Sparse denoising and adaptive estimation enhances the resolution and contrast of fluorescence emission difference microscopy based on an array detector.

An array detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high-resolution image by linearly combining sub-detector images, especially the fluorescence emission difference (FED) technique. To improve the resolution and contrast of FED microscopy based on an array detector, we propose to associate sparse denoising with spatial adaptive estimation. We show on both calibration slides and real data that our approach applied to the full stack of spatially reassigned detector signals, enables us to achieve a higher reconstruction performance in terms of resolution, image contrast, and noise reduction.

Encapsulation of Luminescent Gold Nanoclusters into Synthetic Vesicles.

Gold nanoclusters (Au NCs) are attractive luminescent nanoprobes for biomedical applications. In vivo biosensing and bioimaging requires the delivery of the Au NCs into subcellular compartments. In this view, we explore here the possible encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. Different methods were investigated to prepare vesicles containing Au NCs in their lumen. The efficiency of the process was correlated to the structural and morphological aspect of the Au NCs' encapsulating vesicles thanks to complementary analyses by SAXS, cryo-TEM, and confocal microscopy techniques. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Furthermore, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS.

Targeted Laser Ablation in the Embryo of Saccharina latissima.

In Saccharina latissima, the embryo develops as a monolayered cell sheet called the lamina or the blade. Each embryo cell is easy to observe, readily distinguishable from its neighbors, and can be individually targeted. For decades, laser ablation has been used to study embryo development. Here, a protocol for cell-specific laser ablation was developed for early embryos of the brown alga S. latissima. The presented work includes: (1) the preparation of Saccharina embryos, with a description of the critical parameters, including culture conditions, (2) the laser ablation settings, and (3) the monitoring of the subsequent growth of the irradiated embryo using time-lapse microscopy. In addition, details are provided on the optimal conditions for transporting the embryos from the imaging platform back to the lab, which can profoundly affect subsequent embryo development. Algae belonging to the order Laminariales display embryogenesis patterns similar to Saccharina; this protocol can thus be easily transferred to other species in this taxon.

Label-free microscopy of mitotic chromosomes using the polarization orthogonality breaking technique.

We report how a recently developed polarization imaging technique, implementing micro-wave photonics and referred to as orthogonality-breaking (OB) imaging, can be adapted on a classical confocal fluorescence microscope, and is able to provide informative polarization images from a single scan of the cell sample. For instance, the comparison of the images of various cell lines at different cell-cycle stages obtained by OB polarization microscopy and fluorescence confocal images shows that an endogenous polarimetric contrast arizes with this instrument on compacted chromosomes during cell division.

The Ouzo effect: A tool to elaborate high-payload nanocapsules.

We investigate the encapsulation in hybridosomes®, a type of capsules unique regarding their structure and method of elaboration. Hybridosomes® are made of a single shell of inorganic nanoparticles (~5 nm) crosslinked with a polymer and are easily obtained via spontaneous emulsification in a ternary mixture THF/water/butylated hydroxytoluene (BHT). Our main finding is that an exceptionally high concentration of a hydrophobic model dye can be loaded in the hybridosomes®, up to 0.35 mol.L-1 or equivalently 170 g.L-1 or 450,000 molecules/capsule. The detailed investigation of the encapsulation mechanism shows that the dye concentrates in the droplets during the emulsification step simultaneously with capsule formation. Then it precipitates inside the capsules during the course of solvent evaporation. In vitro fluorescence measurements show that the nano-precipitated cargo can be transferred from the core of the hybridosomes® to the membrane of liposomes. In vivo studies suggest that the dye diffuses through the body during several days. The released dye tends to accumulate in body-fat, while the inorganic nanoparticles remain trapped into the liver and the spleen macrophages.

Poly(ADP-ribose)-dependent chromatin unfolding facilitates the association of DNA-binding proteins with DNA at sites of damage.

The addition of poly(ADP-ribose) (PAR) chains along the chromatin fiber due to PARP1 activity regulates the recruitment of multiple factors to sites of DNA damage. In this manuscript, we investigated how, besides direct binding to PAR, early chromatin unfolding events controlled by PAR signaling contribute to recruitment to DNA lesions. We observed that different DNA-binding, but not histone-binding, domains accumulate at damaged chromatin in a PAR-dependent manner, and that this recruitment correlates with their affinity for DNA. Our findings indicate that this recruitment is promoted by early PAR-dependent chromatin remodeling rather than direct interaction with PAR. Moreover, recruitment is not the consequence of reduced molecular crowding at unfolded damaged chromatin but instead originates from facilitated binding to more exposed DNA. These findings are further substantiated by the observation that PAR-dependent chromatin remodeling at DNA lesions underlies increased DNAse hypersensitivity. Finally, the relevance of this new mode of PAR-dependent recruitment to DNA lesions is demonstrated by the observation that reducing the affinity for DNA of both CHD4 and HP1α, two proteins shown to be involved in the DNA-damage response, strongly impairs their recruitment to DNA lesions.

Staphylococcus aureus induces DNA damage in host cell.

Staphylococcus aureus causes serious medical problems in human and animals. Here we show that S. aureus can compromise host genomic integrity as indicated by bacteria-induced histone H2AX phosphorylation, a marker of DNA double strand breaks (DSBs), in human cervix cancer HeLa and osteoblast-like MG-63 cells. This DNA damage is mediated by alpha phenol-soluble modulins (PSMα1-4), while a specific class of lipoproteins (Lpls), encoded on a pathogenicity island in S. aureus, dampens the H2AX phosphorylation thus counteracting the DNA damage. This DNA damage is mediated by reactive oxygen species (ROS), which promotes oxidation of guanine forming 7,8-dihydro-8-oxoguanine (8-oxoG). DNA damage is followed by the induction of DNA repair that involves the ATM kinase-signaling pathway. An examination of S. aureus strains, isolated from the same patient during acute initial and recurrent bone and joint infections (BJI), showed that recurrent strains produce lower amounts of Lpls, induce stronger DNA-damage and prompt the G2/M transition delay to a greater extent that suggest an involvement of these mechanisms in adaptive processes of bacteria during chronicization. Our findings redefine our understanding of mechanisms of S. aureus-host interaction and suggest that the balance between the levels of PSMα and Lpls expression impacts the persistence of the infection.

Endotoxin regulates matrix genes increasing reactive oxygen species generation by intercellular communication between palmitate-treated hepatocyte and stellate cell.

Previous studies have shown that gut-derived bacterial endotoxins contribute in the progression of simple steatosis to steatohepatitis, although the mechanism(s) remains inaccurate to date. As hepatic stellate cells (HSC) play a pivotal role in the accumulation of excessive extracellular matrix (ECM), leading to collagen deposition, fibrosis, and perpetuation of inflammatory response, an in vitro model was developed to investigate the crosstalk between HSC and hepatocytes (human hepatoma cell) pretreated with palmitate. Bacterial lipopolysaccharide (LPS) stimulated HSC with phosphorylation of the p38 mitogen-activated protein kinase/NF-κB pathway, while several important pro-inflammatory cytokines were upregulated in the presence of hepatocyte-HSC. Concurrently, fibrosis-related genes were regulated by palmitate and the inflammatory effect of endotoxin where cells were more exposed or sensitive to reactive oxygen species (ROS). This interaction was accompanied by increased expression of the mitochondrial master regulator, proliferator-activated receptor gamma coactivator alpha, and a cytoprotective effect of the agent N-acetylcysteine suppressing ROS production, transforming growth factor-ß1, and tissue inhibitor of metalloproteinase-1. In summary, our results demonstrate that pro-inflammatory mediators LPS-induced promote ECM rearrangement in hepatic cells transcriptionally committed to the regulation of genes encoding enzymes for fatty acid metabolism in light of differences that might require an alternative therapeutic approach targeting ROS regulation.

A targeted 3D EM and correlative microscopy method using SEM array tomography.

Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography for asymmetric samples and model organisms (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio). We modified a diamond knife to simplify serial section array acquisition with minimal artifacts. After array acquisition, the arrays were transferred to a glass coverslip or silicon wafer support. Using light microscopy, the arrays were screened rapidly for initial recognition of global anatomical features (organs or body traits). Then, using SEM, an in-depth study of the cells and/or organs of interest was performed. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.

Bioconjugated fluorescent organic nanoparticles targeting EGFR-overexpressing cancer cells.

The field of optical bioimaging has considerably flourished with the advent of sophisticated microscopy techniques and ultra-bright fluorescent tools. Fluorescent organic nanoparticles (FONs) have thus recently appeared as very attractive labels for their high payload, absence of cytotoxicity and eventual biodegradation. Nevertheless, their bioconjugation to target specific receptors with high imaging contrast is scarcely performed. Moreover, assessing the reality of bioconjugation represents high challenges given the sub-nanomolar concentrations resulting from the commonly adopted nanoprecipitation fabrication process. Here, we describe how the combination of a magnetic shell allows us to easily generate red-emitting FONs conjugated with the epidermal growth factor ligand (EGF), a small protein promoting cancer cell proliferation by activating the EGF receptor (EGFR) pathway. Dual color fluorescence correlation spectroscopy combined with immunofluorescence is originally harnessed in its time trace mode to unambiguously demonstrate covalent attachment between the FON and EGF at sub-nanomolar concentrations. Strong asymmetric clustering of EGF-conjugated FONs is observed at the membrane of MDA-MB-468 human breast cancer cells overexpressing EGF receptors using super-resolution fluorescence microscopy. Such high recruitment of EGF-conjugated FONs is attributed to their EGF multivalency (4.7 EGF per FON) which enables efficient EGFR activation and subsequent phosphorylation. The large hydrodynamic diameter (DH ∼ 301 nm) of EGF-conjugated FONs prevents immediate engulfment of the sequestered receptors, which provides very bright and localized spots in less than 30 minutes. The reported bioconjugated nanoassemblies could thus serve as ultra-bright probes of breast cancer cells with EGFR-overexpression that is often associated with poor prognosis.

Propionibacterium freudenreichii Surface Protein SlpB Is Involved in Adhesion to Intestinal HT-29 Cells.

Propionibacterium freudenreichii is a beneficial bacterium traditionally used as a cheese ripening starter and more recently for its probiotic abilities based on the release of beneficial metabolites. In addition to these metabolites (short-chain fatty acids, vitamins, and bifidogenic factor), P. freudenreichii revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. This effect is, however, highly strain-dependent. Local action of metabolites and of immunomodulatory molecules is favored by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. In the present study, we showed different adhesion rates to cultured human intestinal cells, among strains of P. freudenreichii. The most adhesive one was P. freudenreichii CIRM-BIA 129, which is known to expose surface-layer proteins. We evidenced here the involvement of these proteins in adhesion to cultured human colon cells. We then aimed at deciphering the mechanisms involved in adhesion. Adhesion was inhibited by antibodies raised against SlpB, one of the surface-layer proteins in P. freudenreichii CIRM-BIA 129. Inactivation of the corresponding gene suppressed adhesion, further evidencing the key role of slpB product in cell adhesion. This work confirms the various functions fulfilled by surface-layer proteins, including probiotic/host interactions. It opens new perspectives for the understanding of probiotic determinants in propionibacteria, and for the selection of the most efficient strains within the P. freudenreichii species.

Neurite analyzer: An original Fiji plugin for quantification of neuritogenesis in two-dimensional images.

BACKGROUND: In life sciences, there is a growing need for new informatics tools designed to provide automated solutions in order to analyze big amounts of images obtained from high-throughput imaging systems. Among the most widely used assays in neurotoxicity, endocrinology and brain diseases, the neurite outgrowth assay is popular. NEW METHOD: Cell-to-cell quantification of the main morphological features of neurite outgrowth assays remains very challenging. Here, we provide a new pipeline developed on Fiji software for analysis of series of two-dimensional images. It allows the automated analysis of most of these features. RESULTS: We tested the accuracy and usefulness of the software by confirming the effects of estradiol and hypoxia on in vitro neuronal differentiation, previously published by different authors with manual analysis methods. With this new method, we highlighted original interesting data. COMPARISON WITH EXISTING METHOD(S): The innovation brought by this plugin lies in the fact that it can process multiple images at the same time, in order to obtain: the number of nuclei, the number of neurites, the length of neurites, the number of neurites junctions, the number of neurites branches, the length of each branch, the position of the branch in the image, the angle of each branch, but also the area of each cell and the number of neurites per cell. CONCLUSIONS: This plugin is easy to use, highly sensitive, and allows the experimenter to acquire ready-to-use data coming from a vast amount of images.

MNK1 kinase activity is required for abscission.

MNK1 is a serine/threonine kinase identified as a target for MAP kinase pathways. Using chemical drug, kinase-dead expression or knockdown by RNA interference, we show that inhibition of MNK1 induces the formation of multinucleated cells, which can be rescued by expressing a form of MNK1 that is resistant to RNA interference. We found that the active human form of MNK1 localises to centrosomes, spindle microtubules and the midbody. Time-lapse recording of MNK1-depleted cells displays cytokinesis defects, as daughter cells fuse back together. When MNK1 activity was inhibited, no microtubule defect at the midbody was detected, however, anchorage of the membrane vesicle at the midbody was impaired as lumenal GFP-positive vesicles did not accumulate at the midbody. At the molecular level, we found that centriolin localisation was impaired at the midbody in MNK1-depleted cells. As a consequence, endobrevin - a v-SNARE protein implicated in the abscission step - was not properly localised to the midbody. Altogether, our data show that MNK1 activity is required for abscission.

Monoclonal antibodies against human translation termination factor eRF3 and their utilization for sub-cellular localization of eRF3.

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. eRF1 recognizes the stop codon and promotes nascent peptide chain release, while eRF3 facilitates this peptide release in a GTP-dependent manner. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay. Despite extensive investigation, the complete understanding of eRF3 function have been hampered by the lack of specific anti-eRF3 monoclonal antibodies (Mabs). The purpose of the study was production of recombinant eRF3a/GSPT1, development of anti-eRF3a/GSPT1 Mabs and their utilization for eRF3a/GSPT1 sub-cellular localization. Plasmid encoding C-terminal part of human GSPT1/eRF3a was constructed. Purified protein, which was predominantly present in the inclusion bodies, was used for the development of Mabs. Characterization of the regions recognized by Mabs using GSPT1/eRF3a mutants and its visualization in the 3D space suggested that Mabs recognize different epitopes. Consistent with its function in translational termination, immunostaining of the cells with developed Mabs revealed that the endogenous GSPT1/eRF3a localized in endoplasmic reticulum. Taking into account the important role of eRF3 for the fundamental research one can suggests that developed Mabs have great prospective to be used as a research reagent in a wide range of applications.

Localization of aurora A and aurora B kinases during interphase: role of the N-terminal domain.

Aurora kinases possess a conserved catalytic domain (CD) and a N-terminal domain (ND) that varies in size and sequence. We have previously reported that the N-terminal domain of AuroraA (AurA) participates in the localization of the kinase to the centrosome in interphase. AuroraB (AurB) is a chromosome passenger protein and its N-terminal domain is not necessary for its localization or function during mitosis. Using various combinations of GFP-AurA and AurB protein domains we show that AurB N-terminal domain is required for nuclear localization in Xenopus XL2 cells in interphase. In human cells, however, we found both AurA and AurB kinases in the nucleus, AurA being mainly cytoplasmic and AurB mainly nuclear. Both proteins are actively excluded from the nucleus by a CRM1 dependent pathway. Interestingly, at a functional level, in interphase, every combination of Aurora kinase domains (ND-CD) rescues histone H3 Serine10 phosphorylation defect induced by AurB knockdown. This clearly indicates the presence of a functional AurA in the nucleus. However, the chimera ND-AurA/CD-AurB was much more efficient than the ND-AurB/ CD-AurA to rescue multinucleation also induced by AurB knockdown. This indicates that the catalytic domain of AurB is required to fulfill specific functions during mitosis that cannot be fulfilled by the catalytic domain of AurA, probably for localization reasons during mitosis.

PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores.

The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre-messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.

The Bloom syndrome helicase is a substrate of the mitotic Cdc2 kinase.

Bloom syndrome (BS) is a rare human autosomal recessive disorder characterized by marked genetic instability associated with greatly increased predisposition to a wide range of cancers affecting the general population. BS arises through mutations in both copies of the BLM gene which encodes a 3'-5' DNA helicase identified as a member of the RecQ family. Several studies support a major role for BLM in the cellular response to DNA damage and stalled replication forks. However, the specific function(s) of BLM remain(s) unclear. The BLM protein is strongly expressed and phosphorylated during mitosis, but very little information is available about the origin and the significance of this phosphorylation. We show here that ATM kinase provides only a limited contribution to the mitotic phosphorylation of BLM. We also demonstrate that BLM is directly phosphorylated at multiple sites in vitro by the mitotic cdc2 kinase, and identify two new sites of mitotic BLM phosphorylation: Ser-714 and Thr-766. Our results identify BLM helicase as a new substrate for cdc2, which may have potential physiological implications for the role of BLM in mitosis.

Liposome-mediated RNA transfection should be used with caution.

Liposome-mediated RNA transfection appears to present a number of advantages for studying the metabolism of reporter mRNAs in mammalian cells. This method is also widely used to transfect siRNAs. Here we describe results indicating that reporter mRNAs introduced into HeLa cells by liposomes do not present the expected behaviors. Namely, the stability of reporter mRNAs was independent of the presence or absence of an AUUUA instability element, a poly(A) tail, or even a 5' methylated cap. Confocal microscopy showed that fluorescent RNAs introduced by liposome-mediated transfection were present in discrete particles. These observations imply that a number of control experiments are required when using liposome to mediated RNA transfection, and the possible consequences are discussed.

The absence of p53 aggravates polyploidy and centrosome number abnormality induced by Aurora-C overexpression.

Aurora-C is the third member of the aurora serine/threonine kinase family and was found only in mammals. Because Aurora-C is overexpressed in many different types of cancer cells we decided to analyze the consequences of Aurora-C overexpression in human cells. We first investigated the subcellular localization of overexpressed GFP-Aurora-C in mitosis and interphase in HeLa cells. As expected, during mitosis, we found that Aurora-C mimics Aurora-B. Surprisingly, in few interphase cells, we found that Aurora-C localized to the centrosome, like Aurora-A. We then examined the phenotype generated by Aurora-C overexpression. Basically it looked similar to the phenotypes observed after overexpression of the other Aurora kinases. We observed an augmentation of polyploid cells containing more than two centrosomes. More interestingly this phenotype was aggravated in the absence of a functional p53. Although the physiological function of Aurora-C in somatic cells remains to be clarified, our results, just like for the two other Aurora kinases, raised the question of a role of Aurora-C in the development and progression of cancer especially in the presence of mutated p53.

Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition.

Aurora-A protein kinase, which is the product of an oncogene, is required for the assembly of a functional mitotic apparatus and the regulation of cell ploidy. Overexpression of Aurora-A in tumour cells has been correlated with cancer susceptibility and poor prognosis. Aurora-A activity is required for the recruitment of CDK1-cyclin B1 to the centrosome prior to its activation and the commitment of the cell to mitosis. In this report, we demonstrate that the CDC25B phosphatase, an activator of cyclin dependent kinases at mitosis, is phosphorylated both in vitro and in vivo by Aurora-A on serine 353 and that this phosphorylated form of CDC25B is located at the centrosome during mitosis. Knockdown experiments by RNAi confirm that the centrosome phosphorylation of CDC25B on S353 depends on Aurora-A kinase. Microinjection of antibodies against phosphorylated S353 results in a mitotic delay whilst overexpression of a S353 phosphomimetic mutant enhances the mitotic inducing effect of CDC25B. Our results demonstrate that Aurora-A phosphorylates CDC25B in vivo at the centrosome during mitosis. This phosphorylation might locally participate in the control of the onset of mitosis. These findings re-emphasise the role of the centrosome as a functional integrator of the pathways contributing to the triggering of mitosis.