CiliateGEM: an open-project and a tool for predictions of ciliate metabolic variations and experimental condition design

Background

The study of cell metabolism is becoming central in several fields such as biotechnology, evolution/adaptation and human disease investigations. Here we present CiliateGEM, the first metabolic network reconstruction draft of the freshwater ciliate Tetrahymena thermophila. We also provide the tools and resources to simulate different growth conditions and to predict metabolic variations. CiliateGEM can be extended to other ciliates in order to set up a meta-model, i.e. a metabolic network reconstruction valid for all ciliates.Ciliates are complex unicellular eukaryotes of presumably monophyletic origin, with a phylogenetic position that is equal from plants and animals. These cells represent a new concept of unicellular system with a high degree of species, population biodiversity and cell complexity. Ciliates perform in a single cell all the functions of a pluricellular organism, including locomotion, feeding, digestion, and sexual processes.

Results

After generating the model, we performed an in-silico simulation with the presence and absence of glucose. The lack of this nutrient caused a 32.1% reduction rate in biomass synthesis. Despite the glucose starvation, the growth did not stop due to the use of alternative carbon sources such as amino acids.

Conclusions

The future models obtained from CiliateGEM may represent a new approach to describe the metabolism of ciliates. This tool will be a useful resource for the ciliate research community in order to extend these species as model organisms in different research fields. An improved understanding of ciliate metabolism could be relevant to elucidate the basis of biological phenomena like genotype-phenotype relationships, population genetics, and cilia-related disease mechanisms.

     

Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape.     

Nix is a selective autophagy receptor for mitochondrial clearance

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.     

Ginkgo biloba responds to herbivory by activating early signaling and direct defenses

Background

Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses.

Methodology/Principal Findings

Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca²⁺]_cyt) and H₂O₂ production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca²⁺]_cyt and H₂O₂. Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides.

Conclusions/Significance

By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this “living fossil” plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.     

3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168). We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.     

Chrysolina herbacea modulates terpenoid biosynthesis of Mentha aquatica L

Interactions between herbivorous insects and plants storing terpenoids are poorly understood. This study describes the ability of Chrysolina herbacea to use volatiles emitted by undamaged Mentha aquatica plants as attractants and the plant's response to herbivory, which involves the production of deterrent molecules. Emitted plant volatiles were analyzed by GC-MS. The insect's response to plant volatiles was tested by Y-tube olfactometer bioassays. Total RNA was extracted from control plants, mechanically damaged leaves, and leaves damaged by herbivores. The terpenoid quantitative gene expressions (qPCR) were then assayed. Upon herbivory, M. aquatica synthesizes and emits (+)-menthofuran, which acts as a deterrent to C. herbacea. Herbivory was found to up-regulate the expression of genes involved in terpenoid biosynthesis. The increased emission of (+)-menthofuran was correlated with the upregulation of (+)-menthofuran synthase.     

Quantitative analysis of herbivore-induced cytosolic calcium by using a Cameleon (YC 3.6) calcium sensor in Arabidopsis thaliana

Ca²⁺ is a key player in plant cell responses to biotic and abiotic stress. Owing to the central role of cytosolic Ca²⁺ ([Ca²⁺]_cyt) during early signaling and the need for precise determination of [Ca²⁺]_cyt variations, we used a Cameleon YC 3.6 reporter protein expressed in Arabidopsis thaliana to quantify [Ca²⁺]_cyt variations upon leaf mechanical damage (MD), herbivory by 3rd and 5th instar larvae of Spodoptera littoralis and S. littoralis oral secretions (OS) applied to MD. YC 3.6 allowed a clear distinction between MD and herbivory and discriminated between the two larvae instars. To our knowledge this is the first report of quantitative [Ca²⁺]_cyt determination upon herbivory using a Cameleon calcium sensor.     

Comparative analysis of the facilitating effects induced on muscular periphery by the microstimulation of various cerebellar nuclei

The facilitatory effects evoked on the motor periphery by the activation of neuronal pools in cerebellar nuclei were analized in 13 cats. The aim of the work was to compare the frequency and the characteristics of the motor facilitations induced on the ipsilateral forelimb by the microstimulation of cerebellar foci in the fastigial (CBM or in the interposital (NIA) nucleus. CBM or NIA sites, previously identified for the motor effects, were microstimulated, together with the contralateral motor cortex, to give evidence of the facilitations. It was observed that 51% of the NIA motor sites, 46% of the rostral and 33% of the caudal CBM ones, were able, when activated, to evoke facilitatory effects on at least one muscle. The most frequent motor pattern observed following NIA microstimulation was the contraction of a proximal muscle and simultaneously the facilitation of a distal one. Similar responses were detected upon activation of neuronal pools in both zones of CBM. A good number of CBM foci (39% in the rostral division and 33% in the caudal one), however, was unable to induce facilitation, eliciting, upon stimulation, only massive axial movements. Distal muscles were involved by facilitatory effects in a higher number of cases following NIA stimulation (61% of all the facilitatory responses) than CBM rostral (39%) or caudal (43%) one. Furthermore, a particular characteristic of a good percentage of CBM facilitating foci (36% in rostral and 28% in caudal CBM) was the capability to elicit motor activity in the contralateral side and simultaneously facilitation in the ipsilateral one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic Bayesian sensitivity analysis of a myocardial metabolic model

Dynamic compartmentalized metabolic models are identified by a large number of parameters, several of which are either non-physical or extremely difficult to measure. Typically, the available data and prior information is insufficient to fully identify the system. Since the models are used to predict the behavior of unobserved quantities, it is important to understand how sensitive the output of the system is to perturbations in the poorly identifiable parameters. Classically, it is the goal of sensitivity analysis to asses how much the output changes as a function of the parameters. In the case of dynamic models, the output is a function of time and therefore its sensitivity is a time dependent function. If the output is a differentiable function of the parameters, the sensitivity at one time instance can be computed from its partial derivatives with respect to the parameters. The time course of these partial derivatives describes how the sensitivity varies in time.When the model is not uniquely identifiable, or if the solution of the parameter identification problem is known only approximately, we may have not one, but a distribution of possible parameter values. This is always the case when the parameter identification problem is solved in a statistical framework. In that setting, the proper way to perform sensitivity analysis is to not rely on the values of the sensitivity functions corresponding to a single model, but to consider the distributed nature of the sensitivity functions, inherited from the distribution of the vector of the model parameters.In this paper we propose a methodology for analyzing the sensitivity of dynamic metabolic models which takes into account the variability of the sensitivity over time and across a sample. More specifically, we draw a representative sample from the posterior density of the vector of model parameters, viewed as a random variable. To interpret the output of this doubly varying sensitivity analysis, we propose visualization modalities particularly effective at displaying simultaneously variations over time and across a sample. We perform an analysis of the sensitivity of the concentrations of lactate and glycogen in cytosol, and of ATP, ADP, NAD⁺ and NADH in cytosol and mitochondria, to the parameters identifying a three compartment model for myocardial metabolism during ischemia.