Illustration of the mating steps regulated by the distal and proximal actions of M-factor mating pheromone in liquid culture.

<p>When haploid cells of the opposite mating types are mixed in nitrogen-free liquid medium, cell agglutination occurs after arrest of cell division. Mating type-dependent cell-to-cell adhesion is induced in cell aggregates. From the contact site, polarized tip growth occurs leading to cell fusion. Pheromone is required for steps of cell agglutination (distal action), polarized growth and cell fusion (proximal action).</p

Distal and Proximal Actions of Peptide Pheromone M-Factor Control Different Conjugation Steps in Fission Yeast

<div><p>Mating pheromone signaling is essential for conjugation between haploid cells of P-type (P-cells) and haploid cells of M-type (M-cells) in <i>Schizosaccharomyces pombe</i>. A peptide pheromone, M-factor, produced by M-cells is recognized by the receptor of P-cells. An M-factor-less mutant, in which the M-factor-encoding genes are deleted, is completely sterile. In liquid culture, sexual agglutination was not observed in the mutant, but it could be recovered by adding exogenous synthetic M-factor, which stimulated expression of the P-type-specific cell adhesion protein, Map4. Exogenous M-factor, however, failed to recover the cell fusion defect in the M-factor-less mutant. When M-factor-less cells were added to a mixture of wild-type P- and M-cells, marked cell aggregates were formed. Notably, M-factor-less mutant cells were also incorporated in these aggregates. In this mixed culture, P-cells conjugated preferentially with M-cells secreting M-factor, and rarely with M-factor-less M-cells. The kinetics of mating parameters in liquid culture revealed that polarized growth commenced from the contact region of opposite mating-type cells. Taken together, these findings indicate that M-factor at a low concentration induces adhesin expression, leading to initial cell-cell adhesion in a type of “distal pheromone action”, but M-factor that is secreted directly in the proximity of the adhered P-cells may be necessary for cell fusion in a type of “proximal pheromone action”.</p> </div

Deletion analysis of upstream <i>cis</i>-acting elements of <i>map4</i>⁺.

<p>(A) Putative promoter sequence of the <i>map4</i>^<i>+</i> gene. Two possible TR-box elements (blue and green) and a TATA-like box (red) are italicized and underlined. The transcription start site was identified by sequencing of several cDNA clones (Nakamura <i>et al</i>, unpublished). (B) β-galactosidase assay of the promoter intensity of a <i>map4</i> promoter-<i>lacZ</i> fusion gene with different deletions of the promoter sequence of the <i>map4</i> gene. A heterothallic <i>h</i>⁺ strain (FS85) was transformed with the multicopy plasmid pTA(map4^PRO-lacZ) or the deletion constructs. Cells of the transformants were precultured in SSL+N without leucine and then transferred to SSL−N medium containing 200 nM of synthetic M-factor (filled bar) or SSL−N alone (open bar). After 6 hr, β-gal activity was assayed.</p

Expression of mating-type-specific adhesin proteins, Map4 and Mam3 (A, left).

<p>A heterothallic <i>h</i>⁺ strain (FS103) carrying a <i>map4</i>-<i>GFP</i> fusion gene integrated at the authentic chromosomal locus was precultured in SSL+N for 18 hr, then shifted to SSL−N with or without 200 nM of M-factor, and cultured for 4 hr. (A, right) A heterothallic <i>h</i>⁻<i>mam2Δ</i> strain (SS1463) carrying a <i>mam3-mCherry</i> fusion gene integrated at the original chromosomal locus was incubated for 4 hr in SSL−N (−N). Scale bar, 50 µm. (B) Quantification of Map4-GFP and Mam3-mCherry fluorescence. The intensity was quantified by image analysis using Image-J software. At least 8 different areas were analyzed. Means of arbitrary units (A.U.) with standard deviations are presented. (C) Induced agglutination by overexpression of Map4. Strain SS1290 ectopically overexpressing <i>map4</i>⁺ driven by the <i>nmt1</i> promoter was precultured in MM+N without thiamine for 12 hr and then cultured in MM−N for 4 hr (Map4OP). The same strain was transformed by the pSLF173 empty vector [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069491#B43" target="_blank">43</a>] as a control.</p

Recombinant frequency assay of wild-type and pheromone-less mutant strains.

<p>Heterothallic haploid strains carrying a <i>natR</i>, <i>kanR</i> or <i>hygR</i> drug-resistant marker were cultured in SSL+N overnight and then mixed in the nitrogen-free conjugation medium (SSL−N) with or without M-factor (200 nM), and cultured at 28^oC with shaking. Two different mixed cultures ran in parallel. Cross-I, P-type strain (FS404; P^WT, <i>natR</i>), M-type strain (FS357; M^WT, <i>kanR</i>) and M-type pheromone-less mutant (FS419; M^phe-less, <i>hygR</i>). Cross-II, P-type strain (FS404; P^WT, <i>natR</i>), M-type strain-1 (FS357; M^WT1, <i>kanR</i>) and M-type strain-2 (FS423; M^WT2, <i>hygR</i>). The same number of cells of the two M-type strains and twice the number of cells of the P-type strain were mixed. After incubation for 2 days, the cultures were diluted and then spread onto YEA plates containing 100 µg/ml of the appropriate drug: YEA+Nat, YEA+Nat+G418, and YEA+Nat+Hyg. After 3 days of incubation, the plates were photographed. The colony number on the plates was counted.</p

Agglutination ability of heterothallic strains harboring deletion of the pheromone receptor gene.

<p>The strains used were L972 (<i>h</i>^– wild type), L975 (<i>h</i>⁺ wild type), FS65 (<i>h</i>^<i>–</i><i>mam2Δ</i>), and FS66 (<i>h</i>^<i>+</i><i>map3Δ</i>). Cells were incubated with gentle shaking in SSL−N for 4 hr. AI was determined for triplicate samples. Means with standard deviations (vertical bars) are presented. Visible agglutination was seen in samples with an AI of more than 1.1.</p

P-cells preferentially mate with wild-type M-cells.

<p>(A) Experimental design. Wild-type P-cells (P_WT) were mixed with an equal number of wild-type M-cells (M_WT) and M-factor-less M-cells (M_mut), marked by the <i>ade6-M210</i> auxotrophic marker, in nitrogen-free medium. If the P-cell chose an <i>ade6</i>^<i>+</i> M-cell, the descendant spore clones would have no <i>ade6-M210</i> allele. If the P-cell chose an <i>ade6-M210</i> M-cell, the allele would be transmitted to half of the descendant spore clones. Thus, the preference of P-cells for type of mating partner could be determined by the frequency of <i>ade6-M210</i> segregants. (B) Experimental results. The following strains were used: L972 (<i>h</i>^– prototrophic), L975 (<i>h</i>⁺ prototrophic), FS120 (<i>h</i>^<i>–</i><i>mfm1,2,3Δ ade6-M210</i>), and FS121 (<i>h</i>^–<i>mfm1,2,3</i>⁺<i>ade6-M210</i>). The mixed cells were allowed to mate, and resulting hybrid diploids were sporulated. Spores were isolated by micromanipulation and grown on nutrient medium with adenine sulfate limitation.</p

Kinetics of sexual agglutination and polar cell growth in liquid culture.

<p>(A) Mating kinetics of a homothallic wild-type strain (L968) cultured in SSL−N liquid medium. At hourly intervals, the intensity of agglutination (AI) was determined. Portions of the culture were subjected to brief sonication and microphotographs were taken. The long (L) and short (S) axes of cells were measured to calculate the L/S ratio for triplicate samples (100 cells each). The frequencies of prezygotes and cells with pointy projections were determined. (B) Microphotographs showing typical cell morphology. Arrows, prezygotes with a pointy mating projection (shown by asterisks). Scale bar, 10 µm. (C, D) Mating kinetics of M-factor-less mutant cells incubated with (C) or without (D) 200 nM M-factor. The experimental procedures are as described in (A). (E) Left, polar cell growth and prezygote formation in M-factor-induced aggregated cells and floating free cells. An M-factor-less strain (FS55) and an M-factor-producing <i>fus1Δ</i> strain (FS123) were incubated with 200 nM M-factor. After 4 hr, aggregates and free cells were isolated. After brief sonication, the L/S ratio was determined (n=1,000 for both strains). Right, frequency of prezygotes formed with <i>fus1Δ</i> (Aggregates <i>vs</i> Free: each <i>p</i>-value was shown, <i>t</i>-test)..</p

M-factor-less cells are incorporated in cell aggregates during co-cultivation with wild-type M-cells.

<p>Wild-type P-cells (FS127), wild-type M-cells (L972) and M-factor-less M-cells (FS120) were mixed in SSL−N in the cell number ratio of 2:1:1. FS127 carries a uracil-auxotrophic marker (<i>ura4-D18</i>), and FS120 harbors an adenine-auxotrophic marker (<i>ade6-M210</i>). After 4 hr of culture with shaking, cell aggregates were obtained after the culture was allowed to stand for a few minutes. Cells were sampled from the precipitated aggregates and the floating fractions separately. The fractions were briefly sonicated to disperse cell clusters, and the cells were spread on SD without leucine but with one-quarter strength adenine-sulfate. P-cells could not form colonies because of uracil depletion. M-cells derived from the M-factor-less strain formed red colonies due to <i>ade6-M210</i>, and those from the wild-type strain formed white colonies.</p

Induction of sexual agglutination by mating pheromone.

<p>(A) Illustration of mating pheromone signaling in <i>S. pombe</i>. The pheromone signal is transmitted through trimeric G-protein α-subunit and the mitogen-activated protein kinase cascade, comprising Byr2, Byr1 and Spk1. The signal transmission pathway is common to both M- and P-cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069491#B22" target="_blank">22</a>]. (B) The defect in sexual agglutination of the M-factor-less mutant was recovered by exogenously added synthetic M-factor. The homothallic strain FS55 was treated with synthetic M-factor for 4 hr in SSL−N. The agglutination index (AI) was determined for triplicate samples. Means with standard deviations are presented. A portion of each culture was incubated for 24 hr, and the frequency of zygotes was counted. At least 1,000 cells were examined for each sample. (C) Exogenous P-factor addition enhanced constitutive agglutination in a homothallic P-factor-less strain (FS251). At least 500 cells were observed for each sample.</p