Article Cite This: Biochemistry 2018, 57, 861−871
pubs.acs.org/biochemistry
Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking Lydia A. Perkins,†,§ Qi Yan,∥ Brigitte F. Schmidt,§,‡ Dmytro Kolodieznyi,§,‡ Saumya Saurabh,⊥ Mads Breum Larsen,# Simon C. Watkins,# Laura Kremer,@ and Marcel P. Bruchez*,†,§,‡ †
Department of Biological Sciences, ‡Department of Chemistry, and §Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ∥ Sharp Edge Laboratories, Pittsburgh, Pennsylvania 15203, United States ⊥ Department of Developmental Biology, Stanford University, Stanford, California 94305, United States # Center for Biologic Imaging, Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, United States @ Institute of Human Genetics, Helmholtz Zentrum München, Munich, Germany S Supporting Information *
ABSTRACT: 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 β2adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling. To tackle these challenges, fluorescent protein (FP)-based sensors have been developed to study intracellular pH changes. (Super)ecliptic pHluorin exhibits a weak fluorescence signal at pH 0.85). The boxplot whiskers represent the 90th and 10th percentiles. The experimental data were also binned to 0.05
endocytosis and recycling conditions (Figures 2a and 3a). The 75th, median, and 25th percentile were calculated at every time point, and the median was fitted to a sigmoidal curve to 864
DOI: 10.1021/acs.biochem.7b01135 Biochemistry 2018, 57, 861−871
Article
Biochemistry
Table 1. Comparison of Dyes (5−8) in the Presence of dL5**, with Reported Percent Quantum Yields (QY) and x-fold Activation, Rmax/Rmin, pKa, and Kd Values quantum yield (%)
x-fold activation
dye
pH 5
pH 8
pH 5
pH 8
Rmax/Rmina
pKa
Kd (nM)
Cy3(S/S)pH−MG (5) Cy3(S/SA)pH−MG (6) Cy3(SA/SA)pH−MG (7) Cy3−MG (8)
21.2 21.8 21.2 25.1
17.4 13 10.4 16.2
321 91 67 136
116 122 49 110
6.6 6.3 7.8 not available
7.5 7 6.6 not available
