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Genetically targeted ratiometric and activated pH indicator complexes (TRApHIC) for receptor trafficking.
Biochemistry 57, 861-871 (2018)
Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged beta 2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Red Fluorescent Protein; Beta(2)-adrenergic Receptor; Surface-proteins; Intracellular Ph; Cell; Probes; Internalization; Endocytosis; Microscopy; Pathways
ISSN (print) / ISBN
0006-2960
e-ISSN
1520-4995
Journal
Biochemistry
Quellenangaben
Volume: 57,
Issue: 5,
Pages: 861-871
Publisher
American Chemical Society (ACS)
Publishing Place
Washington
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Human Genetics (IHG)