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      MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures

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          Abstract

          Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

          Author Summary

          In this paper we present the software package Molecular Interactions in Super Resolution (MIiSR), which provides a series of quantitative analytical tools for measuring molecular interactions and the formation of higher-order molecular complexes in super-resolution microscopy images.

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          Most cited references41

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          Direct stochastic optical reconstruction microscopy with standard fluorescent probes.

          Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ∼20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.
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            Breaking the diffraction barrier: super-resolution imaging of cells.

            Anyone who has used a light microscope has wished that its resolution could be a little better. Now, after centuries of gradual improvements, fluorescence microscopy has made a quantum leap in its resolving power due, in large part, to advancements over the past several years in a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM. Then, we describe recent applications of super-resolution microscopy in cells, which demonstrate how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology. Copyright © 2010 Elsevier Inc. All rights reserved.
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              Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling.

              G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Comput Biol
                PLoS Comput. Biol
                plos
                ploscomp
                PLoS Computational Biology
                Public Library of Science (San Francisco, CA USA )
                1553-734X
                1553-7358
                11 December 2015
                December 2015
                : 11
                : 12
                : e1004634
                Affiliations
                [1 ]The J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada
                [2 ]Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
                [3 ]Department of Physics and Astronomy, The University of Western Ontario, London, Ontario, Canada
                [4 ]Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
                [5 ]Department of Clinical Neurological Sciences, Schulich School of Medicine, The University of Western Ontario, London, Ontario, Canada
                Johns Hopkins University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: BH JRdB JDD SHP SSGF. Performed the experiments: FAC BSD JHKT PCC MG. Analyzed the data: FAC BSD JHKT BH. Contributed reagents/materials/analysis tools: BH JRdB JDD SHP SSGF. Wrote the paper: BH JRdB JDD SHP SSGF FAC BSD.

                Article
                PCOMPBIOL-D-15-00682
                10.1371/journal.pcbi.1004634
                4676698
                26657340
                8b8935bd-2af8-44ee-89e2-a5919c37eaf9
                © 2015 Caetano et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                History
                : 28 April 2015
                : 27 October 2015
                Page count
                Figures: 7, Tables: 1, Pages: 30
                Funding
                This study was funded in part by a Canadian Institutes of Health Research (CIHR) operating grants MOP-123419 (BH), MOP 119437 and MOP 111093 (SSGF), MOP 130465 (JDD) and MOP 82890 (SHP), as well as National Sciences and Engineering Research Counsel of Canada (NSERC) discovery grants (BH, JDD, JRdB). JHKT is funded by an Ontario Graduate Scholarship. MG is funded by a NSERC graduate scholarship. SSGF is a Career Investigator for the Heart and Stroke Foundation of Ontario. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                All molecular position files (Leica .ascii format) used to generate the figures in this paper, as well as the scripts used for modeled data (Matlab .m and .mat files), are available for download from the Scholars Portal Dataverse: http://dataverse.scholarsportal.info/dvn/dv/MIiSR

                Quantitative & Systems biology
                Quantitative & Systems biology

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