Peptide tags for labeling membrane proteins in live cells with multiple fluorophores.
We describe a method to label specific membrane proteins with fluorophores for live imaging. Fusion proteins are generated that incorporate into their extracellular domains short peptide sequences (13-38 amino acids) recognized with high affinity and specificity by protein ligands, alpha-bungarotoxin (BTX), or streptavidin (SA). Many fluorophore- and enzyme-conjugated derivatives of both ligands are commercially available. To demonstrate the general utility of the methods, we tagged a vesicle-associated protein (VAMP2), a receptor tyrosine kinase [muscle-specific kinase (MuSK)], and receptors for three neurotransmitters: acetylcholine (nAChR alpha3), glutamate (mGluR2), and gamma-aminobutyric acid (GABA(A) alpha3). In all cases, we could selectively label surface-exposed proteins without interference from intracellular pools. By successive pulse-labeling with different fluorophore conjugates of a single ligand, we were able to monitor endocytosis of tagged molecules. By combining the two ligands, we could assess co-localization of synaptic components in cells. This strategy for epitope tagging provides a useful adjunct to green fluorescent protein (GFP)-tagging, which fails to distinguish intracellular from extracellular pools, sometimes interferes with protein localization or function, and requires a separate construct for each color.
McCann
Corey M
CM
Harvard University, Cambridge, MA, USA.
Bareyre
Florence M
FM
Lichtman
Jeff W
JW
Sanes
Joshua R
JR
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D014162:Transfection
D017931:DNA Primers; D000939:Epitopes; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010455:Peptides