Growing Yeast into Cylindrical Colonies

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.     

Growing Yeast into Cylindrical Colonies

Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes.     

CONVERSION EFFICIENCIES IN HETEROTROPHIC ORGANISMS

1. The maximum possible efficiency at which living systems are able to convert input nutrients to their own biomass is between 70 and 80 %. 2. Conversion efficiency in bacteria, protozoa and metazoan cells in culture approximates more closely to 60%. 3. Conversion efficiency during embryonic development begins below 60% and rises above this level in the later stages. 4. Very young, post-natal organisms have high net efficiencies; 50 to 70% in homeotherms and 50 to 80 % in poikilotherms. 5. In cellular systems, capable of proliferation, conversion efficiency is independent of food supply. This means that conversion is directly dependent on nutrient supply. 6. Control of growth at the tissue level may occur through the control of the supply of nutrients to the tissues and its entry into the cells. 7. Compensatory growth, after and during undernutrition, involves increased absorption efficiency and reduced metabolic costs.     

Redox tolerance and metabolic reprogramming in solid tumors

Tumor cells need to cope with the host environment for survival and keep growing in hard conditions. This suggests that tumors must acquire characteristics more potent than what is seen for normal tissue cells, without which they are condemned to disruption.  For example, cancer cells have more potent redox tolerance compared with normal cells, which is due to their high adaptation to an oxidative crisis. In addition, increased demand for bioenergetics and biosynthesis can cause a rise in nutrient uptake in tumors. Utilizing nutrients in low nutrient conditions suggests that tumors are also equipped with adaptive metabolic processes. Switching the metabolic demands toward glucose consumption upon exposure to the hypoxic tumor microenvironment, or changing toward using other sources when there is an overconsumption of glucose in the tumor area are examples of fitness metabolic systems in tumors. In fact, cancer cells in cooperation with their nearby stroma (in a process called metabolic coupling) can reprogram their metabolic systems in their favor. This suggests the high importance of stroma for meeting the metabolic demands of a growing tumor, an example in this context is the metabolic symbiosis between cancer-associated fibroblasts with cancer cells. The point is that redox tolerance and metabolic reprogramming are interrelated, and that, without a doubt, disruption of redox tolerance systems by transient exposure to either oxidative or antioxidative loading, or targeting metabolic rewiring by modulation of tumor glucose availability, controlling tumor/stroma interactions, etc. can be effective from a therapeutic standpoint     

Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis

Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.     

Determinants of nutrient limitation in cancer

Proliferation requires that cells accumulate sufficient biomass to grow and divide. Cancer cells within tumors must acquire a variety of nutrients, and tumor growth slows or stops if necessary metabolites are not obtained in sufficient quantities. Importantly, the metabolic demands of cancer cells can be different from those of untransformed cells, and nutrient accessibility in tumors is different than in many normal tissues. Thus, cancer cell survival and proliferation may be limited by different metabolic factors than those that are necessary to maintain noncancerous cells. Understanding the variables that dictate which nutrients are critical to sustain tumor growth may identify vulnerabilities that could be used to treat cancer. This review examines the various cell-autonomous, local, and systemic factors that determine which nutrients are limiting for tumor growth.     

Minimal nutritional requirements for immobilized yeast

The effect of reduced nutritional levels (particularly nitrogen source) for immobilized K. fragilis type yeast were studied using a trickle flow, "differential" plug flow type reactor with cells immobilized by adsorption onto an absorbant packing matrix. Minimizing nutrient levels in a feed stream to an immobilized cell reactor (ICR) might have the benefits of reducing cell growth and clogging problems in the ICR, reducing feed preparation costs, as well as reducing effluent disposal costs. In this study step changes in test feed medium nutrient compositions were introduced to the ICR, followed by a return to a basal medium. Gas evolution rates were monitored and logged on a continuous basis, and effluent cell density was used as an indicator of cell growth rate of the immobilized cell mass. Startup of the reactor using a YEP medium showed a rapid buildup of cells in the reactor during the initial 110 h operation. The population density then stabilized at 1.6 x 10(11) cells/g sponge. A defined medium containing a complex mix of essential nutrients with an inorganic nitrogen source (ammonium sulfate) was able to maintain 90% of the productivity in the ICR as compared to the YEP medium, but proved unable to promote growth of the immobilized cell mass during startup. Experiments on reduced ammonium sulfate in the defined medium, and reduced yeast extract and peptone in YEP medium indicated that stable productivity could be maintained for extended periods (80 h) in the complete absence of any nutrients besides a few salts (potassium phosphate and magnesium sulfate). It was found that productivity rates dropped by 35-65% from maximal values as nitrogenous nutrients were eliminated from the test mediums, while growth rates (as determined by shed cell density from the reactor) dropped by 75-95%. Thus, nutritional deficiencies largely decoupled growth and productivity of the immobilized yeast which suggests productivity is both growth- and non-growth-associated for the immobilized cells. A yeast extract concentration of 0.375 g/L with or without 1 g/L ammonium sulfate was determined to be the minimum level which gave a sustained increase in productivity rates as compared to the nutritionally deficient salt medium. This represents a 94% reduction in complex nitrogenous nutrient levels compared to standard YEP batch medium (3 g/L YE and 3.5 g/L peptone).     

The flexible evolutionary anchorage-dependent Pardee's restriction point of mammalian cells: how its deregulation may lead to cancer

Living cells oscillate between the two states of quiescence and division that stand poles apart in terms of energy requirements, macromolecular composition and structural organization and in which they fulfill dichotomous activities. Division is a highly dynamic and energy-consuming process that needs be carefully orchestrated to ensure the faithful transmission of the mother genotype to daughter cells. Quiescence is a low-energy state in which a cell may still have to struggle hard to maintain its homeostasis in the face of adversity while waiting sometimes for long periods before finding a propitious niche to reproduce. Thus, the perpetuation of single cells rests upon their ability to elaborate robust quiescent and dividing states. This led yeast and mammalian cells to evolve rigorous Start [L.H. Hartwell, J. Culotti, J. Pringle, B.J. Reid, Genetic control of the cell division cycle in yeast, Science 183 (1974) 46-51] and restriction (R) points [A.B. Pardee, A restriction point for control of normal animal cell proliferation, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 1286-1290], respectively, that reduce deadly interferences between the two states by enforcing their temporal insulation though still enabling a rapid transition from one to the other upon an unpredictable change in their environment. The constitutive cells of multi-celled organisms are extremely sensitive in addition to the nature of their adhering support that fluctuates depending on developmental stage and tissue specificity. Metazoan evolution has entailed, therefore, the need for exceedingly flexible anchorage-dependent R points empowered to assist cells in switching between quiescence and division at various times, places and conditions in the same organism. Programmed cell death may have evolved concurrently in specific contexts unfit for the operation of a stringent R point that increase the risk of deadly interferences between the two states (as it happens notably during development). But, because of their innate flexibility, anchorage-dependent R points have also the ability to readily adjust to a changing structural context so as to give mutated cells a chance to reproduce, thereby encouraging tumor genesis. The Rb and p53 proteins, which are regulated by the two products of the Ink4a-Arf locus [C.J. Sherr, The INK4a/ARF network in tumor suppression, Nat. Rev., Mol. Cell Biol. 2 (2001) 731-737], govern separable though interconnected pathways that cooperate to restrain cyclin D- and cyclin E-dependent kinases from precipitating untimely R point transit. The expression levels of the Ink4a and Arf proteins are especially sensitive to changes in cellular shape and adhesion that entirely remodel at the time when cells shift between quiescence and division. The Arf proteins further display an extremely high translational sensitivity and can activate the p53 pathway to delay R point transit, but, only when released from the nucleolus, 'an organelle formed by the act of building a ribosome' [T. Mélèse, Z. Xue, The nucleolus: an organelle formed by the act of building a ribosome, Curr. Opin. Cell Biol. 7 (1995) 319-324]. In this way, the Ink4a/Rb and Arf/p53 pathways emerge as key regulators of anchorage-dependent R point transit in mammalian cells and their deregulation is, indeed, a rule in human cancers. Thus, by selecting the nucleolus to mitigate cell cycle control by the Arf proteins, mammalian cells succeeded in forging a highly flexible R point enabling them to match cell division with a growth rate imposed by factors controlling nucleolar assembling, such as nutrients and adhesion. It is noteworthy that nutrient control of critical size at Start in budding yeast has been shown recently to be governed by a nucleolar protein interaction network [P. Jorgensen, J.L. Nishikawa, B.-J. Breitkreutz, M. Tyers, Systematic identification of pathways that couple cell growth and division in yeast, Science 297 (2002) 395-400].     

Eisosomes at the intersection of TORC1 and TORC2 regulation

Eisosomes are furrows in the yeast plasma membrane that form a membrane domain with distinct lipid and protein composition. Recent studies highlighted the importance of this domain for the regulation of proton‐nutrient symporters. The amino acids and other nutrients, which these transporters deliver to the cytoplasm not only feed into metabolic pathways but also activate the metabolic regulator TORC1. Eisosomes have also been shown to harbor the membrane stress sensors Slm1 and Slm2. Membrane tension caused by hypoosmotic shock results in the redistribution of Slm1/2 from eisosomes to TORC2 which in turn regulates lipid synthesis. Therefore, eisosomes function upstream of both TORC1 and TORC2 regulation.     

Growth patterns of yeast colonies depending on nutrient supply

Common theories of microbial growth and physiology are formulated exclusively in terms of the isolated microorganisms – especially bacteria. This is, however, an inadmissible simplification because it is obvious that the organization of microbial populations and colonies follows certain general rules. Bacterial colonies are able to generate complex interfacial growth patterns similar to those observed during diffusion-limited growth processes in non-living systems. One reason for these patterns is assumed to be the ability of many bacteria to swarm in an active manner on a substrate surface. Therefore the models of bacterial colony growth incorporate “random walkers”, which move actively in response to a gradient in the concentration of nutrients and communicate with each other by means of a chemotactic feedback. A selected number of yeasts were tested with regard to their colony growth patterns depending on the medium parameters such as nutrient concentration. Growth patterns similar to those which were described in literature for bacteria were also found in these experiments. It concerns in particular growth types like compact growth, fractal growth and dense-branching growth. This result allows a hypothesis to be formulated, that – especially in the case of fractal growth patterns – wandering of cells on a substrate surface may be induced by uncontrolled “swimming” on a thin water film caused by the metabolic activity (e.g. respiration) of the cells on the surface of the agar. Furthermore it was found that an interplay between changes in the individual morphology of yeast cells and the morphology transitions takes place. Such growth patterns are known for Candida sp. which are able to form pseudomycel and blastospores.     

The flexible evolutionary anchorage-dependent Pardee's restriction point of mammalian cells. How its deregulation may lead to cancer

Living cells oscillate between the two states of quiescence and division that stand poles apart in terms of energy requirements, macromolecular composition and structural organization and in which they fulfill dichotomous activities. Division is a highly dynamic and energy-consuming process that needs be carefully orchestrated to ensure the faithful transmission of the mother genotype to daughter cells. Quiescence is a low-energy state in which a cell may still have to struggle hard to maintain its homeostasis in the face of adversity while waiting sometimes for long periods before finding a propitious niche to reproduce. Thus, the perpetuation of single cells rests upon their ability to elaborate robust quiescent and dividing states. This led yeast and mammalian cells to evolve rigorous Start [L.H. Hartwell, J. Culotti, J. Pringle, B.J. Reid, Genetic control of the cell division cycle in yeast, Science 183 (1974) 46–51] and restriction (R) points [A.B. Pardee, A restriction point for control of normal animal cell proliferation, Proc. Natl. Acad. Sci. U. S. A. 71 (1974) 1286–1290], respectively, that reduce deadly interferences between the two states by enforcing their temporal insulation though still enabling a rapid transition from one to the other upon an unpredictable change in their environment. The constitutive cells of multicelled organisms are extremely sensitive in addition to the nature of their adhering support that fluctuates depending on developmental stage and tissue specificity. Metazoan evolution has entailed, therefore, the need for exceedingly flexible anchorage-dependent R points empowered to assist cells in switching between quiescence and division at various times, places and conditions in the same organism. Programmed cell death may have evolved concurrently in specific contexts unfit for the operation of a stringent R point that increase the risk of deadly interferences between the two states (as it happens notably during development). But, because of their innate flexibility, anchorage-dependent R points have also the ability to readily adjust to a changing structural context so as to give mutated cells a chance to reproduce, thereby encouraging tumor genesis. The Rb and p53 proteins, which are regulated by the two products of the Ink4a-Arf locus [C.J. Sherr, The INK4a/ARF network in tumor suppression, Nat. Rev., Mol. Cell Biol. 2 (2001) 731–737], govern separable though interconnected pathways that cooperate to restrain cyclin D- and cyclin E-dependent kinases from precipitating untimely R point transit. The expression levels of the Ink4a and Arf proteins are especially sensitive to changes in cellular shape and adhesion that entirely remodel at the time when cells shift between quiescence and division. The Arf proteins further display an extremely high translational sensitivity and can activate the p53 pathway to delay R point transit, but, only when released from the nucleolus, ‘an organelle formed by the act of building a ribosome’ [T. Mélèse, Z. Xue, The nucleolus: an organelle formed by the act of building a ribosome, Curr. Opin. Cell Biol. 7 (1995) 319–324]. In this way, the Ink4a/Rb and Arf/p53 pathways emerge as key regulators of anchorage-dependent R point transit in mammalian cells and their deregulation is, indeed, a rule in human cancers. Thus, by selecting the nucleolus to mitigate cell cycle control by the Arf proteins, mammalian cells succeeded in forging a highly flexible R point enabling them to match cell division with a growth rate imposed by factors controlling nucleolar assembling, such as nutrients and adhesion. It is noteworthy that nutrient control of critical size at Start in budding yeast has been shown recently to be governed by a nucleolar protein interaction network [P. Jorgensen, J.L. Nishikawa, B.-J. Breitkreutz, M. Tyers, Systematic identification of pathways that couple cell growth and division in yeast, Science 297 (2002) 395–400].     

The metabolic cross-talk between cancer and T cells

The metabolic cross-talk between cancer cells and T cells dictates cancer formation and progression. These cells possess metabolic plasticity. Thus, they adapt their metabolic profile to meet their phenotypic requirements. However, the nutrient microenvironment of a tumor is a very hostile niche in which these cells are forced to compete for the available nutrients. The hyperactive metabolism of tumor cells often outcompetes the antitumorigenic CD8⁺ T cells while promoting the protumorigenic exhausted CD8⁺ T cells and T regulatory (T_reg) cells. Thus, cancer cells elude the immune response and spread in an uncontrolled manner. Identifying the metabolic pathways necessary to shift the balance from a protumorigenic to an antitumorigenic immune phenotype is essential to potentiate antitumor immunity.     

Analysis of cell growth in a polymer scaffold using a moving boundary approach

Two mathematical models of chondrocyte generation and nutrient consumption are developed to analyze the behavior of cell growth in a biodegradable polymer matrix. Substrate reaction and diffusion are analyzed in two regions: one consisting of cells and nutrients and the other consisting of only nutrients. A pseudo-steady state approximation for the transport of nutrients in these two regions is utilized. The rate of growth is determined by a moving boundary equation that equates the rate at which the interfacial region between the cells and the void space moves to a substrate dependent growth reaction. The change in the location of this interfacial region with time therefore depicts the rate at which the cells propagate. The two limiting cases discussed in this article represent extremes in how the cells will grow in the polymer matrix; one case assumes that cells grow inward from the external boundary, and the other case assumes that cells grow parallel to the external boundary. The results of both models are compared to experimental data found in the literature. It is found through these comparisons that the model parameters, including the unit cell spacing parameter L, the metabolic rate constant k, the growth rate constant k(G), and external mass transfer coefficient, K, may vary as the thickness of the polymer matrix is changed, however, unrealistic and large changes in the diffusion coefficients were required to account for the full range of experimental data. Furthermore, these results suggest modification of the functional form of the growth kinetics to include substrate or product inhibition, or death terms. Based upon diffusion/reaction concepts, these models for cell growth in a biodegradable polymer give bounds for the upper and lower limits of the cellular growth rate and nutrient consumption in a polymer matrix and will aid in the development of more extensive models. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 422-432, 1997.     

Mathematical modeling of regulatory mechanisms in yeast colony development

In the present study, yeast colony development serves as a model system to study growth of fungal populations with negligible nutrient and signal transport within the mycelium. Mathematical simulations address the question whether colony development is governed by diffusional limitation of nutrients. A hybrid one-dimensional cellular automaton model was developed that describes growth of discrete cells based upon microscopic interaction rules in a continuous field of nutrient and messenger. The model is scaled for the geometry of the experimental setup, cell size, growth- and substrate uptake rates. Therefore, calculated cell density profiles and nutrient distributions can be compared to experimental results and the model assumptions can be verified. In the physiologically relevant parameter range, simulations show an exponentially declining cell density along the median axis of the colonies in case of a diffusion limited growth scenario. These results are in good agreement with cell density profiles obtained in cultivations of the yeast Candida boidinii with glucose as the limiting carbon source but stand in contrast to the constant cell density profile estimated for Yarrowia lipolytica grown under the same conditions. While from the comparison of experimental results and simulations a diffusion limited growth mechanism is proposed for glucose limited C. boidinii colonies, this hypothesis is rejected for the growth of Y. lipolytica. As an alternative, a quorum sensing model was developed that can explain the evolution of constant cell density profiles based on the effect of a not further characterized unstable or volatile messenger.     

Bacterial swarming: a re-examination of cell-movement patterns

Many bacteria simultaneously grow and spread rapidly over a surface that supplies them with nutrient. Called 'swarming', this pattern of movement directs new cells to the edge of the colony. Swarming reduces competition between cells for nutrients, speeding growth. Behind the swarm edge, where the cell density is higher, growth is limited by transport of nutrient from the subsurface to the overlying cells. Despite years of study, the choreography of swarm cell movement, the bacterial equivalent of dancing toward an exit in a very dense crowd of moving bodies, remains a mystery. Swarming can be propelled by rotating flagella, and either by pulling with type IV pili or by pushing with the secretion of slime. By identifying patterns of movement that are common to swarms making use of different engines, a model of swarm choreography can be proposed.     

Lewis lung carcinoma variant with a high sensitivity to antitumor antiangiogenic therapy exhibits a high capacity for autophagy

This paper presents a comparative study of growth characteristics of two variants of Lewis lung carcinoma cells (LLC and LLC/R9) that were cultivated under conditions of nutrient deficiency caused by long-term incubation without changing the medium. It was found that LLC/R9 cells, which are characterized by high sensitivity to antitumor antiangiogenic therapy (AAT) compared to LLC cells, demonstrated a high dependence of cell survival on glucose levels in the growth medium, as well as an ability to activate autophagy under nutrient deprivation. It indicates that tumor sensitivity to AAT may be associated with the ability of tumor cells to induce autophagy under the conditions of nutrient substrate deprivation.     

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays.

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.     

Granulocyte-macrophage colony stimulating factor modulates the production of TNF alpha by differentiated U937 cells infected with Leishmania major

In this work, the production of tumor necrosis factor alpha (TNF alpha) during interaction of human phagocytes with the intracellular parasite Leishmania major was further investigated. The human monocytic cell line U937, differentiated with a combination of 1 alpha, 25 dihydroxyvitamin D3 (VD) and retinoic acid (RA), or with granulocyte macrophage colony stimulating factor (GM-CSF) was used. Differentiated U937 cells were infected with Leishmania major promastigotes, and TNF alpha was assayed in cell culture supernatants. It was found that the cytokine was produced only by U937 cells differentiated with VD/RA and further incubated with GM-CSF and LPS or interferon gamma (IFN gamma). L. major induced TNF alpha production only in the presence of GM-CSF. No direct relationship was found, however, between production of TNF alpha and resistance of differentiated U937 cells to infection with L. major.