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.

• Publikační typ
• časopisecké články MeSH
• přehledy 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.

• Klíčová slova
• Saccharomyces cerevisiae, cell-specific regulation, differentiated colonies, proteasomal degradation, spatially structured populations, transcription factor, yeast,
• MeSH
• protein-serin-threoninkinasy metabolismus   genetika   MeSH
• proteolýza MeSH
• proteosyntéza MeSH
• regulace genové exprese u hub * MeSH
• Saccharomyces cerevisiae - proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• transkripční faktory bZIP * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GCN2 protein, S cerevisiae MeSH Prohlížeč
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• transkripční faktory bZIP * 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.

• Klíčová slova
• Biofilms, Cell differentiation, Colonies, Multicellular yeast structures, Regulation, Spatial community structure,
• Publikační typ
• časopisecké články MeSH
• přehledy 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.

• Klíčová slova
• Saccharomyces cerevisiae, colony development and differentiation, mitochondrial retrograde signaling, proteomic analysis, yeast colonies,
• MeSH
• aminokyseliny metabolismus   MeSH
• analýza jednotlivých buněk MeSH
• biosyntetické dráhy genetika   MeSH
• chromatografie kapalinová MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• proteom genetika   metabolismus   MeSH
• proteomika MeSH
• regulace genové exprese u hub genetika   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• tandemová hmotnostní spektrometrie MeSH
• transkripční faktory BHLH-Zip genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• intracelulární signální peptidy a proteiny MeSH
• MKS1 protein, S cerevisiae MeSH Prohlížeč
• proteom MeSH
• represorové proteiny MeSH
• RTG1 protein, S cerevisiae MeSH Prohlížeč
• RTG2 protein, S cerevisiae MeSH Prohlížeč
• RTG3 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktory BHLH-Zip 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.

• Klíčová slova
• Flo11p adhesin, cell adhesion, cell organization, colonies and biofilms, laboratory and wild Saccharomyces cerevisiae strains, structure development, yeast multicellular structures,
• MeSH
• biofilmy * MeSH
• buněčné dělení MeSH
• membránové glykoproteiny genetika   metabolismus   MeSH
• mikrobiální interakce MeSH
• mutace MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• FLO11 protein, S cerevisiae MeSH Prohlížeč
• membránové glykoproteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Yeast biofilms are complex multicellular structures, in which the cells are well protected against drugs and other treatments and thus highly resistant to antifungal therapies. Colony biofilms represent an ideal system for studying molecular mechanisms and regulations involved in development and internal organization of biofilm structure as well as those that are involved in fungal domestication. We have identified here antagonistic functional interactions between transcriptional regulators Cyc8p and Tup1p that modulate the life-style of natural S. cerevisiae strains between biofilm and domesticated mode. Herein, strains with different levels of Cyc8p and Tup1p regulators were constructed, analyzed for processes involved in colony biofilm development and used in the identification of modes of regulation of Flo11p, a key adhesin in biofilm formation. Our data show that Tup1p and Cyc8p regulate biofilm formation in the opposite manner, being positive and negative regulators of colony complexity, cell-cell interaction and adhesion to surfaces. Notably, in-depth analysis of regulation of expression of Flo11p adhesin revealed that Cyc8p itself is the key repressor of FLO11 expression, whereas Tup1p counteracts Cyc8p's repressive function and, in addition, counters Flo11p degradation by an extracellular protease. Interestingly, the opposing actions of Tup1p and Cyc8p concern processes crucial to the biofilm mode of yeast multicellularity, whereas other multicellular processes such as cell flocculation are co-repressed by both regulators. This study provides insight into the mechanisms regulating complexity of the biofilm lifestyle of yeast grown on semisolid surfaces.

• MeSH
• biofilmy * MeSH
• buněčná adheze fyziologie   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• membránové glykoproteiny genetika   metabolismus   MeSH
• mezibuněčná komunikace fyziologie   MeSH
• regulace genové exprese u hub * MeSH
• represorové proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CYC8 protein, S cerevisiae MeSH Prohlížeč
• FLO11 protein, S cerevisiae MeSH Prohlížeč
• jaderné proteiny MeSH
• membránové glykoproteiny MeSH
• represorové proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• TUP1 protein, S cerevisiae MeSH Prohlížeč

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 - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   genetika   metabolismus   MeSH
• stanovení celkové genové exprese metody   MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Saccharomyces cerevisiae - proteiny 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.

• Klíčová slova
• Cell differentiation, Colony biofilms, Invasive cell subpopulation, Regulation of glycogen metabolism, Saccharomyces cerevisiae, Transcriptomics,
• MeSH
• biofilmy * MeSH
• metabolické sítě a dráhy MeSH
• regulace genové exprese u hub * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Saccharomyces cerevisiae - proteiny MeSH

Nutrient availability controls the landscape of nutrient transporters present at the plasma membrane, notably by regulating their ubiquitylation and subsequent endocytosis. In yeast, this involves the Nedd4 ubiquitin ligase Rsp5 and arrestin-related trafficking adaptors (ARTs). ARTs are targeted by signaling pathways and warrant that cargo ubiquitylation and endocytosis appropriately respond to nutritional inputs. Here, we show that glucose deprivation regulates the ART protein Csr2/Art8 at multiple levels to trigger high-affinity glucose transporter endocytosis. Csr2 is transcriptionally induced in these conditions through the AMPK orthologue Snf1 and downstream transcriptional repressors. Upon synthesis, Csr2 becomes activated by ubiquitylation. In contrast, glucose replenishment induces CSR2 transcriptional shutdown and switches Csr2 to an inactive, deubiquitylated form. This glucose-induced deubiquitylation of Csr2 correlates with its phospho-dependent association with 14-3-3 proteins and involves protein kinase A. Thus, two glucose signaling pathways converge onto Csr2 to regulate hexose transporter endocytosis by glucose availability. These data illustrate novel mechanisms by which nutrients modulate ART activity and endocytosis.

• MeSH
• arrestin genetika   metabolismus   MeSH
• časové faktory MeSH
• endocytóza * MeSH
• genetická transkripce MeSH
• glukosa nedostatek   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• mutace MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinfosfatasa 1 metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• proteiny přenášející monosacharidy genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• represorové proteiny metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• ubikvitinace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arrestin MeSH
• BMH1 protein, S cerevisiae MeSH Prohlížeč
• BMH2 protein, S cerevisiae MeSH Prohlížeč
• Csr2 protein, S cerevisiae MeSH Prohlížeč
• glukosa MeSH
• Hxt6 protein, S cerevisiae MeSH Prohlížeč
• HXT7 protein, S cerevisiae MeSH Prohlížeč
• jaderné proteiny MeSH
• MIG1 protein, S cerevisiae MeSH Prohlížeč
• Mig2 protein, S cerevisiae MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• proteinfosfatasa 1 MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny 14-3-3 MeSH
• proteiny přenášející monosacharidy MeSH
• represorové proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SNF1-related protein kinases MeSH Prohlížeč

Mitochondrial retrograde signaling mediates communication from altered mitochondria to the nucleus and is involved in many normal and pathophysiological changes, including cell metabolic reprogramming linked to cancer development and progression in mammals. The major mitochondrial retrograde pathway described in yeast includes three activators, Rtg1p, Rtg2p and Rtg3p, and repressors, Mks1p and Bmh1p/Bmh2p. Using differentiated yeast colonies, we show that Mks1p-Rtg pathway regulation is complex and includes three branches that divergently regulate the properties and fate of three specifically localized cell subpopulations via signals from differently altered mitochondria. The newly identified RTG pathway-regulated genes ATO1/ATO2 are expressed in colonial upper (U) cells, the cells with active TORC1 that metabolically resemble tumor cells, while CIT2 is a typical target induced in one subpopulation of starving lower (L) cells. The viability of the second L cell subpopulation is strictly dependent on RTG signaling. Additional co-activators of Rtg1p-Rtg3p specific to particular gene targets of each branch are required to regulate cell differentiation.

• Klíčová slova
• ageing and longevity, development and differentiation, mitochondrial retrograde signaling,
• MeSH
• buněčná diferenciace fyziologie   MeSH
• geny hub fyziologie   MeSH
• mitochondrie metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae fyziologie   MeSH
• signální transdukce fyziologie   MeSH
• viabilita buněk fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Saccharomyces cerevisiae - proteiny MeSH