Cell death is a natural part of the development of multicellular organisms and is central to their physiological and pathological states. However, the existence of regulated cell death in unicellular microorganisms, including eukaryotic and prokaryotic microbes, has been a topic of debate. One reason for the continued debate is the lack of obvious benefit from cell death in the context of a single cell. However, unicellularity is relative, as most of these microbes dwell in communities of varying complexities, often with complicated spatial organization. In these spatially organized microbial communities, such as yeast and bacterial colonies and biofilms growing on solid surfaces, cells differentiate into specialized types, and the whole community often behaves like a simple multicellular organism. As these communities develop and age, cell death appears to offer benefits to the community as a whole. This review explores the potential roles of cell death in spatially organized communities of yeasts and draws analogies to similar communities of bacteria. The natural dying processes in microbial cell communities are only partially understood and may result from suicidal death genes, (self-)sabotage (without death effectors), or from non-autonomous mechanisms driven by interactions with other differentiated cells. We focus on processes occurring during the stratification of yeast colonies, the formation of the extracellular matrix in biofilms, and discuss potential roles of cell death in shaping the organization, differentiation, and overall physiology of these microbial structures.

• Publication type
• Journal Article MeSH
• Review MeSH

Gcn4p belongs to conserved AP-1 transcription factors involved in many cellular processes, including cell proliferation, stress response, and nutrient availability in yeast and mammals. AP-1 activities are regulated at different levels, such as translational activation or protein degradation, which increases the variability of regulation under different conditions. Gcn4p activity in unstructured yeast liquid cultures increases upon amino acid deficiency and is rapidly eliminated upon amino acid excess. Gcn2p kinase is the major described regulator of Gcn4p that enables GCN4 mRNA translation via the uORFs mechanism. Here, we show that Gcn4p is specifically active in U cells in the upper regions and inactive in L cells in the lower regions of differentiated colonies. Using in situ microscopy in combination with analysis of mutants and strains with GFP at different positions in the translational regulatory region of Gcn4p, we show that cell-specific Gcn4p activity is independent of Gcn2p or other translational or transcriptional regulation. Genetically, biochemically, and microscopically, we identified cell-specific proteasomal degradation as a key mechanism that diversifies Gcn4p function between U and L cells. The identified regulation leading to active Gcn4p in U cells with amino acids and efficient degradation in starved L cells differs from known regulations of Gcn4p in yeast but shows similarities to the activity of AP-1 ATF4 in mammals during insulin signaling. These findings may open new avenues for understanding the parallel activities of Gcn4p/ATF4 and reveal a novel biological role for cell type-specific regulation of proteasome-dependent degradation.IMPORTANCEIn nature, microbes usually live in spatially structured communities and differentiate into precisely localized, functionally specialized cells. The coordinated interplay of cells and their response to environmental changes, such as starvation, followed by metabolic adaptation, is critical for the survival of the entire community. Transcription factor Gcn4p is responsible for yeast adaptation under amino acid starvation in liquid cultures, and its activity is regulated mainly at the level of translation involving Gcn2p kinase. Whether Gcn4p functions in structured communities was unknown. We show that translational regulation of Gcn4p plays no role in the development of colony subpopulations; the main regulation occurs at the level of stabilization of the Gcn4p molecule in the cells of one subpopulation and its proteasomal degradation in the other. This regulation ensures specific spatiotemporal activity of Gcn4p in the colony. Our work highlights differences in regulatory networks in unorganized populations and organized structures of yeast, which in many respects resemble multicellular organisms.

• Keywords
• Saccharomyces cerevisiae, cell-specific regulation, differentiated colonies, proteasomal degradation, spatially structured populations, transcription factor, yeast,
• MeSH
• Protein Serine-Threonine Kinases metabolism   genetics   MeSH
• Proteolysis MeSH
• Protein Biosynthesis MeSH
• Gene Expression Regulation, Fungal * MeSH
• Saccharomyces cerevisiae Proteins * genetics   metabolism   MeSH
• Saccharomyces cerevisiae * genetics   metabolism   MeSH
• Protein Stability MeSH
• Basic-Leucine Zipper Transcription Factors * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• GCN2 protein, S cerevisiae MeSH Browser
• GCN4 protein, S cerevisiae MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Basic-Leucine Zipper Transcription Factors * MeSH

Yeast populations can undergo diversification during their growth and ageing, leading to the formation of different cell-types. Differentiation into two major subpopulations, differing in cell size and density and exhibiting distinct physiological and metabolic properties, was described in planktonic liquid cultures and in populations of colonies growing on semisolid surfaces. Here, we compare stress resistance, metabolism and expression of marker genes in seven differentiated cell subpopulations emerging during cultivation in liquid fermentative or respiratory media and during colony development on the same type of solid media. The results show that the more-dense cell subpopulations are more stress resistant than the less-dense subpopulations under all cultivation conditions tested. On the other hand, respiratory capacity, enzymatic activities and marker gene expression differed more between subpopulations. These characteristics are more influenced by the lifestyle of the population (colony vs. planktonic cultivation) and the medium composition. Only in the population growing in liquid respiratory medium, two subpopulations do not form as in the other conditions tested, but all cells exhibit a range of characteristics of the more-dense subpopulations. This suggests that signals for cell differentiation may be triggered by prior metabolic reprogramming or by an unknown signal from the structured environment in the colony.

• MeSH
• Cell Differentiation MeSH
• Fermentation MeSH
• Saccharomyces cerevisiae * metabolism   MeSH
• Yeast, Dried * MeSH
• Publication type
• Journal Article MeSH

Single-celled yeasts form spatially structured populations - colonies and biofilms, either alone (single-species biofilms) or in cooperation with other microorganisms (mixed-species biofilms). Within populations, yeast cells develop in a coordinated manner, interact with each other and differentiate into specialized cell subpopulations that can better adapt to changing conditions (e.g. by reprogramming metabolism during nutrient deficiency) or protect the overall population from external influences (e.g. via extracellular matrix). Various omics tools together with specialized techniques for separating differentiated cells and in situ microscopy have revealed important processes and cell interactions in these structures, which are summarized here. Nevertheless, current knowledge is still only a small part of the mosaic of complexity and diversity of the multicellular structures that yeasts form in different environments. Future challenges include the use of integrated multi-omics approaches and a greater emphasis on the analysis of differentiated cell subpopulations with specific functions.

• Keywords
• Biofilms, Cell differentiation, Colonies, Multicellular yeast structures, Regulation, Spatial community structure,
• Publication type
• Journal Article MeSH
• Review MeSH

Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions via changes in nuclear gene expression. The most comprehensive picture of components and regulation of retrograde signaling has been obtained in Saccharomyces cerevisiae, where retrograde-target gene expression is regulated by RTG genes. In this chapter, we describe methods to measure mitochondrial retrograde pathway activation at the level of mRNA and protein products in yeast model systems, including cell suspensions and microcolonies. In particular, we will focus on three major procedures: mRNA levels of RTG-target genes, such as those encoding for peroxisomal citrate synthase (CIT2), aconitase, and NAD+-specific isocitrate dehydrogenase subunit 1 by real-time PCR; expression analysis of CIT2-gene protein product (Cit2p-GFP) by Western blot and fluorescence microscopy; the phosphorylation status of transcriptional factor Rtg1/3p which controls RTG-target gene transcription.

• Keywords
• ACO1, CIT2, IDH1, Mitochondrial retrograde pathway, RTG genes, Yeast,
• MeSH
• Aconitate Hydratase genetics   metabolism   MeSH
• Cell Nucleus genetics   metabolism   MeSH
• Citrate (si)-Synthase genetics   metabolism   MeSH
• Phosphorylation MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Isocitrate Dehydrogenase genetics   metabolism   MeSH
• Mitochondria metabolism   pathology   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Signal Transduction MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aconitate Hydratase MeSH
• Citrate (si)-Synthase MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Isocitrate Dehydrogenase MeSH
• RTG1 protein, S cerevisiae MeSH Browser
• RTG2 protein, S cerevisiae MeSH Browser
• RTG3 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors MeSH

During development of yeast colonies, various cell subpopulations form, which differ in their properties and specifically localize within the structure. Three branches of mitochondrial retrograde (RTG) signaling play a role in colony development and differentiation, each of them activating the production of specific markers in different cell types. Here, aiming to identify proteins and processes controlled by the RTG pathway, we analyzed proteomes of individual cell subpopulations from colonies of strains, mutated in genes of the RTG pathway. Resulting data, along with microscopic analyses revealed that the RTG pathway predominantly regulates processes in U cells, long-lived cells with unique properties, which are localized in upper colony regions. Rtg proteins therein activate processes leading to amino acid biosynthesis, including transport of metabolic intermediates between compartments, but also repress expression of mitochondrial ribosome components, thus possibly contributing to reduced mitochondrial translation in U cells. The results reveal the RTG pathway's role in activating metabolic processes, important in U cell adaptation to altered nutritional conditions. They also point to the important role of Rtg regulators in repressing mitochondrial activity in U cells.

• Keywords
• Saccharomyces cerevisiae, colony development and differentiation, mitochondrial retrograde signaling, proteomic analysis, yeast colonies,
• MeSH
• Amino Acids metabolism   MeSH
• Single-Cell Analysis MeSH
• Biosynthetic Pathways genetics   MeSH
• Chromatography, Liquid MeSH
• Intracellular Signaling Peptides and Proteins genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Proteome genetics   metabolism   MeSH
• Proteomics MeSH
• Gene Expression Regulation, Fungal genetics   MeSH
• Repressor Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Signal Transduction genetics   MeSH
• Tandem Mass Spectrometry MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• MKS1 protein, S cerevisiae MeSH Browser
• Proteome MeSH
• Repressor Proteins MeSH
• RTG1 protein, S cerevisiae MeSH Browser
• RTG2 protein, S cerevisiae MeSH Browser
• RTG3 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors MeSH

Multicellular structures formed by yeasts and other microbes are valuable models for investigating the processes of cell-cell interaction and pattern formation, as well as cell signaling and differentiation. These processes are essential for the organization and development of diverse microbial communities that are important in everyday life. Two major types of multicellular structures are formed by yeast Saccharomyces cerevisiae on semisolid agar. These are colonies formed by laboratory or domesticated strains and structured colony biofilms formed by wild strains. These structures differ in spatiotemporal organization and cellular differentiation. Using state-of-the-art microscopy and mutant analysis, we investigated the distribution of cells within colonies and colony biofilms and the involvement of specific processes therein. We show that prominent differences between colony and biofilm structure are determined during early stages of development and are associated with the different distribution of growing cells. Two distinct cell distribution patterns were identified-the zebra-type and the leopard-type, which are genetically determined. The role of Flo11p in cell adhesion and extracellular matrix production is essential for leopard-type distribution, because FLO11 deletion triggers the switch to zebra-type cell distribution. However, both types of cell organization are independent of cell budding polarity and cell separation as determined using respective mutants.

• Keywords
• Flo11p adhesin, cell adhesion, cell organization, colonies and biofilms, laboratory and wild Saccharomyces cerevisiae strains, structure development, yeast multicellular structures,
• MeSH
• Biofilms * MeSH
• Cell Division MeSH
• Membrane Glycoproteins genetics   metabolism   MeSH
• Microbial Interactions MeSH
• Mutation MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae cytology   metabolism   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• FLO11 protein, S cerevisiae MeSH Browser
• Membrane Glycoproteins MeSH
• Saccharomyces cerevisiae Proteins MeSH

We summarize current knowledge regarding regulatory functions of long noncoding RNAs (lncRNAs) in yeast, with emphasis on lncRNAs identified recently in yeast colonies and biofilms. Potential regulatory functions of these lncRNAs in differentiated cells of domesticated colonies adapted to plentiful conditions versus yeast colony biofilms are discussed. We show that specific cell types differ in their complements of lncRNA, that this complement changes over time in differentiating upper cells, and that these lncRNAs target diverse functional categories of genes in different cell subpopulations and specific colony types.

• MeSH
• Biofilms growth & development   MeSH
• Cell Differentiation MeSH
• Humans MeSH
• RNA, Long Noncoding metabolism   MeSH
• Saccharomyces cerevisiae pathogenicity   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• RNA, Long Noncoding MeSH

We present the spatiotemporal metabolic differentiation of yeast cell subpopulations from upper, lower, and margin regions of colonies of different ages, based on comprehensive transcriptomic analysis. Furthermore, the analysis was extended to include smaller cell subpopulations identified previously by microscopy within fully differentiated U and L cells of aged colonies. New data from RNA-seq provides both spatial and temporal information on cell metabolic reprogramming during colony ageing and shows that cells at marginal positions are similar to upper cells, but both these cell types are metabolically distinct from cells localized to lower colony regions. As colonies age, dramatic metabolic reprogramming occurs in cells of upper regions, while changes in margin and lower cells are less prominent. Interestingly, whereas clear expression differences were identified between two L cell subpopulations, U cells (which adopt metabolic profiles, similar to those of tumor cells) form a more homogeneous cell population. The data identified crucial metabolic reprogramming events that arise de novo during colony ageing and are linked to U and L cell colony differentiation and support a role for mitochondria in this differentiation process.

• MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae cytology   genetics   metabolism   MeSH
• Gene Expression Profiling methods   MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Saccharomyces cerevisiae Proteins MeSH

BACKGROUND: Yeast infections are often connected with formation of biofilms that are extremely difficult to eradicate. An excellent model system for deciphering multifactorial determinants of yeast biofilm development is the colony biofilm, composed of surface ("aerial") and invasive ("root") cells. While surface cells have been partially analyzed before, we know little about invasive root cells. In particular, information on the metabolic, chemical and morphogenetic properties of invasive versus surface cells is lacking. In this study, we used a new strategy to isolate invasive cells from agar and extracellular matrix, and employed it to perform genome wide expression profiling and biochemical analyses of surface and invasive cells. RESULTS: RNA sequencing revealed expression differences in 1245 genes with high statistical significance, indicating large genetically regulated metabolic differences between surface and invasive cells. Functional annotation analyses implicated genes involved in stress defense, peroxisomal fatty acid β-oxidation, autophagy, protein degradation, storage compound metabolism and meiosis as being important in surface cells. In contrast, numerous genes with functions in nutrient transport and diverse synthetic metabolic reactions, including genes involved in ribosome biogenesis, biosynthesis and translation, were found to be important in invasive cells. Variation in gene expression correlated significantly with cell-type specific processes such as autophagy and storage compound accumulation as identified by microscopic and biochemical analyses. Expression profiling also provided indications of cell-specific regulations. Subsequent knockout strain analyses identified Gip2p, a regulatory subunit of type 1 protein phosphatase Glc7p, to be essential for glycogen accumulation in surface cells. CONCLUSIONS: This is the first study reporting genome wide differences between surface and invasive cells of yeast colony biofilms. New findings show that surface and invasive cells display very different physiology, adapting to different conditions in different colony areas and contributing to development and survival of the colony biofilm as a whole. Notably, surface and invasive cells of colony biofilms differ significantly from upper and lower cells of smooth colonies adapted to plentiful laboratory conditions.

• Keywords
• Cell differentiation, Colony biofilms, Invasive cell subpopulation, Regulation of glycogen metabolism, Saccharomyces cerevisiae, Transcriptomics,
• MeSH
• Biofilms * MeSH
• Metabolic Networks and Pathways MeSH
• Gene Expression Regulation, Fungal * MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   physiology   MeSH
• Gene Expression Profiling MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Saccharomyces cerevisiae Proteins MeSH