6 hr. Cells were stained with rhodamine-conjugated phalloidin for F-actin. Bar, 10 µm.

FK506-treated cdc4-8 mutant cells were defective in contractile ring formation:
To investigate further the calcineurin requirement for cdc4⁺ function in cytokinesis, contractile ring formation was analyzed by F-actin staining using rhodamine-conjugated phalloidin. In the absence of FK506, F-actin staining of cdc4-8 cells grown at the permissive temperature resembled that reported previously for wild-type cells (MCCOLLUM et al. 1995 ; Fig 5B). Most strikingly, FK506-treated cdc4-8 cells displayed a pattern of F-actin staining similar to that of cdc4-8 cells at the restrictive temperature (Fig 5B). F-actin staining also appeared in large dots or cables instead of ring structure (arrows). These results indicate that the cdc4-8 mutant requires calcineurin activity to form the contractile ring properly.

Intracellular localization of GFP-Cdc4, GFP-Myo2, and GFP-Myp2:
To further investigate the role of calcineurin in the regulation of contractile ring formation, localization of GFP-Cdc4, GFP-Myo2, and GFP-Myp2 in wild-type and ppb1 cells was examined (Fig 6). In agreement with previous reports (BEZANILLA et al. 2000 ; MOTEGI et al. 2000 ), GFP-Cdc4 and GFP-Myo2 localized to ring structures and GFP-Myp2 localized as spots in the medial regions of wild-type cells (Fig 6A). It should be noted that localization of GFP-Cdc4 and GFP-Myo2 was very similar in wild-type cells. Both of them localized as very clear rings at the medial region and also had a low level of diffuse cytoplasmic fluorescence. Compared with the wild type, ppb1 cells exhibited an approximately threefold increased frequency of medial rings or spots, which were stained by GFP-Cdc4, GFP-Myo2, or GFP-Myp2 (Fig 6A and Fig B). GFP-Cdc4 or GFP-Myp2 in ppb1 cells showed similar staining patterns as those in wild-type cells. On the other hand, GFP-Myo2 rings in ppb1 cells were mostly discontinuous or irregularly shaped. In addition, diffuse cytoplasmic fluorescence of GFP-Myo2 was remarkably reduced in most of the ppb1 cells (Fig 6A). These results suggest that the deletion of calcineurin affects the assembly/disassembly or contraction of the Myo2 ring and results in impaired cell separation and the apparent increase in the number of dividing cells.

View larger version (68K): In this window In a new window Download PPT slide   Figure 6. The effects of calcineurin deletion on the localization of GFP-Cdc4, GFP-Myo2, and GFP-Myp2. (A) Subcellular localization of GFP-Cdc4, GFP-Myo2, and GFP-Myp2 in wild-type and calcineurin-null cells. Wild-type and ppb1 cells carrying pREP41-GFP-Cdc4, pREP81-GFP-Myo2, or pREP81-GFP-Myp2 were grown in the medium without thiamine at 27°. The cells at exponential growth phase were observed under fluorescence or DIC microscopy. Arrowheads indicate the ring-like structure. Arrows indicates the spot-like structure. Bar, 10 µm. (B) Frequency of cells with the medial rings or spots stained by GFP-Cdc4, GFP-Myo2, and GFP-Myp2 was calculated. One hundred cells were examined for each calculation.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In an attempt to identify genes that may have overlapping function with calcineurin, we isolated myp2-c2, a novel allele of fission yeast gene myp2⁺, encoding a type 2 myosin heavy chain. Five lines of evidence show that calcineurin and type 2 myosin coordinately play several functional roles in fission yeast. First, mutation of the myp2⁺, a type 2 myosin heavy chain gene, conferred immunosuppressant sensitivity for the growth; that is, the myp2-c2 mutant was synthetically lethal with the calcineurin deletion. Second, the myp2 deletion caused a cytokinesis defect, which was exacerbated by FK506 treatment. Third, overexpression of a truncated, constitutively active, and Ca²⁺-independent form of calcineurin (ppb1C) suppressed the chloride ion-sensitive defects of myp2-c2 and myp2 cells. Fourth, the essential myosin light chain mutant cdc4-8 required calcineurin activity for its growth and contractile ring formation. Fifth, calcineurin-null cells exhibited aberrant GFP-Myo2 localization.

Calcineurin and Myp2 display a strong genetic interaction:
As described in the previous study, disruption of Ppb1 calcineurin or incubation of wild-type cells with FK506 resulted in the appearance of abnormally septated and branched cells (YOSHIDA et al. 1994 ). From these observations, it has been suggested that the progression of cytokinesis was delayed and the positioning of septation became abnormal in the calcineurin-null mutant (YOSHIDA et al. 1994 ). Likewise, myp2-c2 mutant cells had a septation index approximately three times higher than that seen in wild-type cells and showed a dramatic increase (up to 70%) in the index 8 hr after shifting to the medium containing 0.15 M MgCl₂, whereas the index of wild type was 10% (data not shown). Addition of FK506 to the culture media dramatically increased the septation index of myp2-c2 mutant cells, and the septation index reached nearly 100% 8 hr after the addition of FK506, whereas 30% of wild-type culture displayed septated cells. Branched cells were very frequent upon these medium shifts (data not shown). These results suggest that Myp2 and calcineurin coordinately regulate cytokinesis. myp2 cells also became multiseptated by the addition of MgCl₂ or FK506, although myp2 showed phenotypes less severe than those of the myp2-c2 mutant. These microscopic findings suggest that Myp2 is implicated in the establishment of cytokinesis and cell polarity. Furthermore, aberrant localization of contractile ring-like structures observed in myp2-c2 and myp2 cells suggests that proper coordination between medial ring formation and septum formation is lost in these mutants, further suggesting that calcineurin is also implicated in this proper coordination.

Calcineurin is involved in the regulation of contractile ring formation:
In the study by SLUPSKY et al. 2001 Cdc4 is predicted to associate with Myo2, as well as Myp2, at their IQ motifs. Cdc4 is recruited to the contractile ring prior to cell division (MCCOLLUM et al. 1995 ). Interestingly, this recruitment is independent of Myo2, because Cdc4 is still found in improperly formed rings in myo2-null mutants (NAQVI et al. 1999 ). In the present study, the cdc4-8 (G107S) mutant was shown to be FK506 sensitive, indicating that Cdc4 acts in a pathway overlapping or parallel to calcineurin. Furthermore, the cdc4-8 (G107S) mutant was defective in contractile ring formation in the presence of FK506 even at the permissive temperature, suggesting that calcineurin is involved in this cellular process. However, as shown in the present study, contractile ring formation itself is not defective in myp2-c2 or myp2 mutants treated with FK506. Thus, calcineurin may act to regulate cytokinesis in at least two sites: namely, at the step of contractile ring formation and at linking between contractile ring and septum formation.

Consistent with the previous study, we have found that no GFP-Myo2 ring was detected in the cdc4-8 mutant, suggesting that recruitment of Myo2 to the contractile ring is Cdc4 dependent (data not shown; MOTEGI et al. 2000 ). Thus, the dissociation between the staining patterns of GFP-Myo2 and GFP-Cdc4 in ppb1 cells suggests that interaction of Myo2 and Cdc4 may be impaired by calcineurin deletion.

Recent study showed that Cdc4 interacts with several other proteins besides Myo2, including a putative phosphatidylinositol 4-kinase (DESAUTELS et al. 2001 ). Furthermore, our recent study also showed that calcineurin and phosphatidylinositol-4-phosphate 5-kinase share an essential function in fission yeast cytokinesis, and phosphatidylinositol-4-phosphate 5-kinase was found to be concentrated at the site of cell separation (ZHANG et al. 2000 ). These findings suggest that Cdc4 may be more than just an essential light chain bound to Myo2, as suggested by DESAUTELS et al. 2001 , and that Cdc4 plays an important role in the assembly and function of the contractile ring in fission yeast through its functional interaction with calcineurin- and phosphatidylinositol-mediated pathways.

It has been shown that the regulatory light chain protein Rlc1 also binds to Myo2 in a manner that is dependent on the IQ sequence motif and that Rlc1 is a component of the contractile ring (NAQVI et al. 2000 ). It is of interest to know whether calcineurin affects the interaction between Rlc1 and Myo2.

Myp2 is involved in chloride ion homeostasis:
As shown in the present study, both the myp2-c2 and myp2 cells were hypersensitive to chloride ion. The chloride ion-sensitive growth defects and morphological changes of these mutants were completely suppressed by overexpression of the constitutively active form of calcineurin. These results suggest that Myp2 is involved in the regulation of chloride ion homeostasis and that calcineurin acts in a parallel pathway to enhance the chloride ion tolerance. This is consistent with our previous observation that calcineurin-null mutants are hypersensitive to chloride ion (SUGIURA et al. 1998 ). Alternatively, Myp2 may send a signal that leads to the activation of calcineurin. Increased expression of a constitutively active calcineurin might then compensate for the chloride ion hypersensitivity.

In contrast, cdc4-8 mutant cells were sensitive to FK506, but not hypersensitive to the chloride ion. This suggests that Cdc4 shares an essential function for growth with calcineurin, but is not involved in chloride ion homeostasis. Alternatively, the cdc4-8 mutation could specifically impair the function of Cdc4 that is not related to chloride ion homeostasis. This apparent discrepancy again suggests that Cdc4 has multiple functions and that calcineurin has multiple sites of action to regulate cellular functions related to type 2 myosin.

In conclusion, the present study showed evidence for the first genetic link between calcineurin and type 2 myosin, and our results suggest that calcineurin is involved in the regulation of cytokinesis and chloride ion homeostasis mediated by type 2 myosin in fission yeast. The functional relationship between calcineurin and type 2 myosin demonstrated in this study might be conserved in evolutionarily distant organisms. Thus, elucidation of the mechanisms of interaction between calcineurin and type 2 myosin may be of general interest for an understanding of eukaryotic morphogenetic events.

	ACKNOWLEDGMENTS

We thank Susie O. Sio for critical reading of the manuscript and also thank Mitsuhiro Yanagida (Kyoto University, Japan) and Issei Mabuchi (The University of Tokyo, Japan) for their generous gift of strains and plasmids. This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Hyogo Science and Technology Association.

Manuscript received December 4, 2001; Accepted for publication March 20, 2002.

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