Repetitive reversible labeling of proteins at polyhistidine sequences for single-molecule imaging in live cells.
Sensitive live-cell fluorescence microscopy and single-molecule imaging are severely limited by rapid photobleaching of fluorescent probes. Herein, we show how to circumvent this problem using a novel, generic labeling strategy. Small nickel-nitrilotriacetate fluorescent probes are reversibly bound to oligohistidine sequences of exposed proteins on cell surfaces, permitting selective observation of the proteins by fluorescence microscopy. Photobleached probes are removed by washing and replaced by new fluorophores, thus enabling repetitive acquisition of single-molecule trajectories on the same cell and allowing variation of experimental conditions between acquisitions. This method offers free choice of fluorophores while being minimally perturbing. The strength of the method is demonstrated by labeling engineered polyhistidine sequences of the serotonin-gated 5-HT(3) receptor on the surface of live mammalian cells. Single-molecule microscopy reveals pronounced heterogeneous mobility patterns of the 5-HT(3) receptor. After activating the receptor with serotonin, the number of immobile receptors increases substantially, which might be important for receptor regulation at synapses.
Guignet
Emmanuel G
EG
Laboratory of Physical Chemistry of Polymers and Membranes, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
Segura
Jean-Manuel
JM
Hovius
Ruud
R
Vogel
Horst
H
D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002470:Cell Survival; D004847:Epithelial Cells / Q000378:metabolism*; D005456:Fluorescent Dyes; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D011487:Protein Conformation; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism
D005456:Fluorescent Dyes; D044406:Receptors, Serotonin, 5-HT3; C033223:polyhistidine; D012701:Serotonin; D006639:Histidine