Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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 32Pi/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 32Pi/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 32Pi/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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
Tables
- TABLE 1.
S. pombe strains used in this study
Strain Relevant genotype Source SP870 h90 ade6-210 leu1-32 ura4-D18 D. Beach YMSM108 h90 ade6-210 leu1-32 ura4-D18 psd1::kanMX6 This study YMSM109 h90 ade6-210 leu1-32 ura4-D18 psd2::hphMX6 This study YMSM110 h90 ade6-210 leu1-32 ura4-D18 psd3::ura4 This study YMSM111 h90 ade6-210 leu1-32 ura4-D18 psd1::kanMX6 psd2::hphMX6 This study YMSM112 h90 ade6-210 leu1-32 ura4-D18 psd1::kanMX6 psd3::ura4 This study YMSM113 h90 ade6-210 leu1-32 ura4-D18 psd2::hphMX6 psd3::ura4 This study YMSM114 h90 ade6-210 leu1-32 ura4-D18 psd1::kanMX6 psd2::hphMX6 psd3::ura4 This study - TABLE 2.
Growth of psdΔ mutants relative to that of wild-type S. pombe cells in liquid mediaa
Strain Relative growthb in: YEAU YEAU + EA EMM EMM + EA Wild type +++ +++ +++ +++ psd1 Δ (YMSM108) +++ +++ +++ +++ psd2 Δ (YMSM109) +++ +++ ++ +++ psd3 Δ (YMSM110) +++ +++ +++ +++ psd1 Δ psd2 Δ (YMSM111) +++ +++ + +++ psd1 Δ psd3 Δ (YMSM112) +++ +++ +++ +++ psd2 Δ psd3 Δ (YMSM113) +++ +++ + +++ psd1 Δ psd2 Δ psd3 Δ (YMSM114) + +++ − + ↵ a All strains except YMSM114 were cultured at 30°C with shaking in the indicated liquid medium to mid-log phase, subcultured by dilution into prewarmed medium to a density of 1 × 105 to 4 × 105 cells/ml (slower-growing strains were subcultured to higher densities), and incubated for 24 to 28 h prior to determination of cell densities by hemacytometer counting (final densities ranged from 1.4 × 106 to 6 × 106 cells/ml). Generation times were estimated by calculating the number of cell doublings occurring during 24 to 28 h of growth. YMSM114 cells were precultured to mid-log phase in medium supplemented with 1 mM ethanolamine, washed with non-ethanolamine-supplemented medium, resuspended at about 2 × 105 cells/ml in prewarmed non-ethanolamine-supplemented medium, and incubated for 24 h prior to determination of cell densities by hemacytometer counting. Wild-type cells were cultured under the same conditions for the determination of relative growth rates.
↵ b +++, generation time similar to that of wild-type S. pombe cells; ++, generation time approximately twice that of wild-type cells; +, generation time three times or more that of wild-type cells; −, no growth. EA, ethanolamine.
