title: Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.
abstract: Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process.
authors.affiliation: CNRS, LAAS, 7 avenue du Colonel Roche, 31400, Toulouse, France.|Université de Toulouse, LAAS, ITAV, IPBS, 31400, Toulouse, France.|CNRS, UMR 7565, SRSMC, Vandœuvre-lès-Nancy, France.|Université de Lorraine, UMR 7565, Faculté de Pharmacie, Nancy, France.
authors.forename: C
authors.initials: C
authors.lastname: Formosa
authors.affiliation: CNRS, LAAS, 7 avenue du Colonel Roche, 31400, Toulouse, France.|Université de Toulouse, LAAS, ITAV, IPBS, 31400, Toulouse, France.|Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la Recherche Médicale U1048, Université Toulouse III Paul Sabatier, 31432, Toulouse, France.|CNRS, ITAV, 1 Place Pierre Potier, 31000, Toulouse, France.
authors.forename: V
authors.initials: V
authors.lastname: Lachaize
authors.affiliation: Université de Toulouse, LAAS, ITAV, IPBS, 31400, Toulouse, France.|Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la Recherche Médicale U1048, Université Toulouse III Paul Sabatier, 31432, Toulouse, France.|CNRS, ITAV, 1 Place Pierre Potier, 31000, Toulouse, France.
authors.forename: C
authors.initials: C
authors.lastname: Galés
authors.affiliation: Université de Toulouse, LAAS, ITAV, IPBS, 31400, Toulouse, France.|CNRS, IPBS, UMR 5089, 205 route de Narbonne, 31077, Toulouse, France.
authors.forename: M P
authors.initials: MP
authors.lastname: Rols
authors.affiliation: Université de Toulouse, LAAS, ITAV, IPBS, 31400, Toulouse, France.|INRA, UMR 972 LISBP, Toulouse, France.
authors.forename: H
authors.initials: H
authors.lastname: Martin-Yken
authors.affiliation: Université de Toulouse, LAAS, ITAV, IPBS, 31400, Toulouse, France.|INRA, UMR 972 LISBP, Toulouse, France.
authors.forename: J M
authors.initials: JM
authors.lastname: François
authors.affiliation: CNRS, UMR 7565, SRSMC, Vandœuvre-lès-Nancy, France.|Université de Lorraine, UMR 7565, Faculté de Pharmacie, Nancy, France.|ABC Platform®, Nancy, France.
authors.forename: R E
authors.initials: RE
authors.lastname: Duval
authors.affiliation: CNRS, LAAS, 7 avenue du Colonel Roche, 31400, Toulouse, France.|Université de Toulouse, LAAS, ITAV, IPBS, 31400, Toulouse, France.|CNRS, ITAV, 1 Place Pierre Potier, 31000, Toulouse, France.
authors.forename: E
authors.initials: E
authors.lastname: Dague
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D003412:Cricetulus; D005656:Fungal Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006388:Hemagglutinins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007251:Influenza, Human / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D011941:Receptors, Adrenergic / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C000605690:Ccw12 protein, S cerevisiae; D005656:Fungal Proteins; D006388:Hemagglutinins; D008562:Membrane Glycoproteins; D010455:Peptides; D011941:Receptors, Adrenergic; D029701:Saccharomyces cerevisiae Proteins
keywords: AFM; GPCR; single‐molecule recognition; yeast