RESUMEN
Yeast Rho5 is a small GTPase which mediates the response to nutrient and oxidative stress, and triggers mitophagy and apoptosis. We here studied the rapid translocation of a GFP-tagged Rho5 to mitochondria under such stress conditions by live-cell fluorescence microscopy in the background of strains lacking different mitochondrial outer membrane proteins (MOMP). Fun14, Msp1 and Alo1 were found to be required for efficient recruitment of the GTPase, whereas translocation of Dck1 and Lmo1, the subunits of its dimeric GDP/GTP exchange factor (GEF), remained unaffected. An influence of the voltage-dependent anion channel (VDAC) Por1 on the association of GFP-Rho5 with mitochondria under oxidative stress conditions appeared to be strain-dependent. However, epistasis analyses and bimolecular fluorescence complementation (BiFC) studies indicate a genetic and physical interaction. All four strains lacking a single MOMP were investigated for their effect on mitophagy.
Asunto(s)
Membranas Mitocondriales , Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Unión al GTP rho , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Membranas Mitocondriales/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genética , Transporte de Proteínas , Canales Aniónicos Dependientes del Voltaje/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mitofagia , PorinasRESUMEN
Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.
Asunto(s)
Hanseniaspora , Vino , Humanos , Fermentación , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Hanseniaspora/enzimología , Homocigoto , Eliminación de SecuenciaRESUMEN
Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.
Asunto(s)
Hanseniaspora , Vino , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Hanseniaspora/genética , Reacción en Cadena de la PolimerasaRESUMEN
In this work, the effect of processing conditions created with common meat technology equipment, on the spatial distribution of a green fluorescent protein producing -Escherichia coli in sausage meat was evaluated using confocal fluorescence microscopy and expressed with the help of the dispersion index. The results indicated that the reduction in mean particle size by prolonged comminution improved the distribution of cells in the sausage meat. Furthermore, higher fat content seemed to favor a random distribution, although not significantly. Independent of the any variation of the sausage meat production parameters, Listeria monocytogenes was effectively controlled in fermented sausages, although a theoretically less homogenous distribution of the starter culture in the sausage meat, tended to improve the effect, however, insignificantly. An early onset of the quorum-sensing-driven bacteriocin production in poorly distributed larger colonies may have been the reason for this. No differences in the composition of the microbiome between sausages with poor and good distribution of the starter culture were observed.
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Microbiología de Alimentos , Productos de la Carne , Fermentación , Productos de la Carne/microbiología , Carne , Escherichia coliRESUMEN
The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.
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Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Kluyveromyces/enzimología , Kluyveromyces/genética , Metabolismo Energético , Activación Enzimática , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Prueba de Complementación Genética , Glucólisis , Humanos , Redes y Vías Metabólicas , Estrés Oxidativo , Vía de Pentosa Fosfato , FenotipoRESUMEN
Billionaire philanthropists claim to play a key role in advancing well-being and public goods across the world. One of the most prominent recent expressions of these efforts is the Giving Pledge, created in 2010 by Bill and Melinda Gates in collaboration with Warren Buffett. After a decade of its existence, this analysis of the Giving Pledge population and its commitment letters shows an overall dominance of white, male, and US-based billionaires among the signatories. Tech billionaires are a wealthier and younger subgroup of pledgers than their counterparts in other industries. The pledge letters reveal an emphasis on education and health as dominant philanthropic causes. Among explanations for giving, the four most frequent reasons mentioned are a desire to make a difference, a wish to give back, a sense of personal fulfillment resulting from giving, and references to being socialized into philanthropic giving early in life. While the Giving Pledge is the most prominent global effort to increase philanthropic giving among the wealthy, the voluntary nature and relatively modest commitment goal make it difficult to assess its significance and impact.
RESUMEN
A wealth of data is accumulating on the physiological functions of human Rac1, a member of the Rho GTPase family of molecular switches and substrate of botulinum toxin, which was first identified as a regulator of cell motility through its effect on the actin cytoskeleton. Later on, it was found to be involved in different diseases like cancers, cardiac function, neuronal disorders, and apoptotic cell death. Despite the presence of Rac1 homologues in most fungi investigated so far, including Rho5 in the genetically tractable model yeast Saccharomyces cerevisiae, knowledge on their physiological functions is still scarce, let alone the details of the molecular mechanisms of their actions and interactions. Nevertheless, all functions proposed for human Rac1 seem to be conserved in one or the other fungus. This includes the regulation of MAPK cascades, polarized growth, and actin dynamics. Moreover, both the production and response to reactive oxygen species, as well as the reaction to nutrient availability, can be affected. We here summarize the studies performed on fungal Rac1 homologues, with a special focus on S. cerevisiae Rho5, which may be of use in drug development in medicine and agriculture.
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Proteínas Fúngicas/metabolismo , Hongos/citología , Hongos/fisiología , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Apoptosis , Autofagia , Pared Celular/metabolismo , Senescencia Celular , Citoesqueleto/metabolismo , Metabolismo Energético , Humanos , Concentración Osmolar , Estrés Oxidativo , Unión Proteica , Isoformas de Proteínas , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification, a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid-specific insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.
Asunto(s)
Estrés Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Unión al GTP rho/metabolismo , Prueba de Complementación Genética , Humanos , Dominios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/genéticaRESUMEN
The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 µm and a diameter of 1 µm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure.
Asunto(s)
Proteínas Fúngicas/metabolismo , Saccharomycetales/metabolismo , Cuerpos Polares del Huso/metabolismo , Esporas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Modelos Biológicos , Mutación , Fenotipo , Saccharomycetales/genética , Esporas Fúngicas/genéticaRESUMEN
Rho5 is a small GTPase of Saccharomyces cerevisiae and a homolog of mammalian Rac1. The latter regulates glucose metabolism and actin cytoskeleton dynamics, and its misregulation causes cancer and a variety of other diseases. In yeast, Rho5 has been implicated in different signal transduction pathways, governing cell wall integrity and the responses to high medium osmolarity and oxidative stress. It has also been proposed to affect mitophagy and apoptosis. Here, we demonstrate that Rho5 rapidly relocates from the plasma membrane to mitochondria upon glucose starvation, mediated by its dimeric GDP/GTP exchange factor (GEF) Dck1/Lmo1. A function in response to glucose availability is also suggested by synthetic genetic phenotypes of a rho5 deletion with gpr1, gpa2, and sch9 null mutants. On the other hand, the role of mammalian Rac1 in regulating the action cytoskeleton does not seem to be strongly conserved in S. cerevisiae Rho5. We propose that Rho5 serves as a central hub in integrating various stress conditions, including a crosstalk with the cAMP/PKA (cyclic AMP activating protein kinase A) and Sch9 branches of glucose signaling pathways.
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Glucosa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular/análisis , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Estrés Fisiológico , Proteínas de Unión al GTP rho/análisis , Proteínas de Unión al GTP rho/genéticaRESUMEN
Chitin is mainly formed by the chitin synthase III complex (CSIII) in yeast cells. This complex is considered to be composed of the catalytic subunit Chs3 and the regulatory subunit Chs4, both of which are phosphoproteins and transported to the plasma membrane by different trafficking routes. During cytokinesis, Chs3 associates with Chs4 and other proteins at the septin ring, which results in an active CSIII complex. In this study, we focused on the role of Chs4 as a regulatory subunit of the CSIII complex. We analyzed the dynamic localization and interaction of Chs3 and Chs4 during cell division, and found that both proteins transiently co-localize and physically interact only during bud formation and later in a period during septum formation and cytokinesis. To identify unknown binding partners of Chs4, we conducted different screening approaches, which yielded several novel candidates of Chs4-binding proteins including the septin-associated kinase Gin4. Our further studies confirmed this interaction and provided first evidence that Chs4 phosphorylation is partially dependent on Gin4, which is required for proper localization of Chs4 at the bud neck.
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Quitina Sintasa/genética , Quinasas Ciclina-Dependientes/genética , Proteínas de Saccharomyces cerevisiae/genética , División Celular/genética , Citocinesis/genética , Fosforilación , Saccharomyces cerevisiae/genética , Septinas/genéticaRESUMEN
The NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGpd1) is an important enzyme for maintenance of the cytosolic redox balance in the milk yeast Kluyveromyces lactis. The enzyme is localized in peroxisomes and in the cytosol, indicating its requirement for the oxidation of NADH in both compartments. Klgpd1 mutants grow more slowly on glucose than wild-type cells and do not grow on ethanol as a sole carbon source. We studied the molecular basis of the latter phenotype and found that Gpd1 is required for high expression of KlICL1 and KlMLS1 which encode the key enzymes of the glyoxylate pathway isocitrate lyase and malate synthase, respectively. This regulation is mediated by CSRE elements in the promoters of these genes and the Snf1-regulated transcription factors KlCat8 and KlSip4. To study the transactivation function of these factors we developed a modified yeast one-hybrid system for K. lactis, using the endogenous ß-galactosidase gene LAC4 as a reporter in a lac9 deletion background. In combination with ChIP analyses we discovered that Gpd1 controls both the specific binding of Cat8 and Sip4 to the target promoters and the capacity of these factors to activate the reporter gene expression. We propose a model in which KlGpd1 activity is required for maintenance of the redox balance. In its absence, genes which function in generating redox balance instabilities are not expressed. A comparison of mutant phenotypes further indicates, that this system not only operates in K. lactis, but also in Saccharomyces cerevisiae.
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Etanol/metabolismo , Proteínas Fúngicas/metabolismo , Glicerol-3-Fosfato Deshidrogenasa (NAD+)/metabolismo , Kluyveromyces/enzimología , Factores de Transcripción/antagonistas & inhibidores , Regulación Fúngica de la Expresión Génica , Glioxilatos/metabolismo , Isocitratoliasa/metabolismo , Kluyveromyces/genética , Regiones Promotoras Genéticas , Factores de Transcripción/fisiologíaRESUMEN
Hanseniaspora uvarum (anamorph Kloeckera apiculata) is a predominant yeast on wine grapes and other fruits and has a strong influence on wine quality, even when Saccharomyces cerevisiae starter cultures are employed. In this work, we sequenced and annotated approximately 93% of the H. uvarum genome. Southern and synteny analyses were employed to construct a map of the seven chromosomes present in a type strain. Comparative determinations of specific enzyme activities within the fermentative pathway in H. uvarum and S. cerevisiae indicated that the reduced capacity of the former yeast for ethanol production is caused primarily by an â¼10-fold-lower activity of the key glycolytic enzyme pyruvate kinase. The heterologous expression of the encoding gene, H. uvarumPYK1 (HuPYK1), and two genes encoding the phosphofructokinase subunits, HuPFK1 and HuPFK2, in the respective deletion mutants of S. cerevisiae confirmed their functional homology.IMPORTANCEHanseniaspora uvarum is a predominant yeast species on grapes and other fruits. It contributes significantly to the production of desired as well as unfavorable aroma compounds and thus determines the quality of the final product, especially wine. Despite this obvious importance, knowledge on its genetics is scarce. As a basis for targeted metabolic modifications, here we provide the results of a genomic sequencing approach, including the annotation of 3,010 protein-encoding genes, e.g., those encoding the entire sugar fermentation pathway, key components of stress response signaling pathways, and enzymes catalyzing the production of aroma compounds. Comparative analyses suggest that the low fermentative capacity of H. uvarum compared to that of Saccharomyces cerevisiae can be attributed to low pyruvate kinase activity. The data reported here are expected to aid in establishing H. uvarum as a non-Saccharomyces yeast in starter cultures for wine and cider fermentations.
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Etanol/metabolismo , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Hanseniaspora/genética , Hanseniaspora/metabolismo , Piruvato Quinasa/metabolismo , Vitis/microbiología , Fermentación , Proteínas Fúngicas/genética , Glucólisis , Hanseniaspora/enzimología , Piruvato Quinasa/genéticaRESUMEN
Global health issues vary in the amount of attention and resources they receive. One reason is that the networks of individuals and organizations that address these issues differ in their effectiveness. This article presents key findings from a research project on the emergence and effectiveness of global health networks addressing tobacco use, alcohol harm, maternal mortality, neonatal mortality, tuberculosis and pneumonia. Although networks are only one of many factors influencing priority, they do matter, particularly for shaping the way the problem and solutions are understood, and convincing governments, international organizations and other global actors to address the issue. Their national-level effects vary by issue and are more difficult to ascertain. Networks are most likely to produce effects when (1) their members construct a compelling framing of the issue, one that includes a shared understanding of the problem, a consensus on solutions and convincing reasons to act and (2) they build a political coalition that includes individuals and organizations beyond their traditional base in the health sector, a task that demands engagement in the politics of the issue, not just its technical aspects. Maintaining a focused frame and sustaining a broad coalition are often in tension: effective networks find ways to balance the two challenges. The emergence and effectiveness of a network are shaped both by its members' decisions and by contextual factors, including historical influences (e.g. prior failed attempts to address the problem), features of the policy environment (e.g. global development goals) and characteristics of the issue the network addresses (e.g. its mortality burden). Their proliferation raises the issue of their legitimacy. Reasons to consider them legitimate include their members' expertise and the attention they bring to neglected issues. Reasons to question their legitimacy include their largely elite composition and the fragmentation they bring to global health governance.
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Redes Comunitarias , Eficiencia Organizacional , Salud Global , Investigación , Política de SaludRESUMEN
Smoking and drinking constitute two risk factors contributing to the rising burden of non-communicable diseases in low- and middle-income countries. Both issues have gained increased international attention, but tobacco control has made more sustained progress in terms of international and domestic policy commitments, resources dedicated to reducing harm, and reduction of tobacco use in many high-income countries. The research presented here offers insights into why risk factors with comparable levels of harm experience different trajectories of global attention. The analysis focuses particular attention on the role of dedicated global health networks composed of individuals and organizations producing research and engaging in advocacy on a given health problem. Variation in issue characteristics and the policy environment shape the opportunities and challenges of global health networks focused on reducing the burden of disease. What sets the tobacco case apart was the ability of tobacco control advocates to create and maintain a consensus on policy solutions, expand their reach in low- and middle-income countries and combine evidence-based research with advocacy reaching beyond the public health-centered focus of the core network. In contrast, a similar network in the alcohol case struggled with expanding its reach and has yet to overcome divisions based on competing problem definitions and solutions to alcohol harm. The tobacco control network evolved from a group of dedicated individuals to a global coalition of membership-based organizations, whereas the alcohol control network remains at the stage of a collection of dedicated and like-minded individuals.
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Consumo de Bebidas Alcohólicas/prevención & control , Redes Comunitarias/organización & administración , Salud Global , Prevención del Hábito de Fumar , Humanos , Internacionalidad , Entrevistas como Asunto , Investigación CualitativaRESUMEN
Global efforts to address alcohol harm have significantly increased since the mid-1990 s. By 2010, the World Health Organization (WHO) had adopted the non-binding Global Strategy to Reduce the Harmful Use of Alcohol. This study investigates the role of a global health network, anchored by the Global Alcohol Policy Alliance (GAPA), which has used scientific evidence on harm and effective interventions to advocate for greater global public health efforts to reduce alcohol harm. The study uses process-tracing methodology and expert interviews to evaluate the accomplishments and limitations of this network. The study documents how network members have not only contributed to greater global awareness about alcohol harm, but also advanced a public health approach to addressing this issue at the global level. Although the current network represents an expanding global coalition of like-minded individuals, it faces considerable challenges in advancing its cause towards successful implementation of effective alcohol control policies across many low- and middle-income countries (LMICs). The analysis reveals a need to transform the network into a formal coalition of regional and national organizations that represent a broader variety of constituents, including the medical community, consumer groups and development-focused non-governmental organizations. Considering the growing harm of alcohol abuse in LMICs and the availability of proven and cost-effective public health interventions, alcohol control represents an excellent 'buy' for donors interested in addressing non-communicable diseases. Alcohol control has broad beneficial effects for human development, including promoting road safety and reducing domestic violence and health care costs across a wide variety of illnesses caused by alcohol consumption.
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Consumo de Bebidas Alcohólicas/prevención & control , Redes Comunitarias , Práctica Clínica Basada en la Evidencia , Salud Global , Política de Salud , InternacionalidadRESUMEN
Since 1990 mortality and morbidity decline has been more extensive for some conditions prevalent in low- and middle-income countries than for others. One reason may be differences in the effectiveness of global health networks, which have proliferated in recent years. Some may be more capable than others in attracting attention to a condition, in generating funding, in developing interventions and in convincing national governments to adopt policies. This article introduces a supplement on the emergence and effectiveness of global health networks. The supplement examines networks concerned with six global health problems: tuberculosis (TB), pneumonia, tobacco use, alcohol harm, maternal mortality and newborn deaths. This article presents a conceptual framework delineating factors that may shape why networks crystallize more easily surrounding some issues than others, and once formed, why some are better able than others to shape policy and public health outcomes. All supplement papers draw on this framework. The framework consists of 10 factors in three categories: (1) features of the networks and actors that comprise them, including leadership, governance arrangements, network composition and framing strategies; (2) conditions in the global policy environment, including potential allies and opponents, funding availability and global expectations concerning which issues should be prioritized; (3) and characteristics of the issue, including severity, tractability and affected groups. The article also explains the design of the project, which is grounded in comparison of networks surrounding three matched issues: TB and pneumonia, tobacco use and alcohol harm, and maternal and newborn survival. Despite similar burden and issue characteristics, there has been considerably greater policy traction for the first in each pair. The supplement articles aim to explain the role of networks in shaping these differences, and collectively represent the first comparative effort to understand the emergence and effectiveness of global health networks.
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Redes Comunitarias/organización & administración , Eficiencia Organizacional , Salud Global , Política de Salud , Estudios de Casos OrganizacionalesRESUMEN
The exact function and regulation of the small GTPase Rho5, a putative homolog of mammalian Rac1, in the yeast Saccharomyces cerevisiae have not yet been elucidated. In a genetic screen initially designed to identify novel regulators of cell wall integrity signaling, we identified the homologs of mammalian DOCK1 (Dck1) and ELMO (Lmo1) as upstream components which regulate Rho5. Deletion mutants in any of the encoding genes (DCK1, LMO1, RHO5) showed hyper-resistance to cell wall stress agents, demonstrating a function in cell wall integrity signaling. Live-cell fluorescence microscopy showed that Dck1, Lmo1 and Rho5 quickly relocate to mitochondria under oxidative stress and cell viability assays indicate a role of Dck1/Lmo1/Rho5 signaling in triggering cell death as a response to hydrogen peroxide treatment. A regulatory role in autophagy/mitophagy is suggested by the colocalization of Rho5 with autophagic markers and the decreased mitochondrial turnover observed in dck1, lmo1 and rho5 deletion mutants. Rho5 activation may thus serve as a central hub for the integration of different signaling pathways.
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Regulación Enzimológica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Regulación Fúngica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Estrés Oxidativo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
GTPases of the Rho family are important molecular switches that regulate many basic cellular processes. The function of the Rho2 and Rho5 proteins from Saccharomyces cerevisiae and of their homologs in other species is poorly understood. Here, we report on the analysis of the AgRho2 and AgRho5 proteins of the filamentous fungus Ashbya gossypii. In contrast to S. cerevisiae mutants of both encoding genes displayed a strong morphological phenotype. The Agrho2 mutants showed defects in tip-branching, while Agrho5 mutants had a significantly decreased growth rate and failed to maintain their growth axis. In addition, the Agrho5 mutants had highly defective actin rings at septation sites. We also found that a deletion mutant of a putative GDP-GTP-exchange factor (GEF) that was homologous to a Rac-GEF from other species phenocopied the Agrho5 mutant, suggesting that both proteins act in the same pathway, but the AgRho5 protein has acquired functions that are fulfilled by Rac-proteins in other species.
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Actinas/metabolismo , Ascomicetos/enzimología , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actinas/genética , Ascomicetos/genética , Proteínas Fúngicas/genética , Mutación , Proteínas de Unión al GTP rho/genéticaRESUMEN
Cytokinesis in yeast and mammalian cells is a highly coordinated process mediated by the constriction of an actomyosin ring. In yeasts, it is accompanied by the formation of a chitinous primary septum. Although much is known about the regulation of cytokinesis in budding yeast, overlapping functions of redundant genes complicates genetic analyses. Here, we investigated the effects of various deletion mutants on cytokinesis in the milk yeast Kluyveromyces lactis. To determine the spatiotemporal parameters of cytokinesis components, live-cell imaging of fluorophor-tagged KlMyo1 and a new Lifeact probe for KlAct1 was employed. In contrast to Saccharomyces cerevisiae, where deletion of ScMYO1 is lethal, Klmyo1 deletion was temperature-sensitive. Transmission and scanning electron microscopy demonstrated that the Klmyo1 deletion cells had a defect in the formation of the primary septum and in cell separation; this result was confirmed by FACS analyses. Deletion of KlCYK3 was lethal, whereas in S. cerevisiae a cyk3 deletion is synthetically lethal with hof1 deletion. Growth of Klhof1 mutants was osmoremedial at 25°C, as it is in S. cerevisiae. CYK3 and HOF1 genes cross-complemented in both species, suggesting that they are functional homologs. Inn1, a common interactor for these two regulators, was essential in both yeasts and the encoding genes did not cross-complement. The C2 domain of the Inn1 homologs conferred species specificity. Thus, our work establishes K. lactis as a model yeast to study cytokinesis with less genetic redundancy than S. cerevisiae. The viability of Klmyo1 deletions provides an advantage over budding yeast to study actomyosin-independent cytokinesis. Moreover, the lethality of Klcyk3 null mutants suggests that there are fewer functional redundancies with KlHof1 in K. lactis.