Diploid yeast, like most eukaryotes, can undergo meiotic differentiation to form haploid gametes. Meiotic differentiation and cell growth (proliferation)are mutually exclusive programs, and in yeast the switch between growth and meiosis is controlled by nutritional signals. The signaling pathways that mediate nutritional controls on meiotic initiation fall into three broad classes: those that respond to nutrient starvation, those that respond to non-fermentable carbon sources, and those that respond to glucose. At the onset of meiosis, nutritional signaling pathways converge on transcriptional regulation of two genes: IME1, which encodes a transcription factor;and IME2, which encodes a protein kinase. Transcription of IME1 and IME2 trigger initiation of meiosis, and the expression of these two genes is linked with one other, with expression of later meiotic genes and with early meiotic events such as DNA replication. In addition, the signaling pathways that control IME1 and IME2expression are themselves integrated through a variety of mechanisms. Thus the signal network that controls the switch from growth to meiotic differentiation provides a signaling code that translates different combinations of extracellular signals into appropriate cellular responses.

Glucose inhibits meiosis in Saccharomyces cerevisiae at three different steps (IME1 transcription, IME2 transcription, and entry into late stages of meiosis). Because many of the regulatory effects of glucose in yeast are mediated through the inhibition of Snf1 kinase, a component of the glucose repression pathway, we determined the role of SNF1 in regulating meiosis. Deleting SNF1 repressed meiosis at the same three steps that were inhibited by glucose, suggesting that glucose blocks meiosis by inhibiting Snf1. For example, the snf1⌬ mutant completely failed to induce IME1 transcripts in sporulation medium. Furthermore, even when this block was bypassed by expression of IME1 from a multicopy plasmid, IME2 transcription and meiotic initiation occurred at only 10 to 20% of the levels seen in wild-type cells. The addition of glucose did not further inhibit IME2 transcription, suggesting that Snf1 is the primary mediator of glucose controls on IME2 expression. Finally, in snf1⌬ cells in which both blocks on meiotic initiation were bypassed, early stages of meiosis (DNA replication and commitment to recombination) occurred, but later stages (chromosome segregation and spore formation) did not, suggesting that Snf1 controls later stages of meiosis independently from the two controls on meiotic initiation. Because Snf1 is known to activate the expression of genes required for acetate metabolism, it may also serve to connect glucose and acetate controls on meiotic differentiation.

Multicellular organisms utilize cell-to-cell signals to build patterns of cell types within embryos, but the ability of fungi to form organized communities has been largely unexplored. Here we report that colonies of the yeast Saccharomyces cerevisiae formed sharply divided layers of sporulating and nonsporulating cells. Sporulation initiated in the colony's interior, and this region expanded upward as the colony matured. Two key activators of sporulation, IME1 and IME2, were initially transcribed in overlapping regions of the colony, and this overlap corresponded to the initial sporulation region. The development of colony sporulation patterns depended on cell-to-cell signals, as demonstrated by chimeric colonies, which contain a mixture of two strains. One such signal is alkaline pH, mediated through the Rim101p/PacC pathway. Meiotic-arrest mutants that increased alkali production stimulated expression of an early meiotic gene in neighboring cells, whereas a mutant that decreased alkali production (cit1D) decreased this expression. Addition of alkali to colonies accelerated the expansion of the interior region of sporulation, whereas inactivation of the Rim101p pathway inhibited this expansion. Thus, the Rim101 pathway mediates colony patterning by responding to cell-to-cell pH signals. Cell-to-cell signals coupled with nutrient gradients may allow efficient spore formation and spore dispersal in natural environments.

In the budding yeast Saccharomyces cerevisiae, the cell division cycle and sporulation are mutually exclusive cell fates; glucose, which stimulates the cell division cycle, is a potent inhibitor of sporulation. Addition of moderate concentrations of glucose (0.5%) to sporulation medium did not inhibit transcription of two key activators of sporulation, IME1 and IME2, but did increase levels of Sic1p, a cyclin-dependent kinase inhibitor, resulting in a block to meiotic DNA replication. The effects of glucose on Sic1p levels and DNA replication required Grr1p, a component of the SCF Grr1p ubiquitin ligase. Sic1p is negatively regulated by Ime2p kinase, and several observations indicate that glucose inhibits meiotic DNA replication through SCF Grr1p -mediated destruction of this kinase. First, Ime2p was destabilized in the presence of glucose, and this turnover required Grr1p, a second component of SCF Grr1p , Cdc53p, and an SCF Grr1p -associated E2 enzyme, Cdc34p. Second, Ime2p-ubiquitin conjugates were detected under conditions of rapid Ime2p turnover, and conjugation of Ime2p to ubiquitin required GRR1. Third, a mutant form of Ime2p (Ime2 ⌬PEST ), in which a putative Grr1p-interacting sequence was deleted, was more stable than wild-type Ime2p. Finally, expression of the IME2⌬PEST allele bypassed the block to meiotic DNA replication caused by 0.5% glucose. In addition, Grr1p is required for later events in sporulation independently of its role in Ime2p turnover.

When yeast from the early stages of melosis are transferred from porton to growth medium, they can reenter the mitotic cell cldirectly. In contrast, cells from later stages of meosis (after the initiation of the first nuclear division) will The meiotic fate is triggered by deprivation of glucose and nitrogen. As is true in many types ofcell differentiation, cells in meiosis remain uncommitted through the early stages of the process-i.e., they will abort meiosis and return to mitotic growth if the cells are transferred from sporulation to growth medium (4). Significantly, genetic and cytological analyses have revealed that meiotic DNA replication, synaptonemal complex formation, chromosome pairing, and DNA recombination all occur during this uncommitted period (5-7). In contrast, cells become fillly committed to meiotic development at the initiation of chromosome segregation in meiosis I-i.e., after this point they will complete the two meiotic divisions and form spores despite the addition of glucose and nitrogen. This study presents the unexpected result that when meiotic cells are arrested after either the first or second division, cells are able to return to mitotic growth. Further experiments suggest that commitment results from a transient delay in mitotic growth rather than an irreversible inhibition of the cell cycle during sporulation. MATERIALS AND METHODSStrains. The following diploid strains were used in this study. H65 contains MATa/MATa ADE2/ade2-1 adeSi ADE5 can]/cani CYH2/cyh2 HIS] /hisi his7/HIS7 HOM3/ hom3-10 leu2-3/leu2-1 lysi/LYS) LYS2/lys2 metl4/MET14 pet8/PET8 TRPJ/trpl ura3/ura3. H91 is isogenic to H65 except for the presence of a homozygous disruption/ duplication allele of SP014 designated spol4:URA3:spol4 (8). REE1525 contains MATa/MATa arg4-1/arg4-1 cyhi-li cyhl-l HO/HO met4/met4. SH495 is congenic to REE1525 and is spo3-1/spo3-1.Media. Growth (YPDA) and acetate (YPA) media have been described (8). The sporulation media (SPII-30 and SPII-31) contain 20 g of potassium acetate per liter supplemented with 75 pg (each) ofeither adenine sulfate, L-leucine, and uracil per ml (for strains H65 and H91) or L-arginine and L-methionine (for strains REE1525 and SH493).Assaying nmark Events in Meiosls. Fifty-milliliter cultures were grown for "'20 hr at 300C in YPA to 5.0 x 107. Cells were washed three times, resuspended in 25 ml ofsporulation medium (pH 6.5), and aerated by shaking at constant temperature in a New Brunswick Scientific gyratory water bath shaker at 250 rpm. At intervals, 200-1d samples were fixed by addition of400 y4 of95% ethanol. An aliquot (6 y1) ofthis cell suspension was spotted on a microscope slide and allowed to dry. Nuclei were stained by placing 6 p4 of a solution of 1 pAg of 4',6-diamidino-2-phenylindole dihydrochloride per ml (DAPI, Boehringer Mannheim), 0.1 mg of 1,4-phenylene diamine per ml (Sigma), 50 mM sodium phosphate buffer, and 50%1 glycerol on the dried cells. Cells were visualized by Nomarski/fluorescence microscopy on a Jenalumar microscope. Two hund...

Many microbial communities contain organized patterns of cell types, yet relatively little is known about the mechanism or function of this organization. In colonies of the budding yeast Saccharomyces cerevisiae, sporulation occurs in a highly organized pattern, with a top layer of sporulating cells sharply separated from an underlying layer of nonsporulating cells. A mutant screen identified the Mpk1 and Bck1 kinases of the cell-wall integrity (CWI) pathway as specifically required for sporulation in colonies. The CWI pathway was induced as colonies matured, and a target of this pathway, the Rlm1 transcription factor, was activated specifically in the nonsporulating cell layer, here termed feeder cells. Rlm1 stimulates permeabilization of feeder cells and promotes sporulation in an overlying cell layer through a cell-nonautonomous mechanism. The relative fraction of the colony apportioned to feeder cells depends on nutrient environment, potentially buffering sexual reproduction against suboptimal environments.KEYWORDS cell-wall integrity; cell permeability; cell-cell signaling; Saccharomyces cerevisiae; sporulation A S embryos develop, cells of different fates organize into patterns [reviewed in Kicheva et al. (2012) and Perrimon et al. (2012)] . Intriguingly, even unicellular microbial species form communities in which different cell types are organized into patterns [reviewed in Kaiser et al. (2010), Honigberg (2011, and Loomis (2014)]. For example, colonies of the budding yeast Saccharomyces cerevisiae form an upper layer of larger cells (U cells) overlying a layer of smaller cells (L cells). U and L cells differ in their metabolism, gene expression, and resistance to stress, and U and L layers are separated by a strikingly sharp boundary Vachova et al. 2013). Patterns are also observed in yeast biofilms, where cells closest to the plastic surface grow as ovoid cells, whereas cells further from the surface differentiate into hyphae for Candida species [reviewed in Finkel and Mitchell (2011)] or pseudohyphae and eventually asci for S. cerevisiae (White et al. 2011).Sporulation also occurs in patterns within yeast colonies. Specifically, a narrow horizontal layer of sporulated cells forms through the center of the colony early during colony development. As colonies continue to mature, this layer progressively expands upward to include the top of the colony; this wave is driven by progressive alkalization and activation of the Rim101 signaling pathway . In contrast, cells at the bottom of the colony, i.e., directly contacting the agar substrate, also sporulate at early stages of colony development, but this narrow cell layer does not expand as the colony matures . The same colony sporulation pattern is observed in a range of laboratory yeasts as well as in S. cerevisiae and S. paradoxus isolated from the wild. Indeed, in these wild yeasts, the same colony sporulation pattern forms on a range of fermentable and nonfermentable carbon sources .The mechanism of sporulation patterning and its function remai...

Meiosis can be considered an elaboration of the cell division cycle in the sense that meiosis combines cell-cycle processes with programs specific to meiosis. Each phase of the cell division cycle is driven forward by cell-cycle kinases (Cdk) and coordinated with other phases of the cycle through checkpoint functions. Meiotic differentiation is also controlled by these two types of regulation; however, recent study in the budding yeast S. cerevisiae indicates that progression of meiosis is also controlled by a master regulator specific to meiosis, namely the Ime2p kinase. Below, I describe the overlapping roles of Ime2p and Cdk during meiosis in yeast and speculate on how these two kinases cooperate to drive the progression of meiosis.

The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae, although it is not required for growth. Here we report that in stationary-phase cultures containing low concentration of glucose, cells overexpressing IME1 undergo the early meiotic events, including DNA replication, commitment to recombination, and synaptonemal complex formation and dissolution. In contrast, later meiotic events, such as chromosome segregation, commitment to meiosis, and spore formation, do not occur. Thus, nutrients can repress the late stages of meiosis independently of their block of initiation. Cells arrested at this midpoint in meiosis are relatively stable and can resume meiotic differentiation if transferred to sporulation conditions. Resumption of meiosis does not require repression of IME1 expression, since IME1 RNA levels stay high after transfer of the arrested cells to sporulation medium. These results suggest that meiosis in S. cerevisiae is a paradigm of a differentiation pathway regulated by signal transduction at both early and late stages.