Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast Schizosaccharomyces pombe

Yeast strains, media, and genetic methods.The S. pombe strains used in this study are listed in Table 1. The recombinant PCR gene knockout method described by Krawchuk and Wahls (14) was used to generate the psd1::kanMX6, psd2::hphMX6, and psd3::ura4 deletion cassettes, in which the psd1, psd2, and psd3 open reading frames are replaced by the kanMX6 (kanamycin and G418 resistance), hphMX6 (hygromycin resistance), and ura4 markers, respectively. PCRs used plasmid pFA6a-kanMX6 (2), pFA6a-hphMX6, or pFA6a-ura4 as the template DNA together with the oligonucleotide primer sets psd1-F1 (5′-GGCAGTATGCTATTAGCAGG-3′)-psd1-R1 (5′-GGGGATCCGTCGACCTGCAGCGTACGAGTTAGATTGTTTTTATGGAA-3′), psd1-F2 (5′-GTTTAAACGAGCTCGAATTCATCGATTAGCAAGCAATGCATATTGG-3′)-psd1-R2 (5′-GTTAGAAGAAGTAAACATTC-3′), psd2-F1 (5′-CTCCATCTTATCAGCTAGAC-3′)-psd2-R1 (5′-GGGGATCCGTCGACCTGCAGCGTACGAATCGGTATAAATTTCCTTAG-3′), psd2-F2 (5′-GTTTAAACGAGCTCGAATTCATCGATCCCATTTGCTTTTGATCTTC-3′)-psd2-R2 (5′-GCCTCTTATAAGTCATCCAC-3′), psd3-F1 (5′-GTTCGTAACAAACTCCATCC-3′)-psd3-R1 (5′-GGGGATCCGTCGACCTGCAGCGTACGATTCCCGTAGTGGAAAA CAGC-3′), and psd3-F2 (5′-GTTTAAACGAGCTCGAATTCATCGATCTTGCTTTTAACGACGACAG-3′)-psd3-R2 (5′-CATCATCCCAAATATCTCCC-3′). The S. pombe haploid strain SP870 was transformed with the resulting psd1::kanMX6, psd2::hphMX6, or psd3::ura4 cassette, and transformants were isolated on selective Edinburgh minimal medium (EMM) (for psd3::ura4 transformants) or on YEAU (0.5% yeast extract, 3% dextrose, 225 mg/liter adenine, 225 mg/liter uracil) containing either G418 (for psd1::kanMX6 transformants) or hygromycin (for psd2::hphMX6 transformants). Transformants carrying psdΔ gene deletions were identified by colony PCR (17). S. pombe psdΔ double and triple deletion mutants were constructed by genetic crosses using standard yeast genetic methods (1, 24). S. pombe cultures were grown in either YEAU or synthetic minimal medium (EMM) with appropriate auxotrophic supplements (1). Where indicated, media were supplemented with 1 mM ethanolamine.

Radiolabeling and phospholipid analysis.For phospholipid composition analysis of S. pombe strains cultured in rich medium, cells were cultured in YEAU containing 20 μCi of ³²P_i/ml at 30°C with shaking to a density of 6 × 10⁶ cells/ml and were harvested by centrifugation for subsequent lipid extraction (see below). Where indicated, YEAU was supplemented with 1 mM ethanolamine. For analyses of strains cultured in minimal medium, cells were grown in YEAU containing 1 mM ethanolamine to mid-log phase, washed three times with EMM containing 1 mM ethanolamine, resuspended at a density of 7.5 × 10⁵ cells/ml in EMM containing 1 mM ethanolamine and 20 μCi/ml ³²P_i, and cultured to a density of 6 × 10⁶ cells/ml. A portion of each culture was harvested by centrifugation for lipid extraction, and the remainder was washed with EMM containing 20 μCi/ml ³²P_i, resuspended at 3 × 10⁶ cells/ml in EMM containing 20 μCi/ml ³²P_i, and incubated for 8 h. For lipid extraction, 1 ml of each labeled cell culture was harvested by centrifugation. The cells were washed with 1 ml sterile water, resuspended in 1 ml 5% trichloroacetic acid, and incubated on ice for 15 min. Cells were washed twice with 1 ml of sterile water and resuspended in 1 ml of extraction solvent I (43.7% water, 39.5% ethanol, 13.9% diethyl ether, 2.8% pyridine, and 0.1% ammonium hydroxide) for 1 h at 60°C with occasional swirling. Samples were centrifuged at 1,800 × g for 3 min, and 1 ml of the resulting supernatant volume was added to 0.25 ml of extraction solvent II (water-chloroform-methanol, 9:60:30). After centrifugation at 200 × g for 2 min, the lower chloroform phase was transferred to the new tube and dried down under nitrogen. The extracted lipids were resuspended in chloroform-methanol (2:1), counted by liquid scintillation, and spotted onto Partisil K6 (60-Å silica gel) thin-layer chromatography plates (Whatman), which were then developed in chloroform-ethanol-water-triethylamine (30:35:7:35). Phospholipids were detected by autoradiography and were quantified using the Quantity One program (Bio-Rad).

Fluorescence staining and microscopy of S. pombe cells.For visualization of S. pombe cell walls by fluorescence microscopy, 1 ml of mid-log-phase culture was pelleted by centrifugation, and the majority of the culture supernatant was removed, leaving a volume of approximately 10 μl. Two microliters of the concentrated cell suspension was mixed on a microscope slide with 2 μl of a calcofluor white stock solution (1 mg/ml H₂O) for a final concentration of 0.5 mg/ml, and the stained cells were imaged by epifluorescence microscopy. To visualize F-actin, rhodamine-phalloidin staining was performed using the method of Sawin and Nurse (25). For time lapse microscopy of cells with deletions in all three psd genes (psd1-3Δ cells), petri dishes containing a thin layer (8 ml) of YEAU agar with 1 mM ethanolamine were prepared. About 10⁴ cells were pipetted onto the agar, overlaid with a coverslip, and imaged at regular intervals by differential interference contrast (DIC) photomicroscopy. DIC and epifluorescence microscopic analyses were carried out using a Nikon 90i automated DIC/epifluorescence microscope system operated using Nikon NIS-Elements software. Images were captured using a CoolSNAP HQ2 monochrome charge-coupled device camera (Photometrics), and the level was adjusted using Adobe PhotoShop CS3 software (Adobe Systems, Inc.).

Identification of fission yeast PSD genes.To identify S. pombe open reading frames encoding proteins structurally homologous to eukaryotic PSD enzymes, we conducted TBLASTN and BLASTP searches of the S. pombe nucleic acid and protein sequence databases, respectively, using the S. cerevisiae Psd1 and Psd2 proteins as the query sequences. Two predicted S. pombe genes were identified, which we have designated psd1 (GenBank locus tag SPBC16E9.18) and psd2 (GenBank locus tag SPAC25B8.03), encoding proteins highly homologous in structure to S. cerevisiae Psd1 (E values, 6 × 10⁻⁸⁵ and 5 × 10⁻⁵¹, respectively). A third predicted gene, which we have designated psd3 (GenBank locus tag SPAC31G5.15), was identified; it encodes a protein highly homologous in structure to S. cerevisiae Psd2 (E value, 9 × 10⁻⁹⁶). Like S. cerevisiae Psd1, the S. pombe psd1 and psd2 gene products share high degrees of structural homology with mammalian PSD enzymes (E values, 10⁻⁷¹ and 10⁻⁴¹, respectively) and moderate homologies with various bacterial PSDs (E values as high as 3 × 10⁻²⁸). In contrast, the S. pombe psd3 gene product as well as its counterpart in S. cerevisiae, encoded by the PSD2 gene, share relatively little sequence similarity with mammalian PSDs (E values, <10⁻¹⁶ and 10⁻⁹, respectively) but exhibit substantial homology with PSDs found in a variety of bacterial organisms (E values as high as 4 × 10⁻⁵²).

Sequence analysis of fission yeast PSDs.The structural organization of S. pombe, S. cerevisiae, and human PSD proteins is depicted in Fig. 1A. S. pombe Psd1 (Psd1-Sp) is most similar in structural organization to mammalian PSDs, represented in Fig. 1 by the human Psid protein (GenBank accession no. CR456540). In both Psd2-Sp and S. cerevisiae Psd1 (Psd1-Sc), the predicted PSD superfamily domain is disrupted by a nonconserved sequence, which is quite extensive in the case of Psd2-Sp (Fig. 1A). Interestingly, both Psd3-Sp and Psd2-Sc contain substantial noncatalytic N-terminal sequences that account for more than two-thirds of the length of each protein (Fig. 1A). Psd3-Sp and Psd2-Sc share relatively little sequence homology within their respective noncatalytic N-terminal domains, except for a predicted protein kinase C conserved region 2 (C2) Ca²⁺-binding domain, which spans amino acid residues 281 to 387 in Psd3-Sp and residues 495 to 602 in Psd2-Sc (Fig. 1A). The C2 domain is a relatively common motif present in a variety of eukaryotic proteins, including protein kinase C, a number of different phospholipases, and synaptotagmins (23, 26). Particular C2 domains have been shown to bind to phospholipids, inositol polyphosphates, and other proteins. While some C2 domains have been shown to bind Ca²⁺, others appear to lack Ca²⁺-binding sites. However, it is noteworthy that the predicted C2 domains of both Psd3-Sp (Fig. 1B) and Psd2-Sc (data not shown) contain all four amino acid residues that constitute the conserved metal binding pocket of the archetypal Ca²⁺-binding C2 domain.

• Open in new tab
• Download powerpoint

FIG. 1.

Sequence organization and phylogenetic relationships of S. pombe, S. cerevisiae, mammalian, and representative bacterial PSD proteins. (A) The structural organizations of the Psd1-Sp, Psd2-Sp, Psd3-Sp, Psd1-Sc, Psd2-Sc, human Psid (PSD-Hs), and Fusobacterium nucleatum PSD (PSD-Fn) proteins were determined by use of the Conserved Domain Database (CDD) (16) integrated with the National Center for Biotechnology Information BLASTP application (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). PSD superfamily domains and C2 domains are indicated. (B) Alignment of the C2 domains of rat synaptotagmin III (SNPG III) and Psd3-Sp. The alignment was generated by the CDD program (16) integrated with the NCBI BLASTP application (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Asterisks mark the four conserved amino acids present in the conserved metal binding pocket of Ca²⁺-binding C2 domains. (C) The phylogenetic relationships of the PSD proteins diagramed in panel A as well as the PSD from the marine bacterium Alcanivorax sp. were determined using the CLUSTAL W application provided by the Protein Information Resource website, hosted by Georgetown University Medical Center (http://pir.georgetown.edu/pirwww ). Branch lengths are drawn to scale.

The phylogenetic relationships of S. pombe, S. cerevisiae, human (Psid), and representative bacterial PSD proteins were determined using the CLUSTAL W program (31). A PSD from the marine bacterium Alcanivorax sp. was identified by a BLASTP search as the bacterial PSD exhibiting the greatest degree of sequence homology with Psd1-Sp (E value, 10⁻²⁶), while a PSD from the bacterium Fusobacterium nucleatum showed the greatest degree of homology to Psd3-Sp (E value, 4 × 10⁻⁵²). As shown in Fig. 1C, the yeast, human, and bacterial PSDs used for predicting phylogenetic relationships lie in three branches, two with multiple nodes, derived from a hypothetical common ancestor. Psd1-Sp and Psd2-Sp are predicted to share a common phylogenetic lineage with mammalian PSDs. Psd3-Sp and Psd2-Sc occupy nodes on a separate branch shared with bacterial PSDs. Interestingly, Psd1-Sc lies in a branch that is phylogenetically separate from those occupied by the other PSDs included in this comparative analysis.

The psd1, psd2, and psd3 gene products share overlapping functions essential for the normal growth of S. pombe cells.To determine the phenotypes resulting from deletion of the psd1-3 genes, we used PCR to construct the psd1::kanMX6, psd2::hphMX6, and psd3::ura4 gene knockout cassettes, in which the psd1 to psd3 protein-coding sequences are each replaced by genes encoding different selectable markers (see Materials and Methods). The psd1-3 knockout cassettes were transformed separately into wild-type S. pombe strain SP870. Transformants were plated onto media selective for the growth of the respective knockout mutants, which were subsequently identified by colony PCR (see Materials and Methods). The resulting psd1Δ, psd2Δ, and psd3Δ deletion mutants were tested for growth on complex rich medium (YEAU) and synthetic defined minimal medium (EMM). As shown in Fig. 2A, the psd1Δ, psd2Δ, and psd3Δ strains each grew about as well as the wild-type parental S. pombe strain on both YEAU and EMM. These results demonstrate that no single psd gene is essential for S. pombe cell growth and proliferation.

• Open in new tab
• Download powerpoint

FIG. 2.

Growth of S. pombe PSD mutants on rich and minimal media. (A) Wild-type S. pombe cells and psd1Δ, psd2Δ, and psd3Δ mutant strains were streaked onto a complex rich medium (YEAU) or a synthetic defined minimal medium (EMM) and incubated at 30°C for 3 or 5 days, respectively. (B) S. pombe psd1Δ psd2Δ, psd1Δ psd3Δ, and psd2Δ psd3Δ double mutants and the psd1-3Δ triple mutant were streaked onto YEAU or EMM, as indicated, and incubated at 30°C for 3 or 5 days, respectively.

To determine whether the psd genes encode proteins that share overlapping or redundant functions necessary for the growth of S. pombe cells, we carried out a series of genetic crosses to generate psd1Δ psd2Δ, psd1Δ psd3Δ, and psd2Δ psd3Δ double mutants, as well as a psd1Δ psd2Δ psd3Δ triple mutant (the psd1-3Δ mutant). Each of the psdΔ mutants was tested for growth on YEAU and EMM. As shown in Fig. 2B, the psd1Δ psd2Δ, psd1Δ psd3Δ, and psd2Δ psd3Δ double mutants grew similarly to wild-type S. pombe cells on both YEAU and EMM agar media. In contrast, the psd1-3Δ triple mutant grew slowly on YEAU medium and not at all on EMM.

Although on YEAU and EMM agar media, as well as in YEAU liquid medium, the psdΔ single and double mutants grew similarly to wild-type S. pombe cells, we noted that in liquid EMM, the psd2Δ mutant as well as the psd1Δ psd2Δ and psd2Δ psd3Δ mutants grew markedly more slowly than wild-type S. pombe cells (Table 2). Consistent with the results of growth assays on YEAU and EMM agar media, the psd1-3Δ triple mutant was found to grow very slowly relative to wild-type S. pombe cells in liquid EMM supplemented with ethanolamine, and its growth was arrested within about 1 generation after transfer from EMM supplemented with ethanolamine to EMM lacking ethanolamine (Table 2). Taken together, the results of these assays analyzing the growth of PSD mutants in rich and defined minimal media indicate that the psd1 to psd3 gene products share substantially overlapping but not completely redundant functions essential for S. pombe cell growth and proliferation.

The S. pombe psd1-3Δ mutant is partially rescued by supplementation of growth media with ethanolamine but not with choline.The S. pombe genome contains genes encoding proteins that share structural homology with enzymes comprising the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway (Y. Matsuo and S. Marcus, unpublished data), which in budding yeast and mammalian cells are used to convert ethanolamine to PE and choline to phosphatidylcholine (PC), respectively (15, 34, 36). Previous studies of S. pombe PS synthase (pps1Δ) and phospholipid methyltransferase (cho1Δ) mutants suggested that both branches of the Kennedy pathway are functional in S. pombe (13, 17). We therefore performed experiments to determine whether supplementation of media with ethanolamine or choline can rescue the growth defect of the psd1-3Δ mutant. We found that supplementation of media with 1 mM ethanolamine strongly suppressed the growth-defective phenotype of the psd1-3Δ mutant on YEAU medium (Fig. 3A) but only partially rescued the growth defect of the mutant on EMM (Fig. 3B). In contrast, choline supplementation, even at a concentration as high as 100 mM, did not rescue the growth-defective phenotype of the psd1-3Δ mutant to any extent and, in fact, was inhibitory to the growth of this mutant on YEAU medium (Fig. 3C). Taken together, these results indicate that PE is essential for S. pombe cell growth and, further, that the CDP-ethanolamine branch of the Kennedy pathway can only partially compensate for the essential functions conferred by S. pombe PSDs in synthetic defined minimal medium.

• Open in new tab
• Download powerpoint

FIG. 3.

The growth defect of psd1-3Δ cells is rescued by ethanolamine but not by choline. (A) Wild-type and psd1-3Δ cells were grown in YEAU containing 1 mM ethanolamine (+EA) to mid-log phase, harvested by centrifugation, and resuspended in YEAU or EMM at 10⁷ cells/ml. The cell suspensions were serially diluted (1:10); 4 μl of each dilution was spotted onto YEAU medium without ethanolamine (−EA) or with 1 mM ethanolamine as indicated; and the plates were incubated for 3 days at 30°C. (B) Serially diluted suspensions of wild-type and psd1-3Δ cells, prepared as described for panel A, were spotted onto EMM without ethanolamine or containing 1 mM ethanolamine as indicated, and the plates were incubated for 5 days at 30°C. (C) Serially diluted suspensions of wild-type and psd1-3Δ cells, prepared as described for panel A, were spotted onto YEAU containing 1 mM or 100 mM choline as indicated and were incubated for 3 days at 30°C.

The psd1-3 gene products are responsible for the majority of PE synthesis in S. pombe.We next carried out radiolabeling experiments to determine whether S. pombe cells harboring null mutations in all three psd genes have lower levels of PE than wild-type S. pombe cells. For this purpose, wild-type and psd1-3Δ S. pombe strains were labeled to uniformity with ³²P_i in nonsupplemented YEAU (wild-type cells) or YEAU supplemented with 1 mM ethanolamine (wild-type and psd1-3Δ cells). Lipids were extracted from the radiolabeled cells, and individual phospholipids were resolved by high-performance thin-layer chromatography and quantified by autoradiography and densitometry (see Materials and Methods). We found that psd1-3Δ cells cultured in ethanolamine-supplemented YEAU medium had substantially lower relative levels of PE than wild-type S. pombe cells, which displayed similar phospholipid profiles in nonsupplemented YEAU and YEAU containing ethanolamine (Fig. 4A). In addition, we noted that psd1-3Δ cells cultured under these conditions had markedly higher relative levels of PS than wild-type S. pombe cells (Fig. 4A).

• Open in new tab
• Download powerpoint

FIG. 4.

Comparison of relative phospholipid compositions of wild-type and psd1-3Δ S. pombe strains. (A) Wild-type and psd1-3Δ cells were cultured at 30°C to mid-log phase in nonsupplemented (− EA) YEAU containing 20 μCi of ³²P_i/ml (wild type) or in the same medium supplemented with 1 mM ethanolamine (+ EA) (wild-type and psd1-3Δ cells). Lipids were extracted from labeled cells, resolved by high-performance thin-layer chromatography, and quantitated by autoradiography and subsequent densitometric analysis of scanned films (see Materials and Methods). The bar graphs show the relative quantities of the four major phospholipids detected in each strain. (B) Wild-type and psd1-3Δ cells were radiolabeled in EMM + EA containing 20 μCi of ³²P_i/ml as described in Materials and Methods. Lipids were extracted from labeled cells and analyzed as described for panel A. (C) Wild-type and psd1-3Δ cells were radiolabeled in EMM − EA containing 20 μCi of ³²P_i/ml as described in Materials and Methods. Lipids were extracted from labeled cells and analyzed as described for panel A. Bar graphs show average results ± standard errors of the means for two independent determinations.

We next carried out labeling experiments in EMM, which requires supplementation with ethanolamine to support the growth of psd1-3Δ cells. We first analyzed the phospholipid compositions of wild-type and psd1-3Δ cultures labeled with ³²P_i in EMM containing 1 mM ethanolamine. As with ethanolamine-supplemented YEAU medium, we found that psd1-3Δ cells cultured in EMM containing 1 mM ethanolamine had significantly lower PE levels than wild-type cells as well as higher levels of PS (Fig. 4B). Levels of phosphatidylinositol (PI) relative to PC were also higher in both wild-type and psd1-3Δ strains cultured under these conditions.

Next, we carried out labeling experiments to determine the effect of ethanolamine starvation on phospholipid levels in psd1-3Δ cells. For this purpose, wild-type and psd1-3Δ cells were labeled with ³²P_i in EMM containing 1 mM ethanolamine, washed with EMM, resuspended in EMM, and labeled for 8 additional h prior to the harvesting and extraction of lipids from the labeled cells. As shown in Fig. 4C, ethanolamine starvation led to a further reduction of PE levels in psd1-3Δ cells relative to those in wild-type cells. Taken together, the results of these in vivo labeling experiments indicate that PSD activity is responsible for the majority of PE synthesis in S. pombe and that, consistent with the results of the growth assays described above, the activity of the Kennedy pathway cannot fully compensate for the loss of PSD activity with respect to the maintenance of levels of PE relative to those of PS, PI, and PC.

The psd1-3Δ mutant exhibits severe morphology- and cytokinesis-defective phenotypes under ethanolamine-limited growth conditions.Microscopic analyses were carried out to determine whether S. pombe PSD mutants exhibit defects in cell morphology. When cultured in nonsupplemented YEAU medium, psd1Δ, psd2Δ, and psd3Δ single mutants, as well as psd1Δ psd2Δ, psd1Δ psd3Δ, and psd2Δ psd3Δ double mutants, appeared similar to wild-type S. pombe cells in morphology (Fig. 5). These results demonstrate that the loss of any two psd genes does not result in obvious morphological abnormalities in S. pombe cells cultured in rich medium. To determine whether S. pombe cells lacking all three psd genes exhibit morphological abnormalities, the psd1-3Δ mutant was cultured in YEAU medium containing 1 mM ethanolamine to mid-log phase, washed, resuspended in nonsupplemented YEAU or YEAU containing 1 mM ethanolamine, and cultured for several generations prior to microscopic analysis. In YEAU medium supplemented with ethanolamine, psd1-3Δ cells exhibited a rod-shaped morphology (Fig. 6B) indistinguishable from that of wild-type S. pombe cells (Fig. 6A). In stark contrast, in psd1-3Δ cultures grown for 6 to 7 generations in nonsupplemented YEAU medium, we observed high frequencies of morphologically aberrant cells, including elongated, enlarged, bottle-shaped, and bulbous cells (Fig. 6C). Moreover, we noted that about 20% of septated psd1-3Δ cells contained more than one septum (Fig. 6C), including cells with double septa (two closely positioned septa), and that many septated cells exhibited mispositioned septa (not located near the middle of the cell) and/or misoriented septa (not perpendicular to the apparent growth axis of the cell). After culturing for 9 to 10 generations in YEAU medium lacking ethanolamine, we found that more than 70% of cells in psd1-3Δ cultures exhibited bottle-shaped, ovoid, or bulbous morphologies (Fig. 6D). In addition, more than 40% of cells in these cultures were septated, in contrast to about 10% of cells in the corresponding wild-type S. pombe cultures (Fig. 6E). Abnormally septated cells were also common in these cultures, and a low incidence of multiseptated cells was also detected (about 2% of septated cells) (Fig. 6D, inset).

• Open in new tab
• Download powerpoint

FIG. 5.

Microscopic analysis of psdΔ single and double mutants. Wild-type S. pombe cells, psd1Δ, psd2Δ, and psd3Δ single mutants, and psd1Δ psd2Δ, psd1Δ psd3Δ, and psd2Δ psd3Δ double mutants were cultured overnight in YEAU medium to mid-log phase and analyzed by photomicroscopy.

• Open in new tab
• Download powerpoint

FIG. 6.

psd1-3Δ cells exhibit severe morphology- and cytokinesis-defective phenotypes. (A) Photomicrograph of wild-type S. pombe cells cultured to mid-log phase in YEAU medium. (B) Photomicrograph of psd1-3Δ cells cultured to mid-log phase in YEAU containing 1 mM ethanolamine (+ EA). (C and D) psd1-3Δ cells were cultured to mid-log phase in YEAU + EA, washed three times with YEAU, resuspended in nonsupplemented YEAU (− EA), cultured at 30°C, and subjected to photomicroscopy after approximately 7 (C) or 10 (D) generations of growth. (C) Arrows mark cells with highly aberrant septation. (Inset) Cell containing a double septum. (D) (Inset) Cell containing two septa. (E) Wild-type cells cultured in YEAU and psd1-3Δ cells cultured in YEAU − EA or YEAU + EA for approximately 10 generations were analyzed by photomicroscopy to determine the frequency of septated cells. The bar graph shows average results ± standard errors of the means for two independent experiments.

We next sought to determine whether multiseptated cells in psd1-3Δ cultures grown in nonsupplemented YEAU medium would be capable of completing cytokinesis when transferred to medium supplemented with ethanolamine. For this purpose, psd1-3Δ cells were cultured for about 8 generations in YEAU medium, spotted onto a thin layer of YEAU agar containing 1 mM ethanolamine, and monitored by time lapse DIC microscopy. We found that in the majority of samples observed, multiseptated cells were able to complete cytokinesis to produce daughter cells that were themselves able to grow and divide (Fig. 7). These results demonstrate that the multiseptate phenotype of psd1-3Δ cells cultured in ethanolamine-limited medium is not a terminal phenotype. We noted that in some cases the time required for completion of separation of compartments within a multiseptated cell varied greatly, as in the example documented in Fig. 7. In addition, we noted during the course of this experiment that daughter cells produced from the cytokinesis of multiseptated cells were usually at least somewhat abnormal in shape and that progeny produced from these first-generation daughters of multiseptated cells were likewise often abnormal in shape (Fig. 7). These observations indicate that the reversion of psd1-3Δ cells to normal morphological phenotypes after growth under ethanolamine-limited conditions requires several generations of growth after subculturing to medium supplemented with ethanolamine.

• Open in new tab
• Download powerpoint

FIG. 7.

Completion of cytokinesis by a multiseptated psd1-3Δ cell upon transfer to ethanolamine-containing medium. A culture of S. pombe psd1-3Δ cells was grown for about 8 generations in YEAU; then it was spotted onto YEAU agar containing 1 mM ethanolamine and monitored by time lapse DIC microscopy. Panels show a series of time lapse images of a multiseptated cell (0 min) that completes a series of cytokinesis events (30 min to 340 min) to produce viable daughter cells that likewise grow and divide. The arrow points to a septum in the original parent cell that separates well after several other daughter progeny have grown and divided to produce new daughter cells.

Additional analyses revealed that psd1-3Δ cultures grown in EMM containing 1 mM ethanolamine (Fig. 8B) were generally similar in morphology to wild-type S. pombe cells (Fig. 8A), although approximately 10 to 15% of cells in psd1-3Δ cultures exhibited various degrees of morphological aberrancy, including ovoid, bottle-shaped, and, occasionally, bent cells. In contrast, the majority of cells in psd1-3Δ cultures incubated overnight in EMM lacking ethanolamine exhibited ovoid or bottle-shaped morphologies and were necrotic, lysed, and/or shrunken in appearance, a phenotype indicative of a defect in cell integrity (Fig. 8C). Taken together, the results of these microscopic analyses demonstrate that PE is required for proper regulation of cell morphology and cytokinesis in S. pombe and, moreover, that in rich medium, any one of the S. pombe psd gene products is sufficient for normal cytokinesis and maintenance of a normal cell shape.

• Open in new tab
• Download powerpoint

FIG. 8.

Microscopic analysis of psd1-3Δ cells cultured in synthetic minimal medium. (A and B) Wild-type and psd1-3Δ cells were cultured at 30°C overnight in EMM alone (A) and EMM containing 1 mM ethanolamine (+ EA) (B), respectively, to mid-log phase prior to DIC photomicroscopy. (C) psd1-3Δ cells were cultured in EMM + EA to mid-log phase, washed with EMM, resuspended in unsupplemented (− EA) EMM, and incubated at 30°C overnight prior to DIC photomicroscopy.

Analysis of cell wall and actin cytoskeletal organization in psd1-3Δ cells.Given the severe defects in septation and morphology exhibited by psd1-3Δ cells under ethanolamine-limited growth conditions, we carried out experiments to investigate whether the mutant exhibits defects in cell wall and/or actin cytoskeletal organization. For analysis of cell wall organization, wild-type and psd1-3ΔS. pombe cells cultured to mid-log phase in YEAU medium were stained with the fluorescent cell wall binding dye calcofluor and observed by fluorescence microscopy. Not unexpectedly, we found that psd1-3Δ cells exhibited a variety of abnormalities in septum organization and/or structure, including double-walled (Fig. 9B) and deformed (Fig. 9C and D) septa. Despite these defects, the vast majority of calcofluor white-stained psd1-3Δ cells exhibited an intensity of both cell wall and septum wall fluorescence that was similar to that of wild-type S. pombe cells (compare the wild-type cells in Fig. 9A with the psd1-3Δ cells in Fig. 9B to D). We also noted that cultures of psd1-3Δ cells, unlike S. pombe PS synthase (pps1Δ) mutants (17), did not contain significant frequencies of cells with abnormal cell wall aggregates. Moreover, whereas pps1Δ cells are hypersensitive to the growth-inhibitory effects of calcofluor white, we found that psd1-3Δ cells were no more sensitive to calcofluor white than wild-type S. pombe cells (data not shown).

• Open in new tab
• Download powerpoint

FIG. 9.

Analysis of cell wall organization in psd1-3Δ cells. Wild-type (A) and psd1-3Δ (B to D) cells cultured in YEAU at 30°C for 8 to 9 generations to mid-log phase were stained with calcofluor white (see Materials and Methods) and visualized by fluorescence microscopy. Arrows in panels B to D mark psd1-3Δ cells displaying malformed and/or multiple septa.

For analysis of actin cytoskeletal organization, wild-type and psd1-3Δ cells cultured to mid-log phase in YEAU medium were stained with the florescent F-actin binding compound rhodamine-phalloidin and observed by fluorescence microscopy. In wild-type S. pombe cells, F-actin is concentrated as dots or patches at the growing cell tips during interphase, while in dividing cells it is concentrated at the cell division site in the form of a contractile actin ring and, later in M phase, as actin patches on either side of the forming septum (Fig. 10A). Interphase psd1-3Δ cells exhibited actin dots at one or both cell tips that were generally similar in appearance to those detected in wild-type S. pombe cells (Fig. 10B and C). In contrast, we noted that in multiseptated psd1-3Δ cells, contractile ring and septal actin structures frequently formed asynchronously in the two daughter cells produced by an initial septation event (Fig. 10C and D). In addition, significantly misoriented actin rings were detected at a high frequency in multiseptated psd1-3Δ cells (Fig. 10D and E). Interestingly, despite the relatively high frequency of septation-defective phenotypes detected in psd1-3Δ cells, nuclear staining experiments indicated only a very low frequency of cells (<0.2%) exhibiting discernible defects in nuclear segregation (data not shown).

• Open in new tab
• Download powerpoint

FIG. 10.

Analysis of actin cytoskeletal organization in psd1-3Δ cells. Wild-type (A) and psd1-3Δ (B to E) cells cultured in YEAU at 30°C for 8 to 9 generations to mid-log phase were stained with rhodamine-phalloidin and visualized by fluorescence microscopy. (B to E) Arrows mark bottle-shaped interphase cells (B), a cell with two nonsynchronous septation events (C), a cell displaying improperly synchronized actin ring structures (D), and a cell with a severely misoriented actin ring (E).