title: Imaging sub-plasma membrane cAMP dynamics with fluorescent translocation reporters.
abstract: 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 Ca(2+) 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.
authors.affiliation: Department of Medical Cell Biology, Biomedical Centre, Uppsala University, Husargatan 3, Box 571, 751 23, Uppsala, Sweden, anders.tengholm@mcb.uu.se.
authors.forename: Anders
authors.initials: A
authors.lastname: Tengholm
authors.forename: Olof
authors.initials: O
authors.lastname: Idevall-Hagren
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008857:Microscopy, Interference / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein