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A Fluorescent Live Imaging Screening Assay A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways Sandra Lecat, Hans Matthes, Rainer Pepperkok, Jeremy Simpson, Jean-Luc Galzi To cite this version: Sandra Lecat, Hans Matthes, Rainer Pepperkok, Jeremy Simpson, Jean-Luc Galzi. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Pro- teins Implicated in G Protein-coupled Receptor Signaling Pathways. Molecular and Cellular Pro- teomics, American Society for Biochemistry and Molecular Biology, 2015, 14 (5), pp.1385-1399. 10.1074/mcp.M114.046698. hal-02193773 HAL Id: hal-02193773 https://hal.archives-ouvertes.fr/hal-02193773 Submitted on 24 Jul 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Research © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*□S Sandra Lecat‡ʈ, Hans W.D. Matthes‡, Rainer Pepperkok§, Jeremy C. Simpson¶, and Jean-Luc Galzi‡ Several cytoplasmic proteins that are involved in G pro- PLEKHH2 rearranged from individual dots located near tein-coupled receptor signaling cascades are known to the cell-substrate adhesion surface into lines of dots. translocate to the plasma membrane upon receptor acti- The speriolin-like protein, SPATC1L, redistributed to cell- vation, such as beta-arrestin2. Based on this example and cell junctions. The Chloride intracellular Channel protein, in order to identify new cytoplasmic proteins implicated in CLIC2, translocated from actin-enriched plasma mem- the ON-and-OFF cycle of G protein-coupled receptor, a brane bundles to cell-cell junctions upon activation of live-imaging screen of fluorescently labeled cytoplasmic NK2 receptors. CLIC2, and one of its close paralogs, proteins was performed using translocation criteria. The CLIC4, were further shown to respond with the same screening of 193 fluorescently tagged human proteins translocation pattern to muscarinic M3 and lysophos- identified eight proteins that responded to activation of phatidic LPA receptors. This screen allowed us to identify the tachykinin NK2 receptor by a change in their intracel- potential actors in signaling pathways downstream of G lular localization. Previously we have presented the func- protein-coupled receptors and could be scaled-up for high- tional characterization of one of these proteins, REDD1, content screening. Molecular & Cellular Proteomics 14: that translocates to the plasma membrane. Here we re- 10.1074/mcp.M114.046698, 1385–1399, 2015. port the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated web- site. The proteins BAIAP3 and BIN1, partially translocated Eukaryotic cells have evolved to segregate cellular func- to the plasma membrane upon activation of NK2 recep- tions into specialized intracellular membrane compartments. tors. Proteins ARHGAP12 and PKM2 translocated to- Information regarding the precise intracellular localization of a ward membrane blebs. Three proteins that associate poorly characterized protein is thus important when evaluat- with the cytoskeleton were of particular interest : ing its function. However, localization per se, without addi- tional information, is not sufficient to indicate the potential From the ‡GPCRs, Pain and Inflammation Team, UMR7242, function of cytoplasmic, or cytoplasmic and nuclear proteins CNRS-University of Strasbourg, LabEx Medalis, 300 Bvd Se´bastien that account for almost half of the proteome diversity, when Brant, 67412 Illkirch, France; §European Molecular Biology Labora- compared with proteins located in dedicated organelles, such tory, Advanced Light Microscopy Facility, Meyerhofstr 1, 69117 Heidelberg, Germany; ¶¶School of Biology and Environmental Sci- as the Golgi apparatus or lysosomes. To tackle this need for ence and UCD Conway Institute, University College Dublin, Dublin 4, additional information, we have implemented a live-imaging Ireland screening strategy that is based on changes in localization of Received November 20, 2014, and in revised form, February 20, fluorescently labeled cytoplasmic proteins studied upon the 2015 activation of cells. Indeed, cytoplasmic and nuclear proteins Published, MCP Papers in Press, March 10, 2015, DOI 10.1074/ mcp.M114.046698 often respond to the activation of a given signaling cascade Author contributions: H.W.D.M. did cloning experiments, designed by translocating to another cell compartment, for instance by and prepared polyclonal antibodies, R.P. participated in the design of moving from the cytoplasm to the plasma membrane, or to the live-imaging screen, J.C.S. shared in the design and performance the nucleus. Classical signaling proteins that translocate fol- of the live-imaging screen, J.L.G. participated in the design and lowing receptor activation include AKT/PKB (1), raf1 (2), or follow-up of the project and in the writing of the manuscript, SL designed and coordinated the project and experiments, performed NF-KB (3). Therefore, finding that a given cytoplasmic protein research and interpreted the data, prepared the figures and wrote the responds to the activation of a given signaling cascade pro- manuscript. vides further insight into its function. Molecular & Cellular Proteomics 14.5 1385 GPCR-Induced Protein Translocation Studies dealing with the deciphering of intracellular local- origin of complex signaling cascades that depend on the ization of the proteome have mainly used mass-spectrometry conformation adopted by the receptor and on its capacity to based approaches (4, 5). Microscopy techniques have also stimulate intracellular effectors (now referred to as “biased been successfully used and validated: the localization of 97% agonism”) (14). Not all the signaling cascades following acti- of the yeast Saccharomyces cerevisiae proteome was vation have been fully characterized, making these receptors achieved by tagging each protein with the fluorescent GFP good targets to search for undiscovered cytoplasmic proteins protein (6). In human cells, a similar approach with a fluores- involved in their signaling pathway. cently tagged full-length ORF plasmid collection was used for The present article is dedicated to the full description of the the GFP-cDNA localization project (7) and it was also com- results obtained in this live-imaging screen. Previously, we pleted in mice with the Alliance for Cellular Signaling (8). The reported the identification and characterization of one of use of fluorescently tagged overexpressed proteins to probe these translocated proteins, REDD1, as a novel target of intracellular localization was validated by protein correlation GPCR signaling (15). The data are compared with the results profiling although less correlation was observed for cytosolic of phospho-proteome investigations of signaling pathways proteins (9). More recently, the ϽϽprotein AtlasϾϾ project has governed by GPCRs (16–22). found a good correlation between endogenous proteins lo- calization using immunofluorescence techniques compared EXPERIMENTAL PROCEDURES with intracellular localization of the same proteins as deter- Cell Culture—HEK293 cells and clonal cell lines stably expressing mined in the GFP-cDNA localization project (10). Therefore, either human NK2Gly361Glu Receptor, or CLIC2-mYFP or CLIC4- the cDNA library of this GFP-cDNA localization project was mYFP were cultivated as previously described (23). selected for our live-imaging screen: from this library, we Live-imaging Screening by Microscopy as Described in (15)—On screened the proteins that had been identified residing in the day 0, HEK293 cells expressing the non-fluorescent NK2-Gly361Glu receptor (NK2R-HEK cells) were plated into 8-well Labtek chambers cytoplasm or cytoplasm and nucleus. with glass-slides (NUNC). On day 1, 150 ng of individual vectors were The signaling pathway that we selected for our case study transiently transfected using the calcium phosphate technique. Each is governed by the Gq-coupled tachykinin NK2 receptor, a clone was transfected twice, in two separate eight-well chambers. prototypical G protein-coupled receptor (GPCR)1, that trig- Transfection efficiency was of at least 80% unless otherwise stated gers an elevation of intracellular calcium concentration upon (column headed “localization during screening” of the supplemental Table S1 and on the web site http://www.gfpcdnalive-gpcr.cnrs.fr). activation with the neuropeptide, neurokinin A (11). GPCRs Each fluorescent protein was observed after 24 h of expression under share a common overall structure with 7-transmembrane al- the spinning-disk ultraview confocal microscope (Perkin Elmer) pha-helices embedded in the lipid bilayer mostly at the cell equipped
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