Active Ras2 in mitochondria promotes regulated cell death in a cAMP/PKA pathway-dependent manner in budding yeast.

Previously, we showed that an aberrant accumulation of activated Ras in mitochondria correlates with an increase in apoptosis. In this article, we show that lack of trehalose-6P-synthase, known to trigger apoptosis in Saccharomyces cerevisiae, induces localization of active Ras proteins in mitochondria, confirming the above-mentioned correlation. Next, by characterizing the ras1Δ and ras2Δ mutants, we show that active Ras2 proteins, which accumulate in the mitochondria following addition of acetic acid (a pro-apoptotic stimulus), are likely the GTPases involved in regulated cell death, while active Ras1 proteins, constitutively localized in mitochondria, might be involved in a pro-survival molecular machinery. Finally, by characterizing the gpa2Δ and cyr1Δ mutants, in which the cAMP/PKA pathway is compromised, we show that active mitochondrial Ras proteins promote apoptosis through the cAMP/PKA pathway.

An Optimized Workflow for the Analysis of Metabolic Fluxes in Cancer Spheroids Using Seahorse Technology.

Three-dimensional cancer models, such as spheroids, are increasingly being used to study cancer metabolism because they can better recapitulate the molecular and physiological aspects of the tumor architecture than conventional monolayer cultures. Although Agilent Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, United States) is a valuable technology for studying metabolic alterations occurring in cancer cells, its application to three-dimensional cultures is still poorly optimized. We present a reliable and reproducible workflow for the Seahorse metabolic analysis of three-dimensional cultures. An optimized protocol enables the formation of spheroids highly regular in shape and homogenous in size, reducing variability in metabolic parameters among the experimental replicates, both under basal and drug treatment conditions. High-resolution imaging allows the calculation of the number of viable cells in each spheroid, the normalization of metabolic parameters on a per-cell basis, and grouping of the spheroids as a function of their size. Multivariate statistical tests on metabolic parameters determined by the Mito Stress test on two breast cancer cell lines show that metabolic differences among the studied spheroids are mostly related to the cell line rather than to the size of the spheroid. The optimized workflow allows high-resolution metabolic characterization of three-dimensional cultures, their comparison with monolayer cultures, and may aid in the design and interpretation of (multi)drug protocols.

Fast detection of PKA activity in Saccharomyces cerevisiae cell population using AKAR fluorescence resonance energy transfer probes.

In Saccharomyces cerevisiae, the protein kinase A (PKA) plays a central role in the control of metabolism, stress resistance and cell cycle progression. In a previous work, we used a FRET-based A-kinase activity reporter (AKAR3 probe) to monitor changes in PKA activity in vivo in single S. cerevisiae cells. Since this procedure is quite complex and time-consuming, in this work we used the AKAR3 probe (evenly distributed within the cells) and the plate reader Victor-X3™ (Perkin Elmer®) to measure PKA activity in vivo in a whole cell population. We show that in wild type strains, the FRET increases after addition of glucose to glucose-starved cells, while no changes are observed when this sugar is added to strains with either absent or attenuated PKA activity. Moreover, using the pm-AKAR3 probe, mainly expressed at the plasma membrane and partially at the vacuolar membrane, we could monitor PKA activity from the starting site of the signal to internal regions, where the signal is propagated. Finally, we also show evidence for direct activation of PKA by glucose, independent of cAMP. In conclusion, our data show that AKAR3 and pm-AKAR3 probes are useful biosensors to monitor PKA activity in a S. cerevisiae cell population using a plate reader.

Profiling and Targeting of Energy and Redox Metabolism in Grade 2 Bladder Cancer Cells with Different Invasiveness Properties.

Bladder cancer is one of the most prevalent deadly diseases worldwide. Grade 2 tumors represent a good window of therapeutic intervention, whose optimization requires high resolution biomarker identification. Here we characterize energy metabolism and cellular properties associated with spreading and tumor progression of RT112 and 5637, two Grade 2 cancer cell lines derived from human bladder, representative of luminal-like and basal-like tumors, respectively. The two cell lines have similar proliferation rates, but only 5637 cells show efficient lateral migration. In contrast, RT112 cells are more prone to form spheroids. RT112 cells produce more ATP by glycolysis and OXPHOS, present overall higher metabolic plasticity and are less sensitive than 5637 to nutritional perturbation of cell proliferation and migration induced by treatment with 2-deoxyglucose and metformin. On the contrary, spheroid formation is less sensitive to metabolic perturbations in 5637 than RT112 cells. The ability of metformin to reduce, although with different efficiency, cell proliferation, sphere formation and migration in both cell lines, suggests that OXPHOS targeting could be an effective strategy to reduce the invasiveness of Grade 2 bladder cancer cells.

Respiratory metabolism and calorie restriction relieve persistent endoplasmic reticulum stress induced by calcium shortage in yeast.

Calcium homeostasis is crucial to eukaryotic cell survival. By acting as an enzyme cofactor and a second messenger in several signal transduction pathways, the calcium ion controls many essential biological processes. Inside the endoplasmic reticulum (ER) calcium concentration is carefully regulated to safeguard the correct folding and processing of secretory proteins. By using the model organism Saccharomyces cerevisiae we show that calcium shortage leads to a slowdown of cell growth and metabolism. Accumulation of unfolded proteins within the calcium-depleted lumen of the endoplasmic reticulum (ER stress) triggers the unfolded protein response (UPR) and generates a state of oxidative stress that decreases cell viability. These effects are severe during growth on rapidly fermentable carbon sources and can be mitigated by decreasing the protein synthesis rate or by inducing cellular respiration. Calcium homeostasis, protein biosynthesis and the unfolded protein response are tightly intertwined and the consequences of facing calcium starvation are determined by whether cellular energy production is balanced with demands for anabolic functions. Our findings confirm that the connections linking disturbance of ER calcium equilibrium to ER stress and UPR signaling are evolutionary conserved and highlight the crucial role of metabolism in modulating the effects induced by calcium shortage.

Whi5 phosphorylation embedded in the G1/S network dynamically controls critical cell size and cell fate.

In budding yeast, overcoming of a critical size to enter S phase and the mitosis/mating switch--two central cell fate events--take place in the G1 phase of the cell cycle. Here we present a mathematical model of the basic molecular mechanism controlling the G1/S transition, whose major regulatory feature is multisite phosphorylation of nuclear Whi5. Cln3-Cdk1, whose nuclear amount is proportional to cell size, and then Cln1,2-Cdk1, randomly phosphorylate both decoy and functional Whi5 sites. Full phosphorylation of functional sites releases Whi5 inhibitory activity, activating G1/S transcription. Simulation analysis shows that this mechanism ensures coherent release of Whi5 inhibitory action and accounts for many experimentally observed properties of mitotically growing or conjugating G1 cells. Cell cycle progression and transcriptional analyses of a Whi5 phosphomimetic mutant verify the model prediction that coherent transcription of the G1/S regulon and ensuing G1/S transition requires full phosphorylation of Whi5 functional sites.

Overexpression of Far1, a cyclin-dependent kinase inhibitor, induces a large transcriptional reprogramming in which RNA synthesis senses Far1 in a Sfp1-mediated way.

The FAR1 gene encodes an 830 residue bifunctional protein, whose major function is inhibition of cyclin-dependent kinase complexes involved in the G1/S transition. FAR1 transcription is maximal between mitosis and early G1 phase. Enhanced FAR1 transcription is necessary but not sufficient for the pheromone-induced G1 arrest, since FAR1 overexpression itself does not trigger cell cycle arrest. Besides its well established role in the response to pheromone, recent evidences suggest that Far1 may also regulate the mitotic cell cycle progression: in particular, it has been proposed that Far1, together with the G1 cyclin Cln3, may be part of a cell sizer mechanism that controls the entry into S phase. Far1 is an unstable protein throughout the cell cycle except during G1 phase. Far1 levels peak in newborn cells as a consequence of a burst of synthetic activity at the end of the previous cycle, and the amounts per cell remain roughly constant during the G1 phase. Phosphorylation (at serine 87) by Cdk1-Cln complexes primes Far1 for ubiquitin-mediated proteolysis. By coupling a genome-wide transcriptional analysis of FAR1-overexpressing and far1Δ cells grown in ethanol- or glucose-supplemented minimal media with a range of phenotypic analysis, we show that FAR1 overexpression not only coordinately increases RNA and protein accumulation, but induces strong transcriptional remodeling, metabolism being the most affected cellular property, suggesting that the Far1/Cln3 sizer regulates cell growth either directly or indirectly by affecting metabolism and pathways known to modulate ribosome biogenesis. A crucial role in mediating the effect of Far1 overexpression is played by the Sfp1 protein, a key transcriptional regulator of ribosome biogenesis, whose presence is mandatory to allow a coordinated increase in both RNA and protein levels in ethanol-grown cells.

Regulation of hSos1 activity is a system-level property generated by its multi-domain structure.

The multi-domain protein hSos1 plays a major role in cell growth and differentiation through its Ras-specific guanine nucleotide exchange domain whose complex regulation involves intra-molecular, inter-domain rearrangements. We present a stochastic mathematical model describing intra-molecular regulation of hSos1 activity. The population macroscopic effect is reproduced through a Monte-Carlo approach. Key model parameters have been experimentally determined by BIAcore analysis. Complementation experiments of a Saccharomyces cerevisiae cdc25(ts) strain with Sos deletion mutants provided a comprehensive data set for estimation of unknown parameters and model validation. The model is robust against parameter alteration and describes both the behavior of Sos deletion mutants and modulation of activity of the full length molecule under physiological conditions. By incorporating the calculated effect of amino acid changes at an inter-domain interface, the behavior of a mutant correlating with a developmental syndrome could be simulated, further validating the model. The activation state of Ras-specific guanine nucleotide exchange domain of hSos1 arises as an "emergent property" of its multi-domain structure that allows multi-level integration of a complex network of intra- and inter-molecular signals.

Glucose signaling-mediated coordination of cell growth and cell cycle in Saccharomyces cerevisiae.

Besides being the favorite carbon and energy source for the budding yeast Sacchromyces cerevisiae, glucose can act as a signaling molecule to regulate multiple aspects of yeast physiology. Yeast cells have evolved several mechanisms for monitoring the level of glucose in their habitat and respond quickly to frequent changes in the sugar availability in the environment: the cAMP/PKA pathways (with its two branches comprising Ras and the Gpr1/Gpa2 module), the Rgt2/Snf3-Rgt1 pathway and the main repression pathway involving the kinase Snf1. The cAMP/PKA pathway plays the prominent role in responding to changes in glucose availability and initiating the signaling processes that promote cell growth and division. Snf1 (the yeast homologous to mammalian AMP-activated protein kinase) is primarily required for the adaptation of yeast cell to glucose limitation and for growth on alternative carbon source, but it is also involved in the cellular response to various environmental stresses. The Rgt2/Snf3-Rgt1 pathway regulates the expression of genes required for glucose uptake. Many interconnections exist between the diverse glucose sensing systems, which enables yeast cells to fine tune cell growth, cell cycle and their coordination in response to nutritional changes.

Functional coupling of the mammalian EGF receptor to the Ras/cAMP pathway in the yeast Saccharomyces cerevisiae.

Autophosphorylation of tyrosine residues on the cytoplasmic tail of the epidermal growth factor receptor (EGFR) upon ligand binding leads to recruitment of the Grb2/Sos complex to the activated receptor and to activation of the Ras pathway. The major aim of this study was to ascertain to which extent the EGFR module (receptor, Grb2, hSos1) could work in a lower eukaryote, completely devoid of tyrosine kinase receptors but possessing hortologues to mammalian Ras proteins. We show that the EGFR module can be functionally linked to the Ras/cAMP pathway in a Saccharomyces cerevisiae cdc25 ( ts ) strain, as monitored by several independent biological readouts, including drop of budding index, decrease of cAMP level and acquisition of thermotolerance. Autophosphorylation of the receptor is a necessary step for RTK-dependent activation of the yeast Ras pathway, since genetic and pharmacological downregulation of the EGFR catalytic activity abolish coupling with the Ras/cAMP pathway. Thus, our results newly indicate that a RTK-based signal transduction module can be functionally coupled to the yeast Ras/cAMP pathway and that our system can be a valuable tool for the screen of drugs inhibiting the kinase activity of the receptor.

Proteomic analysis of a nutritional shift-up in Saccharomyces cerevisiae identifies Gvp36 as a BAR-containing protein involved in vesicular traffic and nutritional adaptation.

Yeast cells undergoing a nutritional shift-up from a poor to a rich carbon source take several hours to adapt to the novel, richer carbon source. The budding index is a physiologically relevant "global" parameter that reflects the complex links between cell growth and division that are both coordinately and deeply affected by nutritional conditions. We used changes in budding index as a guide to choose appropriate, relevant time points during an ethanol to glucose nutritional shift-up for preparation of samples for the analysis of proteome by two-dimensional electrophoresis/mass spectrometry. About 600 spots were detected. 90 spots, mostly comprising proteins involved in intermediary metabolism, protein synthesis, and response to stress, showed differential expression after glucose addition. Among modulated proteins we identified a protein of previously unknown function, Gvp36, showing a transitory increase corresponding to the drop of the fraction of budded cells. A gvp36Delta strain shares several phenotypes (including general growth defects, heat shock, and high salt sensitivity, defects in polarization of the actin cytoskeleton, in endocytosis and in vacuolar biogenesis, defects in entering stationary phase upon nutrient starvation) with secretory pathway mutants and with mutants in genes encoding the two previously known yeast BAR proteins (RSV161 and RSV167). We thus propose that Gvp36 represents a novel yeast BAR protein involved in vesicular traffic and in nutritional adaptation.

Catalytic competence of the Ras-GEF domain of hSos1 requires intra-REM domain interactions mediated by phenylalanine 577.

The Ras-specific guanine nucleotide exchange region of hSos1 consists of two consecutive domains: the catalytic core (residues 742-1024) contains all residues binding to Ras, including the catalytic hairpin, and an upstream REM domain (residues 553-741), so called because it contains an evolutionary conserved Ras Exchange Motif (REM). We functionally define the boundaries of the REM domain through a combination of in vivo and in vitro assays. We show that an intra-REM domain interaction, mediated by phenylalanine 577, is required to allow interaction of the REM domain with the catalytic core, constraining it in the active conformation.

In Saccharomyces cerevisiae an unbalanced level of tyrosine phosphorylation down-regulates the Ras/PKA pathway.

The role of tyrosyl phosphorylation/dephosphorylation in the budding yeast Saccharomyces cerevisiae, whose genome does not encode typical tyrosine kinases, has long remained elusive. Nevertheless, several protein kinases phosphorylating poly(TyrGlu) substrates have been identified. In this work, we use the expression of the low molecular weight tyrosine phosphatase Stp1 from the distantly related yeast Schizosaccharomyces pombe, as a tool to investigate whether an unbalanced level of protein tyrosine phosphorylation affects S. cerevisiae growth and metabolism. We correlate the previously reported down-regulation of the phosphotyrosine level brought about by overexpression of Stp1 with a large number of phenotypes indicative of down-regulation of the Ras pathway. These phenotypes include reduction in both glucose- and acidification-induced GTP loading of the Ras2 protein and cAMP signaling, impaired growth on a non-fermentable carbon source, alteration of cell cycle parameters, delayed recovery from nitrogen starvation, increased heat-shock resistance, attenuated pseudohyphal and invasive growth. Genetic data suggest that Stp1 acts either at, or above, the level of Ras2, possibly on the Ira proteins. Consistently, Stp1 was found to bind to immunoprecipitated Ira2. Since a catalytically inactive mutant form of Stp1 (Stp1(C11S)) effectively binds to Ira2 without producing any effect on yeast physiology, we conclude that down-regulation of the Ras pathway by Stp1 requires its phosphatase activity. In conclusion, our data suggest a possible cross-talk between tyrosine phosphorylation and the Ras pathway in yeast.