Unraveling the Anti-Aging Properties of Phycocyanin from the Cyanobacterium Spirulina (Arthrospira platensis).

In recent years, marine natural products have become one of the most important resources of novel lead compounds for critical diseases associated with age. Spirulina, a dietary supplement made from blue-green algae (cyanobacteria: scientific name Arthrospira platensis), is particularly rich in phycocyanin, a phycobiliprotein, which accounts for up to 20% of this cyanobacterium's dry weight and is considered responsible for its anti-cancer, anti-inflammatory and antioxidant activities. Although the anti-aging activity of phycocyanin has been investigated, how exactly this compound works against aging remains elusive. The aim of our research is to use the yeast Saccharomyces cerevisiae as a model organism to investigate the anti-aging properties of phycocyanin from A. platensis. Our results show that phycocyanin has a powerful anti-aging effect, greatly extending the chronological life span of yeast cells in a dose-dependent way, as the effect was also pronounced when cells were grown in SD medium under calorie restriction conditions (0.2% glucose). Both ROS and accumulation of dead cells were followed by staining chronologically aged cells with dihydrorhodamine 123 (DHR123) and propidium iodide (PI). Interestingly, we found that most of the aged phycocyanin-treated cells, which were unable to form colonies, were actually ROS+/PI-. Finally, we show that the moment in which phycocyanin is added to the culture does not substantially influence its effectiveness in counteracting chronological aging.

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.

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.

RalGPS2 Interacts with Akt and PDK1 Promoting Tunneling Nanotubes Formation in Bladder Cancer and Kidney Cells Microenvironment.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor for RalA GTPase that is involved in several cellular processes, including cytoskeletal organization. Previously, we demonstrated that RalGPS2 also plays a role in the formation of tunneling nanotubes (TNTs) in bladder cancer 5637 cells. In particular, TNTs are a novel mechanism of cell-cell communication in the tumor microenvironment, playing a central role in cancer progression and metastasis formation. However, the molecular mechanisms involved in TNTs formation still need to be fully elucidated. Here we demonstrate that mid and high-stage bladder cancer cell lines have functional TNTs, which can transfer mitochondria. Moreover, using confocal fluorescence time-lapse microscopy, we show in 5637 cells that TNTs mediate the trafficking of RalA protein and transmembrane MHC class III protein leukocyte-specific transcript 1 (LST1). Furthermore, we show that RalGPS2 is essential for nanotubes generation, and stress conditions boost its expression both in 5637 and HEK293 cell lines. Finally, we prove that RalGPS2 interacts with Akt and PDK1, in addition to LST1 and RalA, leading to the formation of a complex that promotes nanotubes formation. In conclusion, our findings suggest that in the tumor microenvironment, RalGPS2 orchestrates the assembly of multimolecular complexes that drive the formation of TNTs.

In S. cerevisiae hydroxycitric acid antagonizes chronological aging and apoptosis regardless of citrate lyase.

Caloric restriction mimetics (CRMs) are promising molecules to prevent age-related diseases as they activate pathways driven by a true caloric restriction. Hydroxycitric acid (HCA) is considered a bona fide CRM since it depletes acetyl-CoA pools by acting as a competitive inhibitor of ATP citrate lyase (ACLY), ultimately repressing protein acetylation and promoting autophagy. Importantly, it can reduce inflammation and tumour development. In order to identify phenotypically relevant new HCA targets we have investigated HCA effects in Saccharomyces cerevisiae, where ACLY is lacking. Strikingly, the drug revealed a powerful anti-aging effect, another property proposed to mark bona fide CRMs. Chronological life span (CLS) extension but also resistance to acetic acid of HCA treated cells were associated to repression of cell apoptosis and necrosis. HCA also largely prevented cell deaths caused by a severe oxidative stress. The molecule could act widely by negatively modulating cell metabolism, similarly to citrate. Indeed, it inhibited both growth reactivation and the oxygen consumption rate of yeast cells in stationary phase. Genetic analyses on yeast CLS mutants indicated that part of the HCA effects can be sensed by Sch9 and Ras2, two conserved key regulators of nutritional and stress signal pathways of primary importance. Our data together with published biochemical analyses indicate that HCA may act with multiple mechanisms together with ACLY repression and allowed us to propose an integrated mechanistic model as a basis for future investigations.

Lack of SNF1 induces localization of active Ras in mitochondria and triggers apoptosis in the yeast Saccharomyces cerevisiae.

In previous papers we showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type yeast cells growing exponentially on glucose, while an aberrant accumulation of activated Ras in mitochondria correlated to mitochondrial dysfunction, accumulation of ROS and regulated cell death. Here we show that also in a strain lacking Snf1, the homolog of the AMP-activated protein kinase (AMPK) in Saccharomyces cerevisiae, activated Ras proteins accumulate mainly in these organelles, suggesting an antiapoptotic role for this protein, beside its well-known function in glucose repression. Indeed, in this paper we show that Snf1 protects against apoptosis in Saccharomyces cerevisiae. In particular, following treatment with acetic acid, a well-known inducer of apoptosis in this microorganism, snf1Δ cells show a significant reduction in cell survival and a higher level of ROS when compared with wild-type cells. More importantly, untreated snf1Δ cells show a higher percentage of apoptotic cells compared with wild-type cells, which further increases upon treatment with acetic acid. In order to determine whether the role of Snf1 in regulated cell death is dependent on its catalytic activity, we characterized the Snf1-S214E strain, expressing a catalytically inactive form of Snf1. Data on active Ras proteins localization, cell survival, level of ROS and percentage of apoptotic cells are congruent and suggest that the antiapoptotic role of Snf1 is independent on its kinase activity.

Antagonism between salicylate and the cAMP signal controls yeast cell survival and growth recovery from quiescence.

Aspirin and its main metabolite salicylate are promising molecules in preventing cancer and metabolic diseases. S. cerevisiae cells have been used to study some of their effects: (i) salicylate induces the reversible inhibition of both glucose transport and the biosyntheses of glucose-derived sugar phosphates, (ii) Aspirin/salicylate causes apoptosis associated with superoxide radical accumulation or early cell necrosis in MnSOD-deficient cells growing in ethanol or in glucose, respectively. So, treatment with (acetyl)-salicylic acid can alter the yeast metabolism and is associated with cell death. We describe here the dramatic effects of salicylate on cellular control of the exit from a quiescence state. The growth recovery of long-term stationary phase cells was strongly inhibited in the presence of salicylate, to a degree proportional to the drug concentration. At high salicylate concentration, growth reactivation was completely repressed and associated with a dramatic loss of cell viability. Strikingly, both of these phenotypes were fully suppressed by increasing the cAMP signal without any variation of the exponential growth rate. Upon nutrient exhaustion, salicylate induced a premature lethal cell cycle arrest in the budded-G2/M phase that cannot be suppressed by PKA activation. We discuss how the dramatic antagonism between cAMP and salicylate could be conserved and impinge common targets in yeast and humans. Targeting quiescence of cancer cells with stem-like properties and their growth recovery from dormancy are major challenges in cancer therapy. If mechanisms underlying cAMP-salicylate antagonism will be defined in our model, this might have significant therapeutic implications.

Dynamic of VE-cadherin-mediated spermatid-Sertoli cell contacts in the mouse seminiferous epithelium.

Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid-Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity.

Guidelines and recommendations on yeast cell death nomenclature.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.

Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes.

In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour).

Involvement of Aif1 in apoptosis triggered by lack of Hxk2 in the yeast Saccharomyces cerevisiae.

We recently showed that in hxk2Δ cells, showing constitutive localization of active Ras at the mitochondria, addition of acetic acid caused an increase of both apoptotic and necrotic cells compared with the wild-type strain, providing a new role for hexokinase 2 (EC 2.7.1.1) as an anti-apoptotic factor, besides its known role as a glycolytic enzyme and as a regulator of gene transcription of several Mig1-regulated genes. We also demonstrated that apoptosis induced by lack of Hxk2 may not require the activation of Yca1. Here, we show that deletion of HXK2 causes hypersensitivity to H2O2 and that addition of this well-known apoptotic stimulus to hxk2Δ cells causes an increase in the level ROS, apoptosis and mitochondrial membrane potential. We also show that deletion of AIF1 in hxk2Δ cells enhances survival after induction of apoptosis with both H2O2 and acetic acid, rescues the reduction of both growth rate and cell size, abrogates both H2O2 and acetic acid-induced ROS accumulation and decreases cell death, suggesting that Aif1 might be involved in both H2O2 and acetic acid-induced cell death in hxk2Δ cells. Moreover, we show that active Ras proteins relocalize to the plasma membrane and to the nucleus in hxk2Δ aif1Δ cells.

The transcription factor Swi4 is target for PKA regulation of cell size at the G1 to S transition in Saccharomyces cerevisiae.

To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G1 phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G1 arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4(S159A)) and we found that expression of Swi4(S159A) protein in the GG104-Swi4Δ strain did not restore the transient G1 arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G1/S phase transition.

Peptide-nanoparticle ligation mediated by cutinase fusion for the development of cancer cell-targeted nanoconjugates.

The relationship between the positioning of ligands on the surface of nanoparticles and the structural features of nanoconjugates has been underestimated for a long time, albeit of primary importance to promote specific biological recognition at the nanoscale. In particular, it has been formerly observed that a proper molecular orientation can play a crucial role, first optimizing ligand immobilization onto the nanoparticles and, second, improving the targeting efficiency of the nanoconjugates. In this work, we present a novel strategy to afford peptide-oriented ligation using genetically modified cutinase fusion proteins, which combines the presence of a site-directed "capture" module based on an enzymatic unit and a "targeting" moiety consisting of the ligand terminal end of a genetically encoded polypeptide chain. As an example, the oriented presentation of U11 peptide, a sequence specific for the recognition of urokinase plasminogen activator receptor (uPAR), was achieved by enzyme-mediated conjugation with an irreversible inhibitor of cutinase, an alkylphosphonate p-nitrophenol ester linker, covalently bound to the surface of iron oxide nanoparticles. The targeting efficiency of the resulting protein-nanoparticle conjugates was assessed using uPAR-positive breast cancer cells exploiting confocal laser scanning microscopy and quantitative fluorescence analysis of confocal images. Ultrastructural analysis of transmission electron micrographs provided evidence of a receptor-mediated pathway of endocytosis. Our results showed that, despite the small average number of targeting peptides presented on the nanoparticles, our ligand-oriented nanoconjugates proved to be very effective in selectively binding to uPAR and in promoting the uptake in uPAR-positive cancer cells.

Monitoring yeast intracellular Ca2+ levels using an in vivo bioluminescence assay.

This protocol describes the use of the jellyfish Aequorea victoria aequorin protein to measure Ca(2+) levels in living yeast cells. All yeast strains to be analyzed must express the A. victoria apoprotein of the aequorin calcium biosensor, to be reconstituted into fully active aequorin by association with its cofactor, coelenterazine, which cannot be synthesized by yeast itself. The simplest way to achieve reconstitution is to transform yeast cells with a vector driving apoaequorin expression, and then supply commercially available coelenterazine cofactor in the medium. Coelenterazine is a hydrophobic molecule and is able to permeate yeast cells.

Total cellular Ca2+ measurements in yeast using flame photometry.

A photoelectric flame photometer is a device used in inorganic chemical analysis for determining the concentrations of certain metals in solution. It does this by measuring the intensity of the light emitted by the metal when the solution is sprayed under controlled conditions into a nonluminous flame. This protocol describes how to measure total cellular calcium (maximal emission at 622 nm, orange flame) in yeast using this technique.

Measurement of calcium uptake in yeast using 45Ca.

This protocol describes how to measure calcium uptake in yeast by (45)Ca radioactive isotope incorporation.

The deubiquitinating enzyme UBPy/USP8 interacts with TrkA and inhibits neuronal differentiation in PC12 cells.

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPy(C748A) caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

Evidence for adenylate cyclase as a scaffold protein for Ras2-Ira interaction in Saccharomyces cerevisie.

Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.

Yeast as a model for Ras signalling.

For centuries yeast species have been popular hosts for classical biotechnology processes, such as baking, brewing, and wine making, and more recently for recombinant proteins production, thanks to the advantages of unicellular organisms (i.e., ease of genetic manipulation and rapid growth) together with the ability to perform eukaryotic posttranslational modifications. Moreover, yeast cells have been used for few decades as a tool for identifying the genes and pathways involved in basic cellular processes such as the cell cycle, aging, and stress response. In the budding yeast S. cerevisiae the Ras/cAMP/PKA pathway is directly involved in the regulation of metabolism, cell growth, stress resistance, and proliferation in response to the availability of nutrients and in the adaptation to glucose, controlling cytosolic cAMP levels and consequently the cAMP-dependent protein kinase (PKA) activity. Moreover, Ras signalling has been identified in several pathogenic yeasts as a key controller for virulence, due to its involvement in yeast morphogenesis. Nowadays, yeasts are still useful for Ras-like proteins investigation, both as model organisms and as a test tube to study variants of heterologous Ras-like proteins.

Methods to study the Ras2 protein activation state and the subcellular localization of Ras-GTP in Saccharomyces cerevisiae.

Ras proteins were highly conserved during evolution. They function as a point of convergence for different signalling pathways in eukaryotes and are involved in a wide range of cellular responses (shift from gluconeogenic to fermentative growth, breakdown of storage carbohydrates, stress resistance, growth control and determination of life span, morphogenesis and development, and others). These proteins are members of the small GTPase superfamily, which are active in the GTP-bound form and inactive in the GDP-bound form. Given the importance of studies on the Ras protein activation state to understand the detailed mechanism of Ras-mediated signal transduction, we provide here a simple, sensitive, and reliable method, based on the high affinity interaction of Ras-GTP with the Ras binding domain (RBD) of Raf1, to measure the level of Ras2-GTP on total Ras2 in Saccharomyces cerevisiae. Moreover, to study the localization of Ras-GTP in vivo in single S. cerevisiae cells, we expressed a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3), which was proven to be a useful live-cell biosensor for Ras-GTP in mammalian cells.