The CryoCapsule : Simplifying Correlative Light to Electron Microscopy

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin

Ubiquitylation of the nuclear pore complex controls nuclear migration during mitosis in S. cerevisiae.

International audienceNuclear pore complexes (NPCs) correspond to large protein transport complexes responsible for selective nucleocytoplasmic exchange. Although research has revealed much about the molecular architecture and roles of the NPC subcomplexes, little is known about the regulation of NPC functions by posttranslational modifications. We used a systematic approach to show that more than half of NPC proteins were conjugated to ubiquitin. In particular, Nup159, a nucleoporin exclusively located on the cytoplasmic side of the NPC, was monoubiquitylated by the Cdc34/SCF (Skp1-Cdc53-F-box E3 ligase) enzymes. Preventing this modification had no consequences on nuclear transport or NPC organization but strongly affected the ability of Nup159 to target the dynein light chain to the NPC. This led to defects in nuclear segregation at the onset of mitosis. Thus, defining ubiquitylation of the yeast NPC highlights yet-unexplored functions of this essential organelle in cell division

Use of micro-rotation imaging to study JNK-mediated cell survival in Theileria parva-infected B-lymphocytes

Lymphocytes infected with the protozoan parasite Theileria parva are transformed to permanently proliferating cells, an event underlying the pathology of the disease. However, the molecular signalling mediating this process is complex and poorly understood. Here, we show that down-regulation of JNK signalling by transient over expression of a dominant-negative mutant of JNK (JNK-APF) significantly increases Annexin-V-phycoerythrin (V-PE) labelling on infected B cell populations observed using flow cytometry. To establish whether this increase was specifically due to apoptosis, we used a novel single-cell imaging method: micro-rotation (MR)-imaging, designed to allow high-resolution 3-dimensional imaging of single cells in suspension. With this method we visualized subcellular patterns of V-PE uptake and chromatin organization in lymphocytes co-transfected with JNK-APF and GFP-tagged histone-H2B. This single-cell approach allowed us to clearly reveal characteristic apoptotic phenotypes, whose patterns reflected progressive states of programmed cell death due to JNK down-regulation. Our results strongly suggest a role for JNK in the survival of Theileria-infected B cells, and demonstrate the powerful utility of a new and unique 3-dimensional imaging method for living cells in suspension.Peer Reviewe

A 2-D spectral analysis method to estimate the modulation parameters in structured illumination microscopy

International audienceStructured illumination microscopy is a recent imaging technique that aims at going beyond the classical optical resolution limits by reconstructing a high-resolution image from several low-resolution images acquired through modulation of the transfer function of the microscope. A precise knowledge of the sinusoidal modulation parameters is necessary to enable the super-resolution effect expected after reconstruction. In this work, we investigate the retrieval of these parameters directly from the acquired data, using a novel 2-D spectral estimation method

Nonsmooth Convex Optimization for Structured Illumination Microscopy Image Reconstruction

International audienceIn this paper, we propose a new approach for structured illumination microscopy image reconstruction. We first introduce the principles of this imaging modality and review its properties in various conditions. We then propose the minimization of nonsmooth convex functionals for the recovery of the unknown image and investigate several data–fitting and regularization terms in order to tackle reconstruction of noisy data. More specifically, we consider an original approach based on sparse local patch dictionaries for the regularization of the estimate. We demonstrate the good performance of the proposed approach on a test benchmark and perform some test experiments on images acquired on two different microscopes

TRAIL protein localization in human primary T cells by 3D microscopy using 3 3D interactive surface plot: A new method to visualize plasma membrane

Submitted by sandra infurna ([email protected]) on 2016-01-26T14:29:39Z No. of bitstreams: 1 manana_gandini_etal_IOC_2013.pdf: 1462953 bytes, checksum: 22b537ab238684e0c3e99ee139a17e93 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-01-26T14:41:55Z (GMT) No. of bitstreams: 1 manana_gandini_etal_IOC_2013.pdf: 1462953 bytes, checksum: 22b537ab238684e0c3e99ee139a17e93 (MD5)Made available in DSpace on 2016-01-26T14:41:55Z (GMT). No. of bitstreams: 1 manana_gandini_etal_IOC_2013.pdf: 1462953 bytes, checksum: 22b537ab238684e0c3e99ee139a17e93 (MD5) Previous issue date: 2013CNRS UMR 8147. Université Paris Descartes. Paris, France.CNRS UMR 8601. Université Paris Descartes. Paris, France.Cell and Tissue Imaging Core Facility (PICT-IBiSA) and Nikon Imaging Centre@Institut Curie-CNRS, UMR144, Institut Curie. Centre de Recherche. Paris, France / The PICT-IBiSA Nikon Imaging Facility Institut Curie-CNRS, Paris, France.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Viral. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Viral. Rio de Janeiro, RJ, Brasil.CNRS UMR 8147. Université Paris Descartes. Paris, France.The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4+ T cells is not known. Here we investigated whether primary CD4+ T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4+ T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images

Spindle pole cohesion requires glycosylation-mediated localization of NuMA

Glycosylation is critical for the regulation of several cellular processes. One glycosylation pathway, the unusual O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) has been shown to be required for proper mitosis, likely through a subset of proteins that are O-GlcNAcylated during metaphase. As lectins bind glycosylated proteins, we asked if specific lectins interact with mitotic O-GlcNAcylated proteins during metaphase to ensure correct cell division. Galectin-3, a small soluble lectin of the Galectin family, is an excellent candidate, as it has been previously described as a transient centrosomal component in interphase and mitotic epithelial cells. In addition, it has recently been shown to associate with basal bodies in motile cilia, where it stabilizes the microtubule-organizing center (MTOC). Using an experimental mouse model of chronic kidney disease and human epithelial cell lines, we investigate the role of Galectin-3 in dividing epithelial cells. Here we find that Galectin-3 is essential for metaphase where it associates with NuMA in an O-GlcNAcylation-dependent manner. We provide evidence that the NuMA-Galectin-3 interaction is important for mitotic spindle cohesion and for stable NuMA localization to the spindle pole, thus revealing that Galectin-3 is a novel contributor to epithelial mitotic progress

Myosin 1b and F-actin are involved in the control of secretory granule biogenesis

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Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

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Myosin 1b and F-actin are involved in the control of secretory granule biogenesis

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.This work was supported by Institut National de la Santé et de la Recherche Médicale, the University of Rouen Normandy, the Conseil Régional de Normandie and the Ministère de l’Enseignement Supérieur et de la RecherchePeer Reviewe