Calnexin Is Essential for Survival under Nitrogen Starvation and Stationary Phase in Schizosaccharomyces pombe

Cell fate is determined by the balance of conserved molecular mechanisms regulating death (apoptosis) and survival (autophagy). Autophagy is a process by which cells recycle their organelles and macromolecules through degradation within the vacuole in yeast and plants, and lysosome in metazoa. In the yeast Schizosaccharomyces pombe, autophagy is strongly induced under nitrogen starvation and in aging cells. Previously, we demonstrated that calnexin (Cnx1p), a highly conserved transmembrane chaperone of the endoplasmic reticulum (ER), regulates apoptosis under ER stress or inositol starvation. Moreover, we showed that in stationary phase, Cnx1p is cleaved into two moieties, L_Cnx1p and S_Cnx1p. Here, we show that the processing of Cnx1p is regulated by autophagy, induced by nitrogen starvation or cell aging. The cleavage of Cnx1p involves two vacuolar proteases: Isp6, which is essential for autophagy, and its paralogue Psp3. Blocking autophagy through the knockout of autophagy-related genes (atg) results in inhibition of both, the cleavage and the trafficking of Cnx1p from the ER to the vacuole. We demonstrate that Cnx1p is required for cell survival under nitrogen-starvation and in chronological aging cultures. The death of the mini_cnx1 mutant (overlapping S_cnx1p) cells is accompanied by accumulation of high levels of reactive-oxygen species (ROS), a slowdown in endocytosis and severe cell-wall defects. Moreover, mutant cells expressing only S_Cnx1p showed cell wall defects. Co-expressing mutant overlapping the L_Cnx1p and S_Cnx1p cleavage products reverses the death, ROS phenotype and cell wall defect to wild-type levels. As it is involved in both apoptosis and autophagy, Cnx1p could be a nexus for the crosstalk between these pro-death and pro-survival mechanisms. Ours, and observations in mammalian systems, suggest that the multiple roles of calnexin depend on its sub-cellular localization and on its cleavage. The use of S. pombe should assist in further shedding light on the multiple roles of calnexin.     

Ssd1 is required for thermotolerance and Hsp104-mediated protein disaggregation in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Hsp104-mediated disaggregation of protein aggregates is essential for thermotolerance and to facilitate the maintenance of prions. In humans, protein aggregation is associated with neuronal death and dysfunction in many neurodegenerative diseases. Mechanisms of aggregation surveillance that regulate protein disaggregation are likely to play a major role in cell survival after acute stress. However, such mechanisms have not been studied. In a screen using the yeast gene deletion library for mutants unable to survive an aggregation-inducing heat stress, we find that SSD1 is required for Hsp104-mediated protein disaggregation. SSD1 is a polymorphic gene that plays a role in cellular integrity, longevity, and pathogenicity in yeast. Allelic variants of SSD1 regulate the level of thermotolerance and cell wall remodeling. We have shown that Ssd1 influences the ability of Hsp104 to hexamerize, to interact with the cochaperone Sti1, and to bind protein aggregates. These results provide a paradigm for linking Ssd1-mediated cellular integrity and Hsp104-mediated disaggregation to ensure the survival of cells with fewer aggregates.     

Survival Kinetics of Starving Bacteria Is Biphasic and Density-Dependent

In the lifecycle of microorganisms, prolonged starvation is prevalent and sustaining life during starvation periods is a vital task. In the literature, it is commonly assumed that survival kinetics of starving microbes follows exponential decay. This assumption, however, has not been rigorously tested. Currently, it is not clear under what circumstances this assumption is true. Also, it is not known when such survival kinetics deviates from exponential decay and if it deviates, what underlying mechanisms for the deviation are. Here, to address these issues, we quantitatively characterized dynamics of survival and death of starving E. coli cells. The results show that the assumption – starving cells die exponentially – is true only at high cell density. At low density, starving cells persevere for extended periods of time, before dying rapidly exponentially. Detailed analyses show intriguing quantitative characteristics of the density-dependent and biphasic survival kinetics, including that the period of the perseverance is inversely proportional to cell density. These characteristics further lead us to identification of key underlying processes relevant for the perseverance of starving cells. Then, using mathematical modeling, we show how these processes contribute to the density-dependent and biphasic survival kinetics observed. Importantly, our model reveals a thrifty strategy employed by bacteria, by which upon sensing impending depletion of a substrate, the limiting substrate is conserved and utilized later during starvation to delay cell death. These findings advance quantitative understanding of survival of microbes in oligotrophic environments and facilitate quantitative analysis and prediction of microbial dynamics in nature. Furthermore, they prompt revision of previous models used to analyze and predict population dynamics of microbes.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.     

Induction of Hsp104 synthesis in Saccharomyces cerevisiae in the stationary growth phase is inhibited by the petite mutation

The elevation of Hsp104 (heat shock protein) content under heat stress plays a key role in the development of thermotolerance in yeast (Saccharomyces cerevisiae) cells. Hsp104 synthesis is increased under heat stress and in the stationary growth phase. The loss of mitochondrial DNA (petite mutation) was shown to inhibit the induction of Hsp104 synthesis under heat stress (39°C) and during the transition to the stationary growth phase. Also, the petite mutation suppressed the increase in activity of antioxidant enzymes in the stationary phase, which accompanied by decrease in thermotolerance. At the same time, mutation inhibited production of reactive oxygen species and prevented cell death under heat shock in the logarithmic growth phase. The results of this study suggest that disruption of the mitochondrial functional state suppresses the expression of yeast nuclear genes upon upon entry into the stationary growth phase.     

Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi

The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2)mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087), was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-α3 and Smr) with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2)mbn cells (p<0.05) following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival.     

Gadd45�� activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens.     

Testing for a Gap Junction-Mediated Bystander Effect in Retinitis Pigmentosa: Secondary Cone Death Is Not Altered by Deletion of Connexin36 from Cones

Retinitis pigmentosa (RP) relates to a group of hereditary neurodegenerative diseases of the retina. On the cellular level, RP results in the primary death of rod photoreceptors, caused by rod-specific mutations, followed by a secondary degeneration of genetically normal cones. Different mechanisms may influence the spread of cell death from one photoreceptor type to the other. As one of these mechanisms a gap junction-mediated bystander effect was proposed, i.e., toxic molecules generated in dying rods and propagating through gap junctions induce the death of healthy cone photoreceptors. We investigated whether disruption of rod-cone coupling can prevent secondary cone death and reduce the spread of degeneration. We tested this hypothesis in two different mouse models for retinal degeneration (rhodopsin knockout and rd1) by crossbreeding them with connexin36-deficient mice as connexin36 represents the gap junction protein on the cone side and lack thereof most likely disrupts rod-cone coupling. Using immunohistochemistry, we compared the progress of cone degeneration between connexin36-deficient mouse mutants and their connexin36-expressing littermates at different ages and assessed the accompanied morphological changes during the onset (rhodopsin knockout) and later stages of secondary cone death (rd1 mutants). Connexin36-deficient mouse mutants showed the same time course of cone degeneration and the same morphological changes in second order neurons as their connexin36-expressing littermates. Thus, our results indicate that disruption of connexin36-mediated rod-cone coupling does not stop, delay or spatially restrict secondary cone degeneration and suggest that the gap junction-mediated bystander effect does not contribute to the progression of RP.     

Extracellular methylglyoxal toxicity in Saccharomyces cerevisiae: role of glucose and phosphate ions

Aim: The purpose of this study was to investigate the behaviour of Saccharomyces cerevisiae in response to extracellular methylglyoxal. Methods and Results: Cell survival to methylglyoxal and the importance of phosphates was investigated. The role of methylglyoxal detoxification systems and methylglyoxal‐derived protein glycation were studied and the relation to cell survival or death was evaluated. Extracellular methylglyoxal decreased cell viability, and the presence of phosphate enhanced this effect. d ‐glucose seems to exert a protective effect towards this toxicity. Methylglyoxal‐induced cell death was not apoptotic and was not related to intracellular glycation processes. The glyoxalases and aldose reductase were important in methylglyoxal detoxification. Mutants lacking glyoxalase I and II showed increased sensitivity to methylglyoxal, while strains overexpressing these genes had increased resistance. Conclusions: Extracellular methylglyoxal induced non‐apoptotic cell death, being unrelated to glycation. Inactivation of methylglyoxal‐detoxifying enzymes by phosphate is one probable cause. Phosphate and d ‐glucose may also act through their complex involvement in stress response mechanisms. Significance and Impact of the Study: These findings contribute to elucidate the mechanisms of cell toxicity by methylglyoxal. This information could be useful to on‐going studies using yeast as a eukaryotic cell model to investigate methylglyoxal‐derived glycation and its role in neurodegenerative diseases.     

Mitochondrial involvement in cell death of non-mammalian eukaryotes

Although mitochondria are essential organelles for long-term survival of eukaryotic cells, recent discoveries in biochemistry and genetics have advanced our understanding of the requirements for mitochondria in cell death. Much of what we understand about cell death is based on the identification of conserved cell death genes in Drosophila melanogaster and Caenorhabditis elegans. However, the role of mitochondria in cell death in these models has been much less clear. Considering the active role that mitochondria play in apoptosis in mammalian cells, the mitochondrial contribution to cell death in non-mammalian systems has been an area of active investigation. In this article, we review the current research on this topic in three non-mammalian models, C. elegans, Drosophila, and Saccharomyces cerevisiae. In addition, we discuss how non-mammalian models have provided important insight into the mechanisms of human disease as they relate to the mitochondrial pathway of cell death. The unique perspective derived from each of these model systems provides a more complete understanding of mitochondria in programmed cell death. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Genome-wide analysis of yeast stress survival and tolerance acquisition to analyze the central trade-off between growth rate and cellular robustness

All organisms have evolved to cope with changes in environmental conditions, ensuring the optimal combination of proliferation and survival. In yeast, exposure to a mild stress leads to an increased tolerance for other stresses. This suggests that yeast uses information from the environment to prepare for future threats. We used the yeast knockout collection to systematically investigate the genes and functions involved in severe stress survival and in the acquisition of stress (cross-) tolerance. Besides genes and functions relevant for survival of heat, acid, and oxidative stress, we found an inverse correlation between mutant growth rate and stress survival. Using chemostat cultures, we confirmed that growth rate governs stress tolerance, with higher growth efficiency at low growth rates liberating the energy for these investments. Cellular functions required for stress tolerance acquisition, independent of the reduction in growth rate, were involved in vesicular transport, the Rpd3 histone deacetylase complex, and the mitotic cell cycle. Stress resistance and acquired stress tolerance in Saccharomyces cerevisiae are governed by a combination of stress-specific and general processes. The reduction of growth rate, irrespective of the cause of this reduction, leads to redistribution of resources toward stress tolerance functions, thus preparing the cells for impending change.     

Identification of gene products that control lipid droplet size in yeast using a high-throughput quantitative image analysis

Lipid droplets (LDs) are important organelles involved in energy storage and expenditure. LD dynamics has been investigated using genome-wide image screening methods in yeast and other model organisms. For most studies, genes were identified using two-dimensional images with LD enlargement as readout. Due to imaging limitation, reduction of LD size is seldom explored. Here, we aim to set up a screen that specifically utilizes reduced LD size as the readout. To achieve this, a novel yeast screen is set up to quantitatively and systematically identify genes that regulate LD size through a three-dimensional imaging-based approach. Cidea which promotes LD fusion and growth in mammalian cells was overexpressed in a yeast knockout library to induce large LD formation. Next, an automated, high-throughput image analysis method that monitors LD size was utilized. With this screen, we identified twelve genes that reduced LD size when deleted. The effects of eight of these genes on LD size were further validated in fld1 null strain background. In addition, six genes were previously identified as LD-regulating genes. To conclude, this methodology represents a promising strategy to screen for players in LD size control in both yeast and mammalian cells to aid in the investigation of LD-associated metabolic diseases.     

High-throughput knockout screen in fission yeast

We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.     

Identification of the essential Brucella melitensis porin Omp2b as a suppressor of Bax-induced cell death in yeast in a genome-wide screening

Background

Inhibition of apoptosis is one of the mechanisms selected by numerous intracellular pathogenic bacteria to control their host cell. Brucellae, which are the causative agent of a worldwide zoonosis, prevent apoptosis of infected cells, probably to support survival of their replication niche.

Methodology/Principal Findings

In order to identify Brucella melitensis anti-apoptotic effector candidates, we performed a genome-wide functional screening in yeast. The B. melitensis ORFeome was screened to identify inhibitors of Bax-induced cell death in S. cerevisiae. B. melitensis porin Omp2b, here shown to be essential, prevents Bax lethal effect in yeast, unlike its close paralog Omp2a. Our results based on Omp2b size variants characterization suggest that signal peptide processing is required for Omp2b effect in yeast.

Conclusion/Significance

We report here the first application to a bacterial genome-wide library of coding sequences of this “yeast-rescue” screening strategy, previously used to highlight several new apoptosis regulators. Our work provides B. melitensis proteins that are candidates for an anti-apoptotic function, and can be tested in mammalian cells in the future. Hypotheses on possible molecular mechanisms of Bax inhibition by the B. melitensis porin Omp2b are discussed.     

Functional genomic analysis of apoptotic DNA degradation in C. elegans

Chromosomal DNA degradation is critical for cell death execution and is a hallmark of apoptosis, yet little is known about how this process is executed. Using an RNAi-based functional genomic approach, we have identified seven additional cell death-related nucleases (crn genes), which along with two known nucleases (CPS-6 and NUC-1) comprise at least two independent pathways that contribute to cell killing, and likely signaling for phagocytosis, by degrading chromosomal DNA. Several crn genes have human homologs that are important for RNA processing, protein folding, DNA replication, and DNA damage repair, suggesting dual roles for CRN nucleases in cell survival and cell death. It should now be possible to systematically decipher the mechanisms of apoptotic DNA degradation.     

Arginylation-Dependent Neural Crest Cell Migration Is Essential for Mouse Development

Coordinated cell migration during development is crucial for morphogenesis and largely relies on cells of the neural crest lineage that migrate over long distances to give rise to organs and tissues throughout the body. Recent studies of protein arginylation implicated this poorly understood posttranslational modification in the functioning of actin cytoskeleton and in cell migration in culture. Knockout of arginyltransferase (Ate1) in mice leads to embryonic lethality and severe heart defects that are reminiscent of cell migration–dependent phenotypes seen in other mouse models. To test the hypothesis that arginylation regulates cell migration during morphogenesis, we produced Wnt1-Cre Ate1 conditional knockout mice (Wnt1-Ate1), with Ate1 deletion in the neural crest cells driven by Wnt1 promoter. Wnt1-Ate1 mice die at birth and in the first 2–3 weeks after birth with severe breathing problems and with growth and behavioral retardation. Wnt1-Ate1 pups have prominent defects, including short palate and altered opening to the nasopharynx, and cranial defects that likely contribute to the abnormal breathing and early death. Analysis of neural crest cell movement patterns in situ and cell motility in culture shows an overall delay in the migration of Ate1 knockout cells that is likely regulated by intracellular mechanisms rather than extracellular signaling events. Taken together, our data suggest that arginylation plays a general role in the migration of the neural crest cells in development by regulating the molecular machinery that underlies cell migration through tissues and organs during morphogenesis.