Saul M. Honigberg

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.

Ime2p and Cdc28p: Co‐pilots driving meiotic development

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 (Hartwell and Weinert [1989]: Science. 246:629–634). Meiotic differentiation is also controlled by these two types of regulation (Murakami and Nurse [2000]: Biochem J. 349:1–12; Roeder and Bailis [2000]: Trends Genet. 16:395–403); 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 (Benjamin et al. [2003]: Genes Dev. 17:1–16; Schindler et al. [2003]: Mol Cell Biol 23:8718–8728). 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.

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.

Cell Signals, Cell Contacts, and the Organization of Yeast Communities

ABSTRACT Even relatively simple species have evolved mechanisms to organize individual organisms into communities, such that the fitness of the group is greater than the fitness of isolated individuals. Within the fungal kingdom, the ability of many yeast species to organize into communities is crucial for their growth and survival, and this property has important impacts both on the economy and on human health. Over the last few years, studies of Saccharomyces cerevisiae have revealed several fundamental properties of yeast communities. First, strain-to-strain variation in the structures of these groups is attributable in part to variability in the expression and functions of adhesin proteins. Second, the extracellular matrix surrounding these communities can protect them from environmental stress and may also be important in cell signaling. Finally, diffusible signals between cells contribute to community organization so that different regions of a community express different genes and adopt different cell fates. These findings provide an arena in which to view fundamental mechanisms by which contacts and signals between individual organisms allow them to assemble into functional communities.

Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast

BackgroundA valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids.ResultsWe have modified the existing method for introducing deletions in the yeast (S. cerevisiae) genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG) mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL) mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis) or is synthesized by error-prone PCR (RDL mutagenesis). In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated.ConclusionSSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

Meiotic differentiation during colony maturation in Sacchatomyces cerevisiae

As yeast colonies ceased growth, cells at the edge of these colonies transited from the cell division cycle into meiosis at high efficiency. This transition occurred remarkably synchronously and only at late stages of colony maturation. The transition occurred on medium containing acetate or low concentrations of glucose, but not on medium containing high glucose. The repression by high glucose was overcome when IME1 was overexpressed from a plasmid. Experiments with different growth media imply that meiosis in colonies is triggered by changes in the nutrient environment as colonies mature. HAP2 is required to sporulate in any carbon source, whereas GRR1 is required for glucose repression of sporulation. CLN3 is required to repress meiosis in colonies but not in liquid cultures, indicating that the regulators that mediate the transition to meiosis in colonies are not identical to the regulators that mediate this transition in liquid cultures.

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.

Cell Size and Cln-Cdc28 Complexes Mediate Entry into Meiosis by Modulating Cell Growth

In the yeast Saccharomyces cerevisiae, mitotic cell cycle progression depends upon the G1-phase cyclin-dependent kinase Cln-Cdc28 and cell growth to a minimum cell size. In contrast,Cln-Cdc28 inhibits entry into meiosis, and a cell growth requirement for sporulation has not beenestablished. Here, we report that entry in meiosis is also dependent upon cell growth. Moreover,sporulation and cell growth rates were proportional to cell size; large cells grew rapidly andsporulated sooner while smaller cells grew slowly and sporulated later. In addition, Cln2 proteinlevels were higher in smaller cells suggesting that Cln-Cdc28 activity represses meiosis insmaller cells by preventing cell growth. In support of this hypothesis, loss of Clns, or thepresence of a cdc28 mutation increased cell growth in smaller cells and accelerated meiosis inthese cells. Finally, over-expression of CLNs repressed meiosis in smaller cells, but not in largecells. Taken together, these results demonstrate that Cln-Cdc28 represses entry into meiosis inpart by inhibiting cell growth.