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      Imaging Polarized Secretory Traffic at the Immune Synapse in Living T Lymphocytes

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          Abstract

          Immune synapse (IS) formation by T lymphocytes constitutes a crucial event involved in antigen-specific, cellular and humoral immune responses. After IS formation by T lymphocytes and antigen-presenting cells, the convergence of secretory vesicles toward the microtubule-organizing center (MTOC) and MTOC polarization to the IS are involved in polarized secretion at the synaptic cleft. This specialized mechanism appears to specifically provide the immune system with a fine strategy to increase the efficiency of crucial secretory effector functions of T lymphocytes, while minimizing non-specific, cytokine-mediated stimulation of bystander cells, target cell killing and activation-induced cell death. The molecular bases involved in the polarized secretory traffic toward the IS in T lymphocytes have been the focus of interest, thus different models and several imaging strategies have been developed to gain insights into the mechanisms governing directional secretory traffic. In this review, we deal with the most widely used, state-of-the-art approaches to address the molecular mechanisms underlying this crucial, immune secretory response.

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          Most cited references65

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          T cell receptor antagonist peptides induce positive selection.

          We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic beta 2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These peptide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive selection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this receptor. One peptide tested, E1, induced positive selection on the beta 2M(-/-) background but negative selection on the beta 2M(+/-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy ligands mediate positive selection.
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            A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.

            The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.
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              Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations

              Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm. Meanwhile, for data sets similar to those obtained in PALM or STORM imaging, SRRF achieves resolutions approaching those of standard single-molecule localization analysis. The broad applicability of SRRF and its performance at low signal-to-noise ratios allows super-resolution using modern widefield, confocal or TIRF microscopes with illumination orders of magnitude lower than methods such as PALM, STORM or STED. We demonstrate this by super-resolution live-cell imaging over timescales ranging from minutes to hours.
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                Author and article information

                Contributors
                Journal
                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                1664-3224
                06 April 2018
                2018
                : 9
                : 684
                Affiliations
                Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM , Madrid, Spain
                Author notes

                Edited by: Jorge Bernardino De La Serna, Science and Technology Facilities Council, United Kingdom

                Reviewed by: Noa B. Martin-Cofreces, Instituto de Investigación Sanitaria del Hospital Universitario de La Princesa, Spain; Dylan Myers Owen, University of New South Wales, Australia

                *Correspondence: Manuel Izquierdo, mizquierdo@ 123456iib.uam.es

                Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology

                Article
                10.3389/fimmu.2018.00684
                5897431
                29681902
                82621721-8955-4673-9f70-90c6fafc7c87
                Copyright © 2018 Calvo and Izquierdo.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 20 December 2017
                : 20 March 2018
                Page count
                Figures: 1, Tables: 1, Equations: 0, References: 90, Pages: 13, Words: 11181
                Funding
                Funded by: Ministerio de Economía y Competitividad 10.13039/501100003329
                Award ID: SAF2016-77561-R
                Categories
                Immunology
                Review

                Immunology
                t lymphocytes,immune synapse,secretory granules,multivesicular bodies,exosomes,cytotoxic activity,cell death

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