Releasable SNAP-tag probes for studying endocytosis and recycling.
Site-specific labeling of cellular proteins with chemical probes is a powerful tool for live cell imaging of biological processes. One popular system, known as the SNAP-tag, is based on an engineered variant of the 20-kDa DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (AGT) that covalently reacts with O(6)-benzylguanine (BG) and can be derivatized with a number of reporter groups. For studying the endocytosis and recycling of cell surface proteins, the covalent nature of BG binding to the SNAP-tag is problematic, since removing excess noninternalized probe from the cell surface is not feasible. Here we describe a modification of the SNAP-tag technology that permits the rapid release of fluorescently labeled probes from the cell surface without affecting the population of labeled molecules sequestered within endosomes. This simple yet effective approach allows quantitative measurements of endocytosis and recycling in both imaging and biochemical assays and is especially useful when studying endosomal dynamics in live cells.
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, United States.
Nelson B
NB
Cole
Julie G
JG
Donaldson
D002460:Cell Line; D002470:Cell Survival; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D008565:Membrane Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase