Imaging Sub-plasma Membrane cAMP Dynamics with Fluorescent Translocation Reporters
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Imaging cAMP dynamics in single cells and tissues can provide important insights into the regulation of a variety of cellular processes. In recent years, a large number of tools for cAMP measurements have been developed. While most cAMP reporters are designed to undergo changes in fluorescence resonance energy transfer (FRET), there are alternative techniques with advantages for certain applications. Here, we describe protocols for cAMP measurements in the sub-plasma membrane space based on the detection of the cAMP-induced translocation of engineered fluorescent protein-tagged subunits of protein kinase A between the cytoplasm and the plasma membrane. Total internal reflection fluorescence (TIRF) imaging of the changes in reporter localization yields robust signal changes and has contributed to the discovery of cAMP oscillations in the sub-plasma membrane space of insulin-secreting β-cells stimulated with glucose and gluco-incretin hormones. We also demonstrate how the technique can be combined with measurements of the cytosolic Ca2+ concentration or with recordings of the subcellular localization of the cAMP effector protein Epac2. The translocation reporter approach provides a valuable complement to other methods for imaging sub-membrane cAMP dynamics in various types of cells.
KeywordscAMP oscillations Plasma membrane Protein kinase A Translocation Total internal reflection fluorescence
We thank Drs Hongyan Shuai and Geng Tian for their help with the preparation of Figs. 3 and Fig. 4. The authors’ work is supported by grants from the European Foundation for the Study of Diabetes, the family Ernfors Foundation, the Novo Nordisk Foundation, the Swedish Diabetes Association, and the Swedish Research Council (67X-14643, 67P-21262, 325-2012-6778, 524-2013-298).