A cellular system for spatial signal decoding in chemical gradients.

Directional cell growth requires that cells read and interpret shallow chemical gradients, but how the gradient directional information is identified remains elusive. We use single-cell analysis and mathematical modeling to define the cellular gradient decoding network in yeast. Our results demonstrate that the spatial information of the gradient signal is read locally within the polarity site complex using double-positive feedback between the GTPase Cdc42 and trafficking of the receptor Ste2. Spatial decoding critically depends on low Cdc42 activity, which is maintained by the MAPK Fus3 through sequestration of the Cdc42 activator Cdc24. Deregulated Cdc42 or Ste2 trafficking prevents gradient decoding and leads to mis-oriented growth. Our work discovers how a conserved set of components assembles a network integrating signal intensity and directionality to decode the spatial information contained in chemical gradients.

PKA antagonizes CLASP-dependent microtubule stabilization to re-localize Pom1 and buffer cell size upon glucose limitation.

Cells couple growth with division and regulate size in response to nutrient availability. In rod-shaped fission yeast, cell-size control occurs at mitotic commitment. An important regulator is the DYRK-family kinase Pom1, which forms gradients from cell poles and inhibits the mitotic activator Cdr2, itself localized at the medial cortex. Where and when Pom1 modulates Cdr2 activity is unclear as Pom1 medial cortical levels remain constant during cell elongation. Here we show that Pom1 re-localizes to cell sides upon environmental glucose limitation, where it strongly delays mitosis. This re-localization is caused by severe microtubule destabilization upon glucose starvation, with microtubules undergoing catastrophe and depositing the Pom1 gradient nucleator Tea4 at cell sides. Microtubule destabilization requires PKA/Pka1 activity, which negatively regulates the microtubule rescue factor CLASP/Cls1/Peg1, reducing CLASP's ability to stabilize microtubules. Thus, PKA signalling tunes CLASP's activity to promote Pom1 cell side localization and buffer cell size upon glucose starvation.

Environmental control of the Pom1-dependent cell-size regulation pathway in fission yeast

Cells couple their growth and division rate in response to nutrient availability to maintain a constant size. This co-ordination happens either at the G1-S or the G2-M transition of the cell cycle. In the rod-shaped fission yeast, size regulation happens at the G2-M transition prior to mitotic commitment. Recent studies have focused on the role of the DYRK-family protein kinase Pom1, which forms gradients emanating from cell poles and inhibits the mitotic activator kinase Cdr2, present at the cell middle. Pom1 was proposed to inhibit Cdr2 until cells reached a critical size before division. However when and where Pom1 inhibits Cdr2 is not clear as medial Pom1 levels do not change during cell elongation. Here I show that Pom1 gradients are susceptible to environmental changes in glucose. Specifically, upon glucose limitation, Pom1 re-localizes from the poles to the cell sides where it delays mitosis through regulating Cdr2. This re-localization occurs due to microtubule de- stabilization and lateral catastrophes leading to transient deposition of the Pom1 gradient nucleator Tea4 along the cell cortex. As Tea4 localization to cell sides is sufficient to recruit Pom1, this explains the mechanism of Pom1 re-localization. Microtubule destabilization and consequently Tea4 and Pom1 spread depends on the activity of the cAMP-dependent Protein Kinase A (PKA/Pka1), as pka1 mutant cells have stable microtubules and retain polar Tea4 and Pom1 under limited glucose. PKA signaling negatively regulates the microtubule rescue factor CLASP/Cls1, thus reducing its ability to stabilize microtubules. Thus PKA signaling tunes CLASP activity to promote microtubule de-stabilization and Pom1 re-localization upon glucose limitation. I show that the side-localized Pom1 delays mitosis and balances the role of the mitosis promoting, mitogen-associated protein kinase (MAPK) protein Sty1. Thus Pom1 re-localization may serve to buffer cell size upon glucose limitation. -- Afin de maintenir une taille constante, les cellules régulent leur croissance ainsi que leur taux de division selon les nutriments disponibles dans le milieu. Dans la levure fissipare, cette régulation de la taille précède l'engagement mitotique et se fait à la transition entre les phases G2 à M du cycle cellulaire. Des études récentes se sont focalisées sur le rôle de la protéine Pom1, membre de la famille des DYRK kinase. Celle-ci forme un gradient provenant des pôles de la cellule et inhibe l'activateur mitotique Cdr2 présent au centre de la cellule. Le model propose que Pom1 inhibe Cdr2 jusqu'à atteindre une taille critique avant la division. Cependant quand et à quel endroit dans la cellulle Pom1 inhibe Cdr2 n'était pas clair car les niveaux médians de Pom1 ne changent pas au cours de la l'élongation des cellules. Dans cette étude, je montre que les gradients de Pom1 sont sensibles aux changements environnementaux du taux de glucose. Plus spécifiquement, en conditions limitantes de glucose, Pom1 se relocalise des pôles de la cellule pour se distribuer sur les côtés de celle-ci. Par conséquent, un délai d'entrée en mitose est observé dû à l'inhibition Cdr2 par Pom1. Cette délocalisation est due à la déstabilisation des microtubules qui va conduire à une déposition transitoire de Tea4, le nucléateur du gradient de Pom1, tout au long du cortex de la cellule. Comme la localisation de Tea4 sur les côtés de la cellule est suffisante pour recruter la protéine Pom1, ceci explique le mécanisme de relocalisation de celle-ci. La déstabilisation des microtubules et par conséquent la diffusion de Tea4 et Pom1 dépendent de l'activité de la protéine kinase A dépendante de l'AMP cyclique (PKA/Pka1). En absence de pka1, la stabilité des microtubules n'est pas affectée ce qui permet la rétention de Tea4 et Pom1 aux pôles de la cellule même en conditions limitantes de glucose. La signalisation via PKA régule négativement le facteur de sauvetage des microtubules CLASP/Cls1 et permet donc de réduire sa fonction de déstabilisation des microtubules. Ainsi la signalisation via PKA affine l'activité des CLASP pour promouvoir la déstabilisation des microtubules et la relocalisation de Pom1 en conditions limitantes de glucose. Je montre que la localisation sur les côtés retarde l'entrée en mitose et compense l'action de la protéine Sty1, connue pour être une MAPK qui induit l'entrée en mitose. Ainsi, la relocalisation de Pom1 pourrait servir à tamponner la taille de la cellule en condition limitantes de glucose. -- Various cell types in the environment such as bacterial, plant or animal cells have a distinct cellular size. Maintaining a constant cell size is important for fitness in unicellular organisms and for diverse functions in multicellular organisms. Cells regulate their size by coordinating their growth rate to their division rate. This coupling is important otherwise cells would get progressively smaller or larger after each successive cell cycle. In their natural environment cells may face fluctuations in the available nutrient supply. Thus cells have to coordinate their division rate to the variable growth rates shown under different nutrient conditions. During my PhD, I worked with a single-celled rod shaped yeast called the fission yeast. These cells are longer when the nutrient supply is abundant and shorter when the nutrient supply is scarce. A protein that senses changes in the external carbon source (glucose) is called Protein Kinase A (PKA). The rod shape of fission yeast cells is maintained thanks to a structural backbone called the cytoskeleton. One of the components of this backbone is called microtubules, which are small tube like structures spanning the length of the cell. They transport a protein called Tea4, which in turn is important for the proper localization of another protein Pom1 to the cell ends. Pom1 helps to maintain proper shape and size of these rod shaped yeast cells. My thesis work showed that upon reduction in the external nutrient (glucose) levels, microtubules become less stable and show an alteration in their organization. A significant percentage of the microtubules contact the side of the cell instead of touching only the cell tip. This leads to the spreading of the protein Pom1 away from the tips all around the cell periphery. This helps fission yeast cells to maintain the proper size required under these conditions of limited glucose supply. I further showed that the protein PKA regulates microtubule stability and organization and thus Pom1 spreading and maintenance of proper cell size. Thus my work led to the discovery of a novel pathway by which fission yeast cells maintain their size under limited supply of glucose. -- Divers types cellulaires dans l'environnement tels que les bactéries, les plantes ou les cellules animales ont une taille précise. Le maintien d'une taille cellulaire constante est importante pour le fitness des organismes unicellulaire ainsi que pour multiples fonctions dans les organismes multicellulaires. Les cellules régulent leur taille en coordonnant le taux de croissance avec le taux de division. Ce couplage est essentiel sinon les cellules deviendraient progressivement plus petites ou plus grandes après chaque cycle cellulaire. Dans leur habitat naturels les cellules peuvent faire face a des fluctuations dans le taux de nutriment disponible. Les cellules doivent donc coordonner leur taux de division aux taux variables de croissances perçus dans les différentes conditions nutritionnels. Pendant ma thèse, j'ai travaillée sur une levure unicellulaire, en forme de bâtonnet, nommé levure fissipare ou levure de fission. La taille de ces cellules est plus grande quand le taux de nutriments est grand et plus courte quand celui-ci est plus faible. Une protéine qui perçoit les changements dans le taux externe de la source de carbone (glucose) est nommée PKA pour protéine kinase A. La forme en bâtonnet de la cellule est due aux caractères structuraux du cytosquelette. Une composante importante de ce cytosquelette sont les microtubules, dont la structures ressemble à des petit tubes qui vont d'un bout à l'autre de la cellule. Ces microtubules transportent une protéine importante nommée Tea4 qui à leur tour importante pour la bonne localisation d'une autre protéine Pom1 aux extrémités de la cellule. La protéine Pom1 aide à maintenir la taille appropriée des levures fissipares. Mon travail de thèse a montré qu'en présence de taux faible de nutriments (glucose) les microtubules deviennent de moins en moins stables et montrent une désorganisation globale. Un pourcentage significatif des microtubules touche les côtés de la cellule aux lieu d'atteindre uniquement les extrémités. Ceci a pour conséquence une diffusion de Pom1 tout au long du cortex de la cellule. Ceci aide les levures fissipares à maintenir la taille appropriée pendant ce stress nutritionnel. De plus, je montre que PKA régule la stabilité et l'organisation des microtubules et par conséquent la diffusion de Pom1 et le maintien d'une taille constante. En conclusion, mon travail a conduit à la découverte d'un nouveau mécanisme par lequel la levure fissipare maintient sa taille dans des conditions limitantes en glucose.

Adaptation of Root Function by Nutrient-Induced Plasticity of Endodermal Differentiation.

Plant roots forage the soil for minerals whose concentrations can be orders of magnitude away from those required for plant cell function. Selective uptake in multicellular organisms critically requires epithelia with extracellular diffusion barriers. In plants, such a barrier is provided by the endodermis and its Casparian strips-cell wall impregnations analogous to animal tight and adherens junctions. Interestingly, the endodermis undergoes secondary differentiation, becoming coated with hydrophobic suberin, presumably switching from an actively absorbing to a protective epithelium. Here, we show that suberization responds to a wide range of nutrient stresses, mediated by the stress hormones abscisic acid and ethylene. We reveal a striking ability of the root to not only regulate synthesis of suberin, but also selectively degrade it in response to ethylene. Finally, we demonstrate that changes in suberization constitute physiologically relevant, adaptive responses, pointing to a pivotal role of the endodermal membrane in nutrient homeostasis.

Fertilizing Methods and Nutrient Balance at the End of Traditional Organic Agriculture in the Mediterranean Bioregion: Catalonia (Spain) in the 1860s

By reconstructing the nutrient balance of a Catalan v illage circa 1861-65 we examine the sustainability of organic agricultural sy stems in the northwest Mediterranean bioregion prior to the green rev olution and the question of whether the nutrients extracted f rom the soil were replenished. With a population density of 59 inhabitants per square km, similar to other northern European rural areas at that time, and a lower liv estock density per cropland unit, this v illage experienced a manure shortage. The gap was f illed by other labour-intensiv e way s of transf erring nutrients f rom uncultiv ated areas into the cropland. Key elements in this agricultural sy stem were v iney ards because they hav e f ew nutrient requirements, and woodland and scrublands as sources of relev ant amounts of nutrients collected in sev eral ways.

The Drosophila TNF Eiger Is an Adipokine that Acts on Insulin-Producing Cells to Mediate Nutrient Response.

Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. Here we show that Eiger, the Drosophila TNF-α, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-α converting enzyme (TACE) family is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, we have identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-α that mediates adaptive response to nutrient deprivation.

Evolutionary Ecology of Mountain Birch in Subarctic Stress Gradients: Interplay of Biotic and Abiotic Factors in Plant-Plant Interactions and Evolutionary Processes

In nature, variation for example in herbivory, wind exposure, moisture and pollution impact often creates variation in physiological stress and plant productivity. This variation is seldom clear-cut, but rather results in clines of decreasing growth and productivity towards the high-stress end. These clines of unidirectionally changing stress are generally known as ‘stress gradients’. Through its effect on plant performance, stress has the capacity to fundamentally alter the ecological relationships between individuals, and through variation in survival and reproduction it also causes evolutionary change, i.e. local adaptations to stress and eventually speciation. In certain conditions local adaptations to environmental stress have been documented in a matter of just a few generations. In plant-plant interactions, intensities of both negative interactions (competition) and positive ones (facilitation) are expected to vary along stress gradients. The stress-gradient hypothesis (SGH) suggests that net facilitation will be strongest in conditions of high biotic and abiotic stress, while a more recent ‘humpback’ model predicts strongest net facilitation at intermediate levels of stress. Plant interactions on stress gradients, however, are affected by a multitude of confounding factors, making studies of facilitation-related theories challenging. Among these factors are plant ontogeny, spatial scale, and local adaptation to stress. The last of these has very rarely been included in facilitation studies, despite the potential co-occurrence of local adaptations and changes in net facilitation in stress gradients. Current theory would predict both competitive effects and facilitative responses to be weakest in populations locally adapted to withstand high abiotic stress. This thesis is based on six experiments, conducted both in greenhouses and in the field in Russia, Norway and Finland, with mountain birch (Betula pubescens subsp. czerepanovii) as the model species. The aims were to study potential local adaptations in multiple stress gradients (both natural and anthropogenic), changes in plant-plant interactions under conditions of varying stress (as predicted by SGH), potential mechanisms behind intraspecific facilitation, and factors confounding plant-plant facilitation, such as spatiotemporal, ontogenetic, and genetic differences. I found rapid evolutionary adaptations (occurring within a time-span of 60 to 70 years) towards heavy-metal resistance around two copper-nickel smelters, a phenomenon that has resulted in a trade-off of decreased performance in pristine conditions. Heavy-metal-adapted individuals had lowered nickel uptake, indicating a possible mechanism behind the detected resistance. Seedlings adapted to heavy-metal toxicity were not co-resistant to others forms of abiotic stress, but showed co-resistance to biotic stress by being consumed to a lesser extent by insect herbivores. Conversely, populations from conditions of high natural stress (wind, drought etc.) showed no local adaptations, despite much longer evolutionary time scales. Due to decreasing emissions, I was unable to test SGH in the pollution gradients. In natural stress gradients, however, plant performance was in accordance with SGH, with the strongest host-seedling facilitation found at the high-stress sites in two different stress gradients. Factors confounding this pattern included (1) plant size / ontogenetic status, with seedling-seedling interactions being competition dominated and host-seedling interactions potentially switching towards competition with seedling growth, and (2) spatial distance, with competition dominating at very short planting distances, and facilitation being strongest at a distance of circa ¼ benefactor height. I found no evidence for changes in facilitation with respect to the evolutionary histories of plant populations. Despite the support for SGH, it may be that the ‘humpback’ model is more relevant when the main stressor is resource-related, while what I studied were the effects of ‘non-resource’ stressors (i.e. heavy-metal pollution and wind). The results have potential practical applications: the utilisation of locally adapted seedlings and plant facilitation may increase the success of future restoration efforts in industrial barrens as well as in other wind-exposed sites. The findings also have implications with regard to the effects of global change in subarctic environments: the documented potential by mountain birch for rapid evolutionary change, together with the general lack of evolutionary ‘dead ends’, due to not (over)specialising to current natural conditions, increase the chances of this crucial forest-forming tree persisting even under the anticipated climate change.

Consequences of warming and resource quality on the stoichiometry and nutrient cycling of a stream shredder

As a result of climate change, streams are warming and their runoff has been decreasing in most temperate areas. These changes can affect consumers directly by increasing their metabolic rates and modifying their physiology and indirectly by changing the quality of the resources on which organisms depend. In this study, a common stream detritivore (Echinogammarus berilloni Catta) was reared at two temperatures (15 and 20°C) and fed Populus nigra L. leaves that had been conditioned either in an intermittent or permanent reach to evaluate the effects of resource quality and increased temperatures on detritivore performance, stoichiometry and nutrient cycling. The lower quality (i.e., lower protein, soluble carbohydrates and higher C:P and N:P ratios) of leaves conditioned in pools resulted in compensatory feeding and lower nutrient retention capacity by E. berilloni. This effect was especially marked for phosphorus, which was unexpected based on predictions of ecological stoichiometry. When individuals were fed pool-conditioned leaves at warmer temperatures, their growth rates were higher, but consumers exhibited less efficient assimilation and higher mortality. Furthermore, the shifts to lower C:P ratios and higher lipid concentrations in shredder body tissues suggest that structural molecules such as phospholipids are preserved over other energetic C-rich macromolecules such as carbohydrates. These effects on consumer physiology and metabolism were further translated into feces and excreta nutrient ratios. Overall, our results show that the effects of reduced leaf quality on detritivore nutrient retention were more severe at higher temperatures because the shredders were not able to offset their increased metabolism with increased consumption or more efficient digestion when fed pool-conditioned leaves. Consequently, the synergistic effects of impaired food quality and increased temperatures might not only affect the physiology and survival of detritivores but also extend to other trophic compartments through detritivore-mediated nutrient cycling.

Erratum: Global pressures, specific responses: effects of nutrient enrichment in streams from different biomes

We assessed the effects of nutrient enrichment on three stream ecosystems running through distinct biomes (Mediterranean, Pampean and Andean). We increased the concentrations of N and P in the stream water 1.6–4-fold following a before–after control–impact paired series (BACIPS) design in each stream, and evaluated changes in the biomass of bacteria, primary producers, invertebrates and fish in the enriched (E) versus control (C) reaches after nutrient addition through a predictive-BACIPS approach. The treatment produced variable biomass responses (2–77% of explained variance) among biological communities and streams. The greatest biomass response was observed for algae in the Andean stream (77% of the variance), although fish also showed important biomass responses (about 9–48%). The strongest biomass response to enrichment (77% in all biological compartments) was found in the Andean stream. The magnitude and seasonality of biomass responses to enrichment were highly site specific, often depending on the basal nutrient concentration and on windows of ecological opportunity (periods when environmental constraints other than nutrients do not limit biomass growth). The Pampean stream, with high basal nutrient concentrations, showed a weak response to enrichment (except for invertebrates), whereas the greater responses of Andean stream communities were presumably favored by wider windows of ecological opportunity in comparison to those from the Mediterranean stream. Despite variation among sites, enrichment globally stimulated the algal-based food webs (algae and invertebrate grazers) but not the detritus-based food webs (bacteria and invertebrate shredders). This study shows that nutrient enrichment tends to globally enhance the biomass of stream biological assemblages, but that its magnitude and extent within the food web are complex and are strongly determined by environmental factors and ecosystem structure

The influence of substratum type and nutrient supply on biofilm organic matter utilization in streams

We investigated the effect of benthic substratum type (sand and rocks) and nutrient supply (N and P) on biofilm structure and heterotrophic metabolism in a field experiment in a forested Mediterranean stream (Fuirosos). Rock and sand colonization and biofilm formation was intensively studied for 44 d at two stream reaches: control and experimental (continuous addition of phosphate, ammonia, and nitrate). Structural (C, N, and polysaccharide content and bacterial and chlorophyll density) and metabolic biofilm parameters (b-glucosidase, peptidase, and phosphatase enzyme activities) were analyzed throughout the colonization process. The epilithic biofilm (grown on rocks) had a higher peptidase activity at the impacted reach, together with a higher algal and bacterial biomass. The positive relationship between the peptidase activity per cell and the N content of the epilithic biofilm suggested that heterotrophic utilization of proteinaceous compounds from within the biofilm was occurring. In contrast, nutrient addition caused the epipsammic biofilm (grown on sand) to exhibit lower b-glucosidase and phosphatase activities, without a significant increase in bacterial and algal biomass. The differential response to nutrient addition was related to different structural characteristics within each biofilm. The epipsammic biofilm had a constant and high C:N ratio (22.7) throughout the colonization. The epilithic biofilm had a higher C:N ratio at the beginning of the colonization (43.2) and evolved toward a more complex structure (high polysaccharide content and low C:N ratio) during later stages. The epipsammic biofilm was a site for the accumulation and degradation of organic matter: polysaccharides and organic phosphorus compounds had higher degradation activities

Global pressures, specific responses: effects of nutrient enrichment in streams from different biomes

We assessed the effects of nutrient enrichment on three stream ecosystems running through distinct biomes (Mediterranean, Pampean and Andean). We increased the concentrations of N and P in the stream water 1.6–4-fold following a before–after control–impact paired series (BACIPS) design in each stream, and evaluated changes in the biomass of bacteria, primary producers, invertebrates and fish in the enriched (E) versus control (C) reaches after nutrient addition through a predictive-BACIPS approach. The treatment produced variable biomass responses (2–77% of explained variance) among biological communities and streams. The greatest biomass response was observed for algae in the Andean stream (77% of the variance), although fish also showed important biomass responses (about 9–48%). The strongest biomass response to enrichment (77% in all biological compartments) was found in the Andean stream. The magnitude and seasonality of biomass responses to enrichment were highly site specific, often depending on the basal nutrient concentration and on windows of ecological opportunity (periods when environmental constraints other than nutrients do not limit biomass growth). The Pampean stream, with high basal nutrient concentrations, showed a weak response to enrichment (except for invertebrates), whereas the greater responses of Andean stream communities were presumably favored by wider windows of ecological opportunity in comparison to those from the Mediterranean stream. Despite variation among sites, enrichment globally stimulated the algal-based food webs (algae and invertebrate grazers) but not the detritus-based food webs (bacteria and invertebrate shredders). This study shows that nutrient enrichment tends to globally enhance the biomass of stream biological assemblages, but that its magnitude and extent within the food web are complex and are strongly determined by environmental factors and ecosystem structure

Organic carbon and nutrient (P, N) concentrations of water and sediment in several aquatic environment types of a Mediterranean coastal wetland (Empordà Wetlands, NE Iberian Peninsula)

Total sediment and water organic carbon and nutrient (nitrogen and phosphorus) concentrations of different environment types of a Mediterranean coastal wetland (temporary and brackish, temporary and freshwater, semi-permanent and brackish, and permanent and brackish basins) were analysed during two hydroperiods. A nitrogen limitation was found for both sediment and water. The total organic carbon concentration of the water was significantly related to the water level, which varies throughout the hydroperiods. In contrast, the total organic carbon concentration of the sediment was not related to water level. However, significant differences in total organic carbon of the sediment were found between hydroperiods. On the other hand, total organic carbon of the sediment varied spatially, being higher in temporary brackish basins with lower sand content, and lower in permanent and semi-permanent brackish basins with higher sand content