Construction of modified α strains

To test the possibility that haploid cell identity in C. neoformans is controlled by pheromones and pheromone receptors, a strain where the pheromone and pheromone receptor genes from one mating type were replaced with those from the opposite mating type was constructed. Specifically, we created an altered α strain by functionally replacing the STE3α receptor gene and the three MFα pheromone genes of an α cell with the STE3a receptor gene and three copies of the MFa1 gene from an a cell (Figure 1).

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Figure 1. α^a cells are α cells that express a pheromone and the pheromone receptor from a cells.

A schematic of pheromones and pheromone receptors produced by a cells (left), α cells (middle), and α^a cells (right) is represented. a cells (pink) produce the Ste3a pheromone receptor (pink half circle/stick structure) and MFa pheromones (pink squares). α cells (blue) produce the Ste3α receptor (blue Y-shaped structure) and MFα pheromones (blue circles). α^a cells produce only the Ste3a pheromone receptor and the MFa1 pheromone.

https://doi.org/10.1371/journal.pgen.1000860.g001

To construct the altered α strain, α cells in which all three copies of the MFα gene had been deleted (α ^mfαΔ), were transformed with a construct to replace the endogenous copy of STE3α with STE3a (Figure 2A) [13]. This replacement resulted in a complete deletion of the STE3α open reading frame and the precise insertion of STE3a under the control of the endogenous STE3α promoter. This strain, designated α^{mfαΔ, STE3a} was confirmed by PCR (data not shown) and Southern analysis (Figure 2A) to contain a single, targeted integration of the STE3a gene at the STE3α locus. This α^{mfαΔ, STE3a} strain was then transformed with a construct expressing MFa1 (under the control of the MFα1 promoter) designed to integrate randomly into the genome (Figure 2B). Multiple transformants of the resulting strain, α^a, were assessed by Southern blot analysis, and several strains containing integrated copies of the MFa construct were carried forward in further analyses. All of the transformants confirmed to contain the MFa construct exhibited identical behaviors in subsequent phenotype analyses. A representative α^a transformant containing three copies of the MFa1 gene is shown in Figure 2B.

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Figure 2. Southern and northern analyses of wild-type and modified α strains.

(A) Schematic of the STE3a/NAT integration event to generate the α^{mfαΔ, STE3a} strain (left). Southern blot analysis confirms a single integration of the STE3a-NAT construct into the STE3α locus (Lane 1: ∼3 kb wild type band. Lane 2: ∼5 kb insertion band, right). (B) Schematic of the MFa1/NEO construct to generate the α^a strain (left). Southern blot analysis reveals that 3 copies of the MFa1 gene integrated into the genome. Lane 1: three wild type bands present in a cells. Lane 2: no MFa signal in α cells. Lane 3: three randomly integrated copies of MFa in α^a cells (right). (C) Northern blot analysis of pheromone receptors (STE3a and STE3α) and pheromones (MFa and MFα). Genotypes of the test strains are indicated over the panels and probes are indicated to the left. A probe to GPD1 was used as a loading and hybridization control. Each lane contains RNA from the following strains or crosses: Lane 1 wild type a, Lane 2 wild type α, Lane 3 α^mfαΔ, Lane 4 α ^{mfαΔ, STE3a}, Lane 5 α^a, and Lane 6 a x α.

https://doi.org/10.1371/journal.pgen.1000860.g002

To assess the expression patterns of pheromones and their receptors in the constructed strains, northern blot analysis was carried out. Haploid strains were grown on V8 medium for 24 hours, and their relative transcript levels were examined (Figure 2C). The STE3a and STE3α transcripts were detected in wild type a and α cells, respectively, and the STE3α transcript was also present in the α^mfαΔ strain. However, as expected, in the α^{mfαΔ, STE3a} strain where the STE3a ORF had replaced the STE3α ORF, only STE3a transcript was observed. In summary, the transcripts detected from the constructed strains were in accordance with the predicted expression patterns for each of the strains tested based on their genotypes.

While pheromone transcript was not detected in either haploid cell type (2C, bottom two panels, lanes 1 and 2), this result was not surprising because pheromone genes are transcribed at low levels in haploid cells grown on minimal medium [13]. Furthermore, pheromone transcript was not observed in strains where these genes had been deleted (α^mfαΔ and α^{mfαΔ, STE3a}) (lanes 3 and 4). However, when the MFa1 gene was introduced into the α^{mfαΔ, STE3a} strain (resulting in the α^a strain), high levels of MFa transcript were detected, and this high level of expression occurred in all of the MFa transformants (lane 5 and data not shown). Because MFa1, in this case, is under the control of the MFα1 promoter (defined here as 1 kb of sequence upstream of the translational start site), a likely explanation for our findings is that normal repression of MFa1 in haploid cells is disrupted. That is, the elements required for wild type levels of expression were likely not included in our construct, resulting in higher levels of MFa1 gene expression. Because pheromones expressed from a non-native promoter do not interfere with development and the mating response, the α^a strain was used in our subsequent analyses [13],[14].

Altered α strains mate with wild-type α cells

To evaluate how the series of modified α strains interacted with mating partners under sexual development conditions, crosses using various test strains were carried out and assessed microscopically for the presence of fusants after 18 hours on V8 medium. The cells used in each cross were stained with either rhodamine (Alexa Fluor 594) or fluorescein (Alexa Fluor 488) prior to mixing with partner cells so that the original mating types could be discerned after cell fusion.

In crosses between wild type a and α strains, fusants were identified as “dumbbell” shaped cells, consisting of one a cell (red) connected to one α cell (green) via a conjugation tube (Figure 3A, panel 1). In contrast, no fusants could be identified in crosses between wild type a cells (red) and any modified α cells (green) (panels 2–4). Specifically, in crosses between a cells and α^mfαΔ cells, the α^mfαΔ cells responded to the presence of a cells by forming conjugation tubes; however, in the absence of α pheromone production, the frequency of fusion with a cells is predicted to be very low, and consistent with this expectation, we did not observe any fusants. Accordingly, in strains no longer harboring the STE3α receptor (α^mfαΔ,STE3a and α^a), the cells appear to neither form conjugation tubes (in the presence of a cells) nor fuse with a cells (panels 3 & 4). These results indicate that modified α strains, in which the STE3α receptor gene has been deleted, do not respond to or mate with a cells.

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Figure 3. α^a strains fuse with wild-type α cells.

(A) Sexual development assays were carried out on V8 media using differentially labeled a and α strains (red or green) to detect the formation of germ tubes (by α cells) and fusants (dumbbell shaped structures). A representative view from the indicated cross is displayed in each panel (1-8). In panels 1–4, a cells are labeled red, and α cells are labeled green. Panel 1: wild type a x α fusant. Panel 2: germ tube from an α^mfαΔ cell; no fusants. Panels 3 and 4: no germ tubes; no fusants. In panel 5, wild type α cells are labeled red or green. In panels 6–8, wild type α cells are labeled red and modified α cells are labeled green. Panels 5–7: no germ tubes; no fusants. Panel 8: wild type α x α^a fusant. (B) α^a cells do not form germ tubes. Wild type α cells were labeled red, and α^a cells were labeled green. Wild type α cells (red) produce germ tubes in response to a cells (A, panel 1) and to α^a cells (A, panel 8). Similar to wild-type a cells, α^a cells do not form germ tubes in response to α cells.

https://doi.org/10.1371/journal.pgen.1000860.g003

In parallel experiments, wild type α strains were crossed with the modified α strains. In a control cross, differentially labeled α cells (either red or green) were crossed and evaluated for the presence of fusants. As expected for wild type cells of the same mating type, α cells did not respond to one another or fuse (panel 5). Fusants were also not detected in crosses between wild type α cells (red) and the modified α^mfαΔ and α^mfαΔ,STE3a (green) strains (panels 6 & 7, respectively). It was not until a wild type α cell (red) was crossed with the altered α^a strain (green) that fusants could be recovered at roughly wild type a x α levels (panel 8). Subsequent quantitative fusion assays confirmed that α x α^a fusion levels are comparable to those of wild type a x α crosses (data not shown). This finding indicates that, as expected, pheromones and pheromone receptors are required for wild type fusion between a and α cells. It also reveals that the altered α^a strain fuses only with wild type α cells, indicating that by simply replacing the α pheromones and receptors with those from a cells, cell identify was switched from α to a. Moreover, that change was sufficient to allow the altered α^a cells to fuse with wild type α cells as though they were of opposite mating types.

In addition, the introduction of pheromone and pheromone receptor from a cells into α cells changes not only their ability to fuse but also the initial mating response. That is, α^a strains do not produce germ tubes in response to wild type α cells, instead appearing to behave like wild type a cells (Figure 3B). These findings indicate that pheromones and pheromone receptors specifically control both the ability to sense a mating partner and the subsequent morphological response.

Complete sexual development between α^a and wild-type α cells occurs only in the presence of both Sxi1α and Sxi2a

To assess the ability of the series of modified α strains to undergo sexual development, crosses were carried out and assessed for the production of filaments, basidia, and spores. In crosses between wild type a and α strains, florid filamentation, basidia formation, and sporulation were all visible at the periphery of the cross (Figure 4A, panel 1). However, when the modified α strains (α^mfαΔ, α^mfαΔ,STE3a, and α^a) were crossed with either wild type a or α cells, none of the combinations resulted in wild type sexual development (panels 2–8). The a x α^mfαΔ cross (panel 2) revealed a significant reduction in sexual development, and this result is consistent with previously published data [13]. In addition, aberrant filaments were observed in the α x α^a cross (panel 8). This limited, mutant filamentation was very close in appearance to crosses between strains containing deletions of SXI2a (panel 9). In crosses between wild type α strains and sxi2aΔ mutants, the strains fuse at wild type levels; however, only aberrant filaments are formed, and they do not progress through sexual development [9]. This phenotype occurs because both Sxi1α and Sxi2a must be present to specify the fused state and initiate either diploid-specific or dikaryon-specific developmental programs. Because the products of an α by α^a cross contain only the Sxi1α developmental regulator (and not the Sxi2a regulator found only in a cells), such crosses would not be expected to progress through sexual development and would be expected to exhibit a sxi2aΔ cross phenotype. This is, in fact, the case. Conversely, if the Sxi proteins, pheromone, and pheromone receptors were necessary and sufficient to specify cellular identify (dikaryon vs. haploid), then simply supplying the SXI2a developmental regulator to an α by α^a cross would result in complete sexual development.

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Figure 4. α x α^a crosses proceed through sexual development only in the presence of Sxi2a.

(A) Sexual development assays were carried out on V8 for 72 hours, and the peripheries of crosses are shown under 200× magnification. Panels: 1) a x α, 2) a x α^mfαΔ, 3) a x α^{mfαΔ, STE3a}, 4) a x α^a, 5) α x α, 6) α x α^mfαΔ, 7) α x α^{mfαΔ, STE3a}, 8) α x α^a, 9) a sxi2aΔ x α, 10) α sxi1αΔ + SXI2a x α^a. Complete sexual development is observed only in panels 1 and 10. (B) Dikaryotic filaments are produced in the α sxi1αΔ + SXI2a x α^a cross. Calcofluor stained filaments appear blue, and Sytox Green strained nuclei appear green. Both an a x α cross (left) and the α sxi1αΔ + SXI2a x α^a cross (right) produce dikaryotic filaments (400× magnification). (C) Basidia and spores are produced in the α sxi1αΔ + SXI2a x α^a cross. High resolution microscopy reveals the formation of basidia and spores from an a x α cross (left), and from the α sxi1αΔ + SXI2a x α^a cross (right) (1000× magnification). (D) Addition of the SXI2a gene to α^a/α diploids results in sexual development. Diploids were incubated on V8 for 72 hours, and test spot peripheries are shown under 200× magnification as follows: wild type a/α diploid (panel 1), α^a/α diploid (panel 2), and α^a/α + SXI2a (panel 3).

https://doi.org/10.1371/journal.pgen.1000860.g004

To test the hypothesis that α x α^a crosses were simply in need of SXI2a to continue through sexual development, we carried out crosses in which exogenous SXI2a was provided. Because strains containing both SXI1α and SXI2a are self-filamentous under sexual development conditions, we crossed the α^a strain with an α sxi1αΔ strain carrying an integrated copy of SXI2a under the control of a constitutive promoter (GPD1). This cross resulted in the restoration of complete sexual development (panel 10). In addition, fluorescence and high-resolution microscopy show that dikaryotic filaments basidia, and spore chains were formed in α^a x α + SXI2a crosses (Figures 4B, 4C). Because complete sexual development occurred between cells with nearly identical genomes (α^a x α + SXI2a), no additional information from a cells is required for this process.

This finding was further supported by the results of assays for diploid formation. Previous studies have shown that a/α diploids are self-filamentous on V8 medium, undergoing sexual development [17]. To test the ability of the α^a strain to form diploids, crosses of α^a by wild type a or α cells were carried out. α^a cells fused to form mononucleate cells (presumed diploids) with wild type α cells, but not with wild type a cells, consistent with α^a cells exhibiting a cell behavior. In this assay, α^a cells (ura5, NEO^R) were mixed with either wild type α or a cells (URA5) and incubated on V8. After 24 hours at room temperature, the co-cultures were plated under double selection (Ura⁻ + G418) at 37°C to select for mononucleate diploids [17]. No colonies grew from the a x α^a crosses, indicating that no a/α^a diploids were formed. In contrast, abundant colonies were recovered from the α x α^a crosses, indicating that α^a/α diploids were formed. The resulting α^a/α strains were determined to be mononucleate (data not shown) and were evaluated for the ability to undergo sexual development by incubation on V8 at room temperature (Figure 4D). As in α x α^a crosses, sexual development was not observed (panel 2); however, the addition of the SXI2a gene to the α^a/α strain resulted in full sexual development on V8, similar to that of a wild type a/α diploid (panels 3 and 1, respectively). Taken together, these results demonstrate that cell identity and sexual development are controlled entirely by three kinds of genes in C. neoformans: pheromones, pheromone receptors, and homeodomain proteins.

The α^a strain undergoes α fruiting

Monokaryotic or α fruiting is another form of sexual development that takes place in C. neoformans; however, this process is specific to α cells, which form filaments, basidia, and spores under severe nutrient limitation and desiccation [10],[18],[19]. Although fruiting is known to be an α specific process, the factors responsible for α fruiting are unknown. To assess whether changing haploid cell identity influences α fruiting, fruiting assays were carried out with wild type a, wild type α, and α^a strains. Strains were incubated on filament agar for 14 days at room temperature in the dark and evaluated for the production of filaments and spores. We observed that the α^a strain undergoes α fruiting that is indistinguishable from wild type α cells (Figure 5), indicating that this α-specific process does not require α-specific pheromones or pheromone receptor, and is not influenced by the presence of a-specific pheromones and pheromone receptor.

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Figure 5. α^a cells undergo α fruiting.

Fruiting assays were carried out on filament agar for 14 days at room temperature in the dark. The peripheries of colonies are shown at 100× magnification. Left panel: wild type a cells. Middle panel: wild type α cells. Right panel: α^a cells.

https://doi.org/10.1371/journal.pgen.1000860.g005

Cell identity in fungi

There is a long history of investigating pheromone/pheromone receptor function in fungi, particularly in S. cerevisiae, and myriad experiments have been carried out to understand the roles of two phylogenetically unrelated receptors STE2 (α factor receptor) and STE3 (a factor receptor). Through altered expression experiments it was found that mating type is controlled by the identity of the expressed surface receptor. Experiments where the receptor expression patterns were reversed in a and α cells led to changes in mating behaviors. For example, α cells that express only STE2 (and not STE3), can sense and respond to α pheromone and therefore mate as a cells and vice versa. Thus, in S. cerevisiae, both a and α cells contain the STE2 and STE3 genes (and both pheromone genes), and it is the actions of transcription factors that impart differential expression patterns that determine cell identity [23],[24].

This is in sharp contrast to other fungi, like U. maydis, in which different cell types encode distinct pheromones and pheromone receptors. Experiments in U. maydis where the pheromones and pheromone receptors have been “swapped” between mating types demonstrate that mis-expression of pheromones and pheromone receptors is sufficient to alter cell identity. In this case, the pheromone receptors have distinct binding specificities but have descended from a common ancestor [4]. This is also the case for other basidiomycetes, including S. commune and C. cinerea. Within these fungi, different mating types encode distinct alleles of pheromones and pheromone receptors; pheromone and pheromone receptors do not govern mating, and cell fusion occurs independently of pheromone signaling. Instead, pheromones and pheromone receptors control post-fusion sexual development [25]–[27]. These examples speak to the wide variety of strategies employed by pheromones and pheromone receptors to control sexual development in fungi and emphasize the need to investigate pheromone/pheromone receptor function on a case-by-case basis.

C. neoformans pheromones and pheromone receptors

In C. neoformans, both a and α cells contain mating type-specific pheromone genes (MFa1, 2, and 3 in a cells, and MFα1, 2, and 3 in α cells), but the regulation of these genes differs between mating types. In a cells, MFa pheromone is highly induced on V8 agar (or under conditions of nutrient limitation) in the absence of a mating partner. In addition to induction under nutrient limiting conditions, MFα pheromone expression is also activated by factors secreted by a cells (MFa pheromone) [13], and expression of the MFα genes is dramatically upregulated upon exposure to synthetic MFa pheromone [12],[13].

In the experiments carried out here, expression of the MFa1 gene in α^a cells is driven by the MFα1 promoter. The MFa1 construct was originally engineered in this fashion to avoid any cell type-specific regulation that might be imposed upon an a-specific promoter in an α cell. However, in α^a strains where MFa1 gene expression was driven by the MFa1 promoter, identical phenotypes and expression patterns were observed.

Attempts to test the roles of the pheromone receptors were not as straight forward, and initially, the STE3a cassette was transformed into a wild type α strain to replace the STE3α gene at its native locus. However, after multiple attempts, no transformants were recovered (Ekena, Giles, and Hull, unpublished data). Transformants were recovered only from strains in which the pheromone genes were deleted previously. These findings, while far from conclusive, intimate that cells expressing both STE3a and α pheromone may engage in autocrine signaling that affects cell growth.

A similar observation has been made in S. cerevisiae, in which a strain that expresses both a pheromone and its cognate receptor undergoes G1 cell cycle arrest [24]. In S. cerevisiae this arrest is transient; however, if a similar arrest is occurring in C. neoformans, the cells do not appear to recover. This is in contrast to U. maydis where cells that have been engineered to express both receptor alleles do not arrest, are fully viable, and are mating competent. It remains to be determined if and how autocrine signaling may be functioning in C. neoformans.

Receptor identity influences mating morphology but not fruiting morphology

In C. neoformans, a and α cells carry out distinct functions to initiate the mating process. For example, in response to nitrogen limitation, a cells secrete MFa pheromone, and α cells respond to the presence of the MFa pheromone by formation of germ tubes. This morphological response is specific to α cells, and only these cells (and not a cells) form germ tubes as an initial mating response [11],[28],[29]. Interestingly, α^a cells not only recognize and fuse with α cells (as would be expected for an a cell), but they also exhibit the morphology of an a cell, and do not produce germ tubes. This finding indicates that the morphological response to pheromone is mediated by the pheromone receptor (not downstream signaling events) and is independent of other mating type-specific factors. Therefore, pheromones and pheromone receptors alone dictate both the detection of a mating partner and the initial morphological response of haploid cells in C. neoformans. Interestingly, however, changes in mating identity did not influence the α-specific process of α fruiting.

Monokaryotic (or α) fruiting is a process that is specific to α cells and was recently shown to be a form of sexual development [19]. While the mechanisms underlying α fruiting are relatively undefined, it is well established that the α fruiting pathway is independent of Sxi1α and Sxi2a, and that this process is attenuated in strains in which STE3α or the MFα genes have been deleted [12],[13],[19]. Our data reveal that although α fruiting is primarily an α-specific process, it occurs regardless of whether pheromones and pheromone receptor are derived from a or α cells.

In summary, while the α fruiting process occurs despite the identity of pheromones and their receptors, these factors are wholly responsible for determining the initial mating response and haploid cell identity. The findings presented here establish a complete cell identity determination profile for C. neoformans and enhance our understanding of the myriad strategies by which fungi specify mating types and undergo sexual development.

Constructing modified α strains

To generate the α^{mfαΔ, STE3a} strain (CHY1900), the α^mfαΔ (WSC17) strain was biolistically transformed with a fragment containing STE3a and a nourseothricin resistance (NAT^R) cassette that was flanked by ∼1 kb of sequence from upstream and downstream of the STE3α open reading frame (ORF) [13]. Nourseothricin resistant transformants were screened by PCR for proper integration of the deletion/insertion construct into the STE3α locus, and putative deletion/insertion strains were confirmed by Southern blot analysis [30]. To generate the α^a strain (CHY1901), the α^{mfαΔ, STE3a} strain was biolistically transformed with a randomly integrating fragment of DNA containing MFa1 (under the control of the MFα1 promoter) and a neomycin resistance (NEO^R) cassette. Neomycin resistant transformants were screened for copy number of the insertion using Southern blot analysis [30]. The α^a/α strain (CHY2049) was generated by crossing the 5-FOA resistant α^a strain (CHY1517) by wild type α cells (JEC21) and selecting for fusants at 37°C on minimal medium containing neomycin. To generate the α^a/α^SXI2a strain (CHY2096), 5-FOA resistant α^a/α cells were biolistically transformed with a randomly integrating fragment of DNA containing the SXI2a (under the control of the GPD1 promoter) and URA5 genes.

Northern blot analysis

RNA was prepared from C. neoformans cells using a hot phenol extraction [30]. Strains were grown on solid V8 medium for 24 hours at room temperature. Northern blots were carried out according to standard protocols with 10 µg of total RNA used for each sample [30]. The glycerol-3-phosphate dehydrogenase gene (GPD1) probe was PCR-generated using CHO651 (CGTCGTTGAATCTACCGGTG) & CHO652 (CACCAGCAATGTAAGAGATG). All other probes were generated by PCR using the following oligonucleotides: CHO2030 (CCCCGACTATCCCTTTTGGAATCTCACTGC) & CHO2031 (GGCGAACAGTTCTTCGGGATATTGTGATACC) for STE3a, CHO2032 (GCACGACCTCAGCCTCGTCATTTTCAGCGG) & CHO2033 (CCGTATCCAGCAGCAATGATCGTCAGC) for STE3α, CHO2052 (GACTTACTCTTGCGTTATTGCTTAAAGTGGG) & CHO1917 (GAAAAGAGGTACGAGTAGAT) for MFa1, and CHO2053 (TTGTGTCATCGCCTAGACCCAACGTCCCC) & CHO2054 (CCATCTAAACAAGTCCCATACGCTTCGTTACC) for MFα1. Radiolabeled probes (Decaprime II kit; Ambion) were used in hybridization reactions as described previously [31]. Hybridizations and washes were carried out at 65°C as described previously [30].

Strain manipulations and media

All strains used were of the serotype D background (Table 1) and were handled using standard techniques and media as described previously [32],[33]. Sexual development assays were conducted on 5% V8 medium at room temperature in the dark for 2–4 days and were evaluated by observing the periphery of test spots. The mating tester strains used were JEC20 (a) and JEC21 (α), and crosses were photographed at 100X magnification [18]. Spores were photographed at 400X magnification. Fruiting assays were carried out by growing cells on filament agar (0.67% yeast nitrogen base (without amino acids or ammonium sulfate) containing 100 nM (NH₄)₂SO₄ at room temperature in the dark for 14 days. The periphery of test spots were photographed at 100X magnification.

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Table 1. Strains.

https://doi.org/10.1371/journal.pgen.1000860.t001

Microscopy and staining

Fluorescent microscopy was carried out on a Zeiss Axioskop 2 fluorescent microscope fitted with an Axiocam MRM REV3 digital camera and corresponding AV4 software. Light microscopy was carried out using a Zeiss Axioplan microscope fitted with a 10X long working distance objective. Photographs were taken with a Nikon Coolpix 5400 camera mounted on the microscope. To label fusants, a and α cells were grown to stationary phase in yeast extract peptone dextrose (YPD) liquid medium and were suspended in 1X phosphate buffered saline (PBS). Carboxylic acid, succinimidyl-ester Alexa Fluor 488 or 594 dyes (Invitrogen, Carlsbad CA) were coupled to a or α yeast cells in 100 mM potassium phosphate buffer pH 7.5 for 30 min at room temperature. After washing, differentially labeled cells were mixed in 1X PBS and spotted onto V8 agar. Spots were incubated at room temperature for 6 hours and were then resuspended into a mounting solution of 1X PBS containing 0.4 µg/ml calcofluor white MR2 (Sigma-Aldrich). For filament staining, crosses were incubated on a thin layer of V8 media on a microscope slide and incubated at room temperature for 24 hours. Cells were fixed with 2.5% gluteraldehyde/4% paraformaldehyde for 30 minutes. Cells were washed twice with 1X PBS containing 0.1% Triton X-100, once with 1X PBS, and incubated in 1X PBS containing 0.4 µg/mL calcofluor white to stain septa, and 1nM Sytox green (Invitrogen) to stain nuclei for 20 minutes. Cells were washed in 5% glycerol with 20% DABCO antifade (Sigma) prior to visualization.