Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation.

International audienceVezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

International audienceDrosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra-and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes

Breaking the Dynamic Instability of the Actomyosin Cortex Triggered by a Single Mechanical Cue Drives Cell Polarity in Non-Adherent Cells

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Fluorescence in situ hybridization (FISH) of Microcystis strains producing microcystin using specific mRNA probes : supplementary materials

Supplementary data related to Zeller et al. 201

Fluorescence in situ hybridization (FISH) of Microcystis strains producing microcystin using specific mRNA probes : supplementary materials

Supplementary data related to Zeller et al. 201

Fluorescence in situ hybridization (FISH) of Microcystis strains producing microcystin using specific mRNA probes : supplementary materials

Supplementary data related to Zeller et al. 201

Nurselike cells sequester B cells in disorganized lymph nodes in chronic lymphocytic leukemia via alternative production of CCL21

International audienceAbstract Tumor microenvironment exerts a critical role in sustaining homing, retention, and survival of chronic lymphocytic leukemia (CLL) cells in secondary lymphoid organs. Such conditions foster immune surveillance escape and resistance to therapies. The physiological microenvironment is rendered tumor permissive by an interplay of chemokines, chemokine receptors, and adhesion molecules as well as by direct interactions between malignant lymphocytes and stromal cells, T cells, and specialized macrophages referred to as nurselike cells (NLCs). To characterize this complex interplay, we investigated the altered architecture on CLL lymph nodes biopsies and observed a dramatic loss of tissue subcompartments and stromal cell networks as compared with nonmalignant lymph nodes. A supplemental high density of CD68+ cells expressing the homeostatic chemokine CCL21 was randomly distributed. Using an imaging flow cytometry approach, CCL21 mRNA and the corresponding protein were observed in single CD68+ NLCs differentiated in vitro from CLL peripheral blood mononuclear cells. The chemokine was sequestered at the NLC membrane, helping capture of CCR7-high-expressing CLL B cells. Inhibiting the CCL21/CCR7 interaction by blocking antibodies or using therapeutic ibrutinib altered the adhesion of leukemic cells. Our results indicate NLCs as providers of an alternative source of CCL21, taking over the physiological task of follicular reticular cells, whose network is deeply altered in CLL lymph nodes. By retaining malignant B cells, CCL21 provides a protective environment for their niching and survival, thus allowing tumor evasion and resistance to treatment. These findings argue for a specific targeting or reeducation of NLCs as a new immunotherapy strategy for this disease

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry.

BACKGROUND: The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface.RESULTS: Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells.CONCLUSION: The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs