Sepal shape variability is robust to cell size heterogeneity in Arabidopsis.

How organisms produce organs with robust shapes and sizes is still an open question. In recent years, the Arabidopsis sepal has been used as a model system to study this question because of its highly reproducible shape and size. One interesting aspect of the sepal is that its epidermis contains cells of very different sizes. Previous reports have qualitatively shown that sepals with more or less giant cells exhibit comparable final size and shape. Here, we investigate this question using quantitative approaches. We find that a mixed population of cell size modestly contribute to the normal width of the sepal but is not essential for its shape robustness. Furthermore, in a mutant with increased cell and organ growth variability, the change in final sepal shape caused by giant cells is exaggerated but the shape robustness is not affected. This formally demonstrates that sepal shape variability is robust to cell size heterogeneity.

Brucella effectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins.

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen.

Lipid anchoring and electrostatic interactions target NOT-LIKE-DAD to pollen endo-plasma membrane.

Phospholipases cleave phospholipids, major membrane constituents. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD, also known as MTL or ZmPLA1) leads to peculiar defects in sexual reproduction, notably the induction of maternal haploid embryos. Contrary to previous reports, NLD does not localize to cytosol and plasma membrane of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. After pollen tube burst, NLD localizes at the apical region of the egg apparatus. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and lipid biosensors indicated that phosphatidylinositol-4,5-bisphosphate is enriched in the endo-PM, uncovering a unique example of how membrane electrostatic properties can define a specific polar domain (i.e., endo-PM), which is critical for plant reproduction and gamete formation.

Xyloglucans and Microtubules Synergistically Maintain Meristem Geometry and Phyllotaxis.

The shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition.

Automated Tracking of Root for Confocal Time-lapse Imaging of Cellular Processes.

Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root over time. The protocol makes possible the image acquisition of multiple growing root tips, therefore increasing the number of recorded mitotic events in a given experiment. The protocol also allows the visualization of more than one fluorescent protein or dye simultaneously, using multiple channel acquisition. We particularly focus on imaging of cytokinesis in Arabidopsis root tip meristem, but this protocol is also suitable to follow root hair growth, pollen tube growth, and other regions of root over time, in various plant species. It may as well be amendable to automatically track non-plant structures with an apical growth.

Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs.

According to the World Health Organization (WHO), at least 50% of emerging viruses endowed with pathogenicity in humans can infect the Central Nervous System (CNS) with induction of encephalitis and other neurologic diseases ( Taylor et al., 2001 ; Olival and Daszak, 2005). While neurological diseases are progressively documented, the underlying cellular and molecular mechanisms involved in virus infection and dissemination within the CNS are still poorly understood (Swanson and McGavern, 2015; Ludlow et al., 2016 ). For example, measles virus (MeV) can infect neural cells, and cause a persistent brain infections leading to lethal encephalitis from several months to years after primary infection with no available treatment (Reuter and Schneider-Schaulies, 2010; Laksono et al., 2016 ). The Organotypic Brain Culture (OBC) is a suitable model for the virology field to better understand the CNS infections. Indeed, it allows not only studying the infection and the dissemination of neurotropic viruses within the CNS but it could also serve as screening model of innovative antiviral strategies or molecules, such as our recently published studies about fusion inhibitory peptides and the HSP90 chaperone activity inhibitor, 17-DMAG ( Welsch et al., 2013 ; Bloyet et al., 2016 ). Based on our previous work, we propose here an optimized method to prepare OBC of hippocampi and cerebellums which are suitable for small rodent models based virus studies, including mice, rats as well as hamsters at a post-natal stage, between P6 to P10. We notably took into account the stress of the slice procedure on the tissue and the subsequent cellular reactions, which is essential to fully characterize the model prior to any use in infectious conditions. With this knowledge, we propose a protocol highlighting the requirements, including potential trouble shootings of the slicing parameters, to consider the variations we observed according to the structure and animal studied. This framework should facilitate the use of OBC for better conclusive studies of neurotropic viruses.

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17ß-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

Effects of a small dose of olanzapine on healthy subjects according to their schizotypy: an ERP study using a semantic categorization and an oddball task.

Delusions and hallucinations are often meaningful. They thus reveal abnormal semantic activations. To start testing whether antipsychotics act by reducing abnormal semantic activations we focused on the N400 event-related brain potential, which is elicited by meaningful stimuli, such as words, and whose distribution on the scalp is known to depend on the semantic category of these stimuli. We used a semantic-categorization task specially designed to reduce the impact of the variations of context processing across subjects' groups and a classical oddball task as a control. Healthy subjects were recruited rather than psychotic patients to ensure that the medication effects could not be secondary to a reduction of symptoms. These participants (n=47) were tested in a double-blind cross-over paradigm where the ERP effects of 2.5mg of olanzapine taken on the eve of the testing were compared to those of the placebo. The amplitudes of the N400s elicited by the target words were greater at anterior scalp sites in the half of the subjects having higher schizotypal scores. Olanzapine reduced these larger N400s and had no effect on the small anterior N400s of the half of the subjects with lower scores. These results are discussed as consistent with the idea that antipsychotics reduce abnormal activations of particular semantic representations. Further studies should thus be done to see if this reduction correlates with and predicts the decrease of psychotic symptoms in patients.

Healthy people with delusional ideation change their mind with conviction.

Emotional distress and reasoning biases are two factors known to contribute to delusions. As a step towards elucidating mechanisms underlying delusions, the main aim of this study was to evaluate a possible "jumping to new conclusions" reasoning bias in healthy people with delusional ideation and its association with emotions. We surveyed 80 healthy participants, measuring levels of depression, anxiety, cognitive error and delusional ideation. Participants completed two versions of the beads task to evaluate their reasoning style. Results showed that people with delusional ideation reached a conclusion after less information, as expected. Interestingly, they also tended to change their conclusions more often than people without delusional ideation and did so with greater conviction. Depression and cognitive errors were strong predictors of delusional ideation but not of reasoning style. We conclude that delusional ideation in non-psychotic individuals is independently predicted by depressive symptoms and by a high conviction in new conclusions.

Paranoid induction reduces N400s of healthy subjects with delusional-like ideation.

A previous study suggests that the amplitude of the N400 event-related potentials (ERPs) of healthy subjects does not vary with their delusional-like ideations. This contrasts with the smaller N400 amplitudes observed in more- than in less-deluded schizophrenia patients. Here, we hypothesize that these smaller N400 amplitudes were related to the paranoid feelings patients had during the ERP recording. We thus induced this type of feelings in healthy subjects. Delusional-like ideation was assessed with the schizotypal personality questionnaire. Thirty-four healthy subjects completed a semantic categorization task. Paranoid feelings were significantly enhanced by the induction. In these conditions, greater delusional-like ideation scores were associated with smaller N400 amplitudes and larger late positive components. Controlling for the two other schizotypal factors strengthened these results. These findings may help us understand why delusions persist.

Schizotypal traits and N400 in healthy subjects.

We examined whether correlations previously found between symptoms of schizophrenia patients and the amplitude of an event-related potential (ERP), the N400, could be also found between schizotypal experiences of healthy subjects and the N400. We chose a semantic categorization task previously used with patients. Schizotypal experiences were measured with the schizotypal personality questionnaire (SPQ). The effects of the other factors were controlled for when assessing the correlations between each SPQ factor and N400s. These correlations were assessed at each electrode site to see whether their distribution on the scalp follows that of the N400 effect. Disorganization and interpersonal scores were found to correlate with ERPs in the N400 time window, as previously reported for the comparable symptoms of patients. However, the scalp distribution of these correlations differed from that of the N400 effect.

Circumventing the deficit of context processing in schizophrenia: an event-related brain potential study.

The deficit in semantic processing observed in schizophrenia patients may be due 1) to a 'genuine' deficit in processing new semantic information and 2) to a deficit in the ability to process and maintain the context in which the new semantic information has to be integrated. In a previous study, we designed a paradigm to study only the processing of new information. The processing of context was eased in the hope that patients would perform it as normals do. The goal of the present work was to determine whether this was the case. To achieve this goal, the event-related brain potentials (ERPs) elicited by the two context words of the new protocol were examined in 35 schizophrenia patients and 30 normal controls. These two context words were 'INACTION' and 'ANIMAL?'. Each trial started by one or the other and continued with a target word. 'INACTION' announced that subjects had no decision to make for the target word. 'ANIMAL?' announced that subjects had to decide whether or not the target word could be integrated into the animal category. Two ERP differences were found between normals and patients. However, the first, for 'INACTION', could be related to patients' difficulties at inhibiting the planning of action and the second, for 'ANIMAL?', to the preparation of their motor responses. No ERP difference was found that could have indexed a problem in encoding or in maintaining context. Thus, we conclude that this particular type of protocol could be helpful to focus on the study of the processing of the new semantic information conveyed by target words.

P300 asymmetry and positive symptom severity: a study in the early stage of a first episode of psychosis.

The amplitude of the P300 event-related potential (ERP) has been reported to be reduced over left compared to right temporal sites in schizophrenia patients. This left temporal P300 reduction has been associated with positive symptom severity and gray matter reduction in the left superior temporal gyrus. We investigated a group of patients with a first episode of schizophrenia spectrum psychosis and a group of normal controls to verify if P300 amplitude asymmetry already exists around the time of presentation for treatment. Relative to normal control subjects, no P300 asymmetry was found in patients. Nevertheless, P300 asymmetry was correlated with the severity of positive symptoms and worse global functioning (GAF), a good predictor of poor outcome.

Delusions and processing of discrepant information: an event-related brain potential study.

One possible explanation for why delusions persist despite the awareness of contradictory information is that the new information fails to be integrated. Interestingly, the amplitude of the N400 event-related brain potential (ERP) has been proposed as an index of the integration of information that is discrepant with expectancies whatever the task in which the potential is found. Thus, delusions may persist because of a deficit in integration as indexed by the N400. To test this hypothesis, ERPs were recorded in 35 schizophrenia patients (mean age=30.5+/-5.6 years) and 26 normal controls during a task in which they either had to decide whether or not each target word could be integrated into the category "animal", or had no decision to make, according to the prompt "animal?" or the prompt "inaction". In these conditions, the amplitudes of the N400s to target words that were discrepant with the category were found to be negatively correlated with delusion severity. The patient group was then dichotomized according to a median split of delusion severity, excluding the 5 patients with delusion scores at the median. Mean age, sex ratio, and severity of conceptual disorganization and hallucinations of the two subgroups differed. Controlling for these 4 covariates, the N400s for discrepant targets were found smaller in the 14 More-Delusional patients than in the 16 Less-Delusional patients. These results support the hypothesis that delusions are associated with smaller N400s in patients. Further studies should thus be done to test whether a deficit of N400 processes could have a causal role in the persistence of delusions.

Role of petal-specific orcinol O-methyltransferases in the evolution of rose scent.

Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominantly in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells' secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses.

Identification of new members of Fertilisation Independent Seed Polycomb Group pathway involved in the control of seed development in Arabidopsis thaliana.

In higher plants, double fertilisation initiates seed development. One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm. The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole. We report that ontogenesis of the posterior pole in Arabidopsis thaliana involves oriented migration of nuclei in the syncytium. We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED (FIS) class, MEDEA (MEA), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM (FIE). A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia. We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1). The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker.