Comparison of two Phaeodactylum tricornutum ecotypes under nitrogen starvation and resupply reveals distinct lipid accumulation strategies but a common degradation process

IntroductionPhaeodactylum tricornutum is a model species frequently used to study lipid metabolism in diatoms. When exposed to a nutrient limitation or starvation, diatoms are known to accumulate neutral lipids in cytoplasmic lipid droplets (LDs). Those lipids are produced partly de novo and partly from the recycle of plastid membrane lipids. Under a nitrogen resupply, the accumulated lipids are catabolized, a phenomenon about which only a few data are available. Various strains of P. tricornutum have been isolated around the world that may differ in lipid accumulation patterns.MethodsTo get further information on this topic, two genetically distant ecotypes of P. tricornutum (Pt1 and Pt4) have been cultivated under nitrogen deprivation during 11 days followed by a resupply period of 3 days. The importance of cytoplasmic LDs relative to the plastid was assessed by a combination of confocal laser scanning microscopy and cell volume estimation using bright field microscopy pictures.Results and discussionWe observed that in addition to a basal population of small LDs (0.005 μm3 to 0.7 μm3) present in both strains all along the experiment, Pt4 cells immediately produced two large LDs (up to 12 μm3 after 11 days) while Pt1 cells progressively produced a higher number of smaller LDs (up to 7 μm3 after 11 days). In this work we showed that, in addition to intracellular available space, lipid accumulation may be limited by the pre-starvation size of the plastid as a source of membrane lipids to be recycled. After resupplying nitrogen and for both ecotypes, a fragmentation of the largest LDs was observed as well as a possible migration of LDs to the vacuoles that would suggest an autophagic degradation. Altogether, our results deepen the understanding of LDs dynamics and open research avenues for a better knowledge of lipid degradation in diatoms

Postnatal Migration of Cerebellar Interneurons

Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders

Contribution à l'étude du couplage stimulus-sécrétion dans les cellules mélanotropes de l'hypophyse : mécanismes d'action de la TRH et du NPY

Dans les cellules mélanotropes, la réponse calcique induite par la TRH résulte de l'influx du Ca2+ via 3 types de canaux Ca2+ (sensibles au Ni2+, L et N) et de la mobilisation du Ca2+ à partir des stocks IP3-dépendants. Le Ca2+ extracellulaire via les canaux de type L et le Ca2+ intracellulaire contribuent avec les PKC et PTK à la libération d'a-MSH induite par la TRH. Le NPY supprime l'effet sécrétoire de la TRH via un récepteur Y1 couplé à une protéine G sensible à la PTX et diminue la libération spontanée d'a-MSH via un récepteur Y5 couplé négativement à l'adénylyl cyclase et aux canaux Ca2+. En conclusion, dans les cellules mélanotropes, le NPY abolit via un récepteur Y1 couplé à une protéine G sensible à la PTX, la libération d'a-MSH induite par la TRH, laquelle résulte d'une augmentation de la [Ca2+]i et de l'activation des PKC et PTK. De plus, le NPY diminue la libération basale d'a-MSH via un récepteur Y5 en inhibant la production d'AMPc et l'influx de Ca2+.In melanotrope cells, activation of TRH receptors induces an increase in [Ca2+]i that results from Ca2+ influx through 3 types of Ca2+ channels (Ni2+-sensitive, L- and N-type Ca2+-channels) and Ca2+ mobilization from IP3-sensitive intracellular pools. Ca2+ entry through LCC and mobilization of intracellular Ca2+, together with PKC and PTK, contribute in TRH-evoked a-MSH release. Melanotrope cells express the mRNAs encoding for Y1 and Y5 receptors. NPY inhibits the spontaneous release of a-MSH through a Y5 receptor coupled to adenylyl cyclase and suppresses TRH-induced a-MSH release through a Y1 receptor coupled to a PTX-sensitive G protein. In conclusion, in frog melanotrope cells, NPY abolishes, through a Y1 receptor coupled to a PTX-sensitive G protein, TRH-induced a-MSH release that results from an increase in [Ca2+]i and activation of protein kinase C et tyrosine kinase. In addition, NPY decreases basal a-MSH release through a Y5 receptor by inhibiting cAMP formation and Ca2+ influx.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Vitamin E but Not GSH Decreases Reactive Oxygen Species Accumulation and Enhances Sperm Production during In Vitro Maturation of Frozen-Thawed Prepubertal Mouse Testicular Tissue

International audienceFreezing-thawing procedures and in vitro culture conditions are considered as a source of stress associated with increased reactive oxygen species (ROS) generation, leading to a damaged cell aerobic metabolism and consequently to oxidative stress. In the present study, we sought to investigate whether vitamin E (Vit E) or reduced glutathione (GSH) enhances sperm production by decreasing ROS accumulation during in vitro maturation of prepubertal mice testes. Testes of prepubertal mice were cryopreserved using a freezing medium supplemented or not supplemented with Vit E and were cultured after thawing. In presence of Rol alone in culture medium, frozen-thawed (F-T) testicular tissues exhibited a higher ROS accumulation than fresh tissue during in vitro culture. However, Vit E supplementation in freezing, thawing, and culture media significantly decreased cytoplasmic ROS accumulation in F-T testicular tissue during in vitro maturation when compared with F-T testicular tissue cultured in the presence of Rol alone, whereas GSH supplementation in culture medium significantly increased ROS accumulation associated with cytolysis and tissue disintegration. Vit E but not GSH promoted a better in vitro sperm production and was a suitable ROS scavenger and effective molecule to improve the yield of in vitro spermatogenesis from F-T prepubertal mice testes. The prevention of oxidative stress in the cytoplasmic compartment should be regarded as a potential strategy for improving testicular tissue viability and functionality during the freeze-thaw procedure and in vitro maturation

Fluorophore-Assisted Click Chemistry through Copper(I) Complexation

International audienceThe copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or biological systems. In order to accelerate the ligation process and ensure efficient formation of conjugates under diluted conditions, external copper(I) ligands or sophisticated copper(I)-chelating azides are used. This latter strategy, however, increases the bulkiness of the triazole linkage, thus perturbing the biological function or dynamic behavior of the conjugates. In a proof-of-concept study, we investigated the use of an extremely compact fluorophore-based copper(I) chelating azide in order to accelerate the CuAAC with concomitant fluorescence labeling; in our strategy, the fluorophore is able to complex copper(I) species while retaining its photophysical properties. It is believed that this unprecedented approach which was applied for the labeling of a short peptide molecule and the fluorescent labeling of live cells, could be extended to other families of nitrogen-based fluorophores in order to tune both the reaction rate and photophysical characteristics

Antibody-Conjugated Nanocarriers for Targeted Antibiotic Delivery: Application in the Treatment of Bacterial Biofilms

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Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

International audienceFluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction

Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion

International audienceIdentification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative, or metabolic diseases. To visualize molecule distribution and dynamics at the nanoscale, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies using transmission electron microscopy, atomic force microscopy, total internal reflection microscopy, and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein or protein-lipid interactions. Furthermore, the potential and the challenges of these different advanced imaging approaches for application in the study of neuroendocrine cell biology are discussed, aiming to guide researchers to select the best approach for their specific purpose around the crucial but not yet fully understood neurosecretion process