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      Tailored co-localization analysis of intracellular microbes and punctum-distributed phagosome–lysosome pathway proteins using ImageJ plugin EzColocalization

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          Abstract

          Immunofluorescence is indispensable to monitor redistribution of proteins involved in phagosome–lysosome association pathway-relevant (P–LApr) proteins. The software digitizing the signals of these proteins in an unbiased and automated manner is generally costly and not widely available. The open-source ImageJ plugin EzColocalization, which is for co-localization analysis of reporters in cells, was not straightforward and sufficient for such analysis. We describe here the input of custom Java code in a novel tailored protocol using EzColocalization to digitize the signals of punctum-distributed P–LApr proteins co-localized with phagosomes and to calculate percentages of phagosomes engaged. We showed that SYBR Gold nucleic acid dye could visualize intracellular mycobacteria that did not express a fluorescent protein. This protocol was validated by showing that IFN-γ enhanced the co-localization of a punctum-distributed P–LApr protein (LC3) with Mycobacterium bovis BCG in the monocyte/macrophage-like RAW264.7 cells and that there was greater co-localization of LC3 with BCG than with M. tuberculosis H37Rv in bone marrow-derived macrophages (BMDMs). Although BCG and a derived strain (rBCG-PA) showed a similarly high degree co-localization with LC3 in BMDMs, in RAW264.7 cells BCG showed much less co-localization with LC3 than rBCG-PA indicating the need for caution in interpreting biological significance from studies in cell lines.

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          Most cited references 23

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          Fiji: an open-source platform for biological-image analysis.

          Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
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            Extracellular M. tuberculosis DNA targets bacteria for autophagy by activating the host DNA-sensing pathway.

            Eukaryotic cells sterilize the cytosol by using autophagy to route invading bacterial pathogens to the lysosome. During macrophage infection with Mycobacterium tuberculosis, a vacuolar pathogen, exogenous induction of autophagy can limit replication, but the mechanism of autophagy targeting and its role in natural infection remain unclear. Here we show that phagosomal permeabilization mediated by the bacterial ESX-1 secretion system allows cytosolic components of the ubiquitin-mediated autophagy pathway access to phagosomal M. tuberculosis. Recognition of extracelluar bacterial DNA by the STING-dependent cytosolic pathway is required for marking bacteria with ubiquitin, and delivery of bacilli to autophagosomes requires the ubiquitin-autophagy receptors p62 and NDP52 and the DNA-responsive kinase TBK1. Remarkably, mice with monocytes incapable of delivering bacilli to the autophagy pathway are extremely susceptible to infection. Our results reveal an unexpected link between DNA sensing, innate immunity, and autophagy and indicate a major role for this autophagy pathway in resistance to M. tuberculosis infection. Copyright © 2012 Elsevier Inc. All rights reserved.
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              A guided tour into subcellular colocalization analysis in light microscopy.

              It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.
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                Author and article information

                Contributors
                litaokc@126.com
                xyfan008@fudan.edu.cn
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                13 January 2021
                13 January 2021
                2021
                : 11
                Affiliations
                [1 ]GRID grid.8547.e, ISNI 0000 0001 0125 2443, Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology of MOE/MOH, , Fudan University, ; Shanghai, 201508 China
                [2 ]TB Center, Shanghai Emerging and Re-emerging Institute, Shanghai, 201508 China
                Article
                79425
                10.1038/s41598-020-79425-5
                7807018
                33441638
                © The Author(s) 2021

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31771004
                Award ID: 81301407
                Award ID: 81770011
                Funded by: Chinese National Mega Science and Technology Program on Infectious Diseases
                Award ID: 2018ZX10302301
                Award ID: 2018ZX10731301
                Categories
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                © The Author(s) 2021

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                cellular microbiology, data processing

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