Sarah Piccirillo

The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.

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 colonys 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 (cit1Δ) 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.

The CLN3/SWI6/CLN2 pathway and SNF1 act sequentially to regulate meiotic initiation in Saccharomyces cerevisiae

Background: IME1, which is required for the initiation of meiosis, is regulated by Cln3:Cdc28 kinase, which activates the G1‐to‐S transition, and Snf1 kinase, which mediates glucose repression. Here we examine the pathway by which Cln3:Cdc28p represses IME1 and the relationship between Cln3:Cdc28p and Snf1p in this regulation.

Sporulation patterning and invasive growth in wild and domesticated yeast colonies

Different cell types can form patterns within fungal communities; for example, colonies of Saccharomyces cerevisiae form two sharply defined layers of sporulating cells separated by an intervening layer of unsporulated cells. Because colony sporulation patterns have only been investigated in a single laboratory strain background (W303), in this report we examined these patterns in other strain backgrounds. Two other laboratory strain backgrounds (SK1 and Sigma1278b) that differ from W303 with respect to colony morphology, invasive growth, and sporulation efficiency nevertheless displayed the same colony sporulation pattern as W303. This pattern was also observed in colonies of wild isolates of S. cerevisiae and Saccharomyces paradoxus. The wild yeast colonies sporulated on a much wider range of carbon sources than did the lab yeast and displayed a similar layered sporulation pattern when grown on either acetate or glucose medium and on either rich or synthetic medium. SK1, Sigma1278b and wild yeast colonies invaded the agar surface. The region of invasion varied between strains with respect to the organization and appearance of cells, but this invasion was always accompanied by sporulation. Thus, sporulation patterns are a general property of S. cerevisiae, and sporulation in colonies can be coordinated with invasive growth.

Glucose inhibits meiotic DNA replication through SCFGrr1p-dependent destruction of Ime2p kinase.

ABSTRACT 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 SCFGrr1p ubiquitin ligase. Sic1p is negatively regulated by Ime2p kinase, and several observations indicate that glucose inhibits meiotic DNA replication through SCFGrr1p-mediated destruction of this kinase. First, Ime2p was destabilized in the presence of glucose, and this turnover required Grr1p, a second component of SCFGrr1p, Cdc53p, and an SCFGrr1p-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 (Ime2ΔPEST), in which a putative Grr1p-interacting sequence was deleted, was more stable than wild-type Ime2p. Finally, expression of the IME2 ΔPEST 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.

Flo11p adhesin required for meiotic differentiation in Saccharomyces cerevisiae minicolonies grown on plastic surfaces.

Saccharomyces cerevisiae grown on plastic surfaces formed organized structures, termed minicolonies, that consisted of a core of round (yeast-like) cells surrounded by chains of filamentous cells (pseudohyphae). Minicolonies had a much higher affinity for plastic than unstructured yeast communities growing on the same surface. Pseudohyphae at the surface of these colonies developed further into chains of asci. These structures suggest that pseudohyphal differentiation and sporulation are sequential processes in minicolonies. Consistent with this idea, minicolonies grown under conditions that stimulated pseudohyphal differentiation contained higher frequencies of asci. Furthermore, a flo11Δ mutant, which fails to form pseudohyphae, yielded normal sporulation in cultures, but was defective for minicolony sporulation. When minicolonies were dispersed in water and cells were then allowed to settle on the plastic surface, these cells sporulated very efficiently. Taken together, our results suggest that sporulation in minicolonies is stimulated by pseudohyphal differentiation because these pseudohyphae are dispersed from the core of the colony.

Phenotypic plasticity within yeast colonies: differential partitioning of cell fates

Across many phyla, a common aspect of multicellularity is the organization of different cell types into spatial patterns. In the budding yeast Saccharomyces cerevisiae, after diploid colonies have completed growth, they differentiate to form alternating layers of sporulating cells and feeder cells. In the current study, we found that as yeast colonies developed, the feeder cell layer was initially separated from the sporulating cell layer. Furthermore, the spatial pattern of sporulation in colonies depended on the colony’s nutrient environment; in two environments in which overall colony sporulation efficiency was very similar, the pattern of feeder and sporulating cells within the colony was very different. As noted previously, under moderately suboptimal conditions for sporulation—low acetate concentration or high temperature—the number of feeder cells increases as does the dependence of sporulation on the feeder-cell transcription factor, Rlm1. Here we report that even under a condition that is completely blocked sporulation, the number of feeder cells still increased. These results suggest broader implications to our recently proposed “Differential Partitioning provides Environmental Buffering” or DPEB hypothesis.

Cell Differentiation and Spatial Organization in Yeast Colonies: Role of Cell-Wall Integrity Pathway

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.

Glucose induction pathway regulates meiosis in Saccharomyces cerevisiae in part by controlling turnover of Ime2p meiotic kinase.

Several components of the glucose induction pathway, namely the Snf3p glucose sensor and the Rgt1p and Mth1p transcription factors, were shown to be involved in inhibition of sporulation by glucose. The glucose sensors had only a minor role in regulating transcript levels of the two key regulators of meiotic initiation, the Ime1p transcription factor and the Ime2p kinase, but a major role in regulating Ime2p stability. Interestingly, Rgt1p was involved in glucose inhibition of spore formation but not inhibition of Ime2p stability. Thus, the glucose induction pathway may regulate meiosis through both RGT1-dependent and RGT1-independent pathways.

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood. One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 μ) useful for light microscopy and thin sections (0.1 μ) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM. The method is based on surrounding colonies with agar, infiltrating them with Spurrs medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

GAL1-SceI directed site-specific genomic (gsSSG) mutagenesis: a method for precisely targeting point mutations in S. cerevisiae

BackgroundPrecise targeted mutations are defined as targeted mutations that do not require the retention of other genetic changes, such as marker genes, near the mutation site. In the yeast, S. cerevisiae, there are several methods for introducing precise targeted mutations, all of which depend on inserting both a counter-selectable marker and DNA bearing the mutation. For example, the marker can first be inserted, and then replaced with either a long oligonucleotide carrying the mutation (delitto perfetto) or a PCR fragment synthesized with one primer containing the mutation (SSG mutagenesis).ResultsA hybrid method for targeting precise mutation into the genomes uses PCR fragments as in SSG mutagenesis together with a CORE cassette devised for delitto perfetto that contains the homing endonuclease SceI. This method, termed gsSSG mutagenesis, is much more efficient than standard SSG mutagenesis, allowing replacements to be identified without extensive screening of isolates. In gsSSG, recombination between the PCR fragment and the genome occurs equally efficiently regardless of the size of the fragment or the distance between the fragment end and the site of marker insertion. In contrast, the efficiency of incorporating targeted mutations by this method increases as the distance between the mutation and the marker insertion site decreases.ConclusiongsSSG is an efficient way of introducing precise mutations into the genome of S. cerevisiae. The frequency of incorporating the targeted mutation remains efficient at least as far as 460 bp from the insertion site meaning that a single insertion can be used to create many different mutants. The overall efficiency of gsSSG can be estimated based on the distance between the mutation and the marker insertion, and this efficiency can be maximized by limiting the number of untargeted mutations. Thus, a single insertion of marker genes plus homing endonuclease cassette can be used to efficiently introduce precise point mutations through a region of > 900 bp.