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Imaging sub-plasma membrane cAMP dynamics with fluorescent translocation reporters
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Tengholm)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
2015 (English)In: cAMP Signaling: Methods And Protocols, Springer-Verlag New York, 2015, Vol. 1294, 85-101 p.Chapter in book (Refereed)
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer-Verlag New York, 2015. Vol. 1294, 85-101 p.
Series
, Methods in Molecular Biology, ISSN 1064-3745 ; 1294
Keyword [en]
cAMP oscillations; Plasma membrane; Protein kinase A; Translocation; Total internal reflection fluorescence
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-300477DOI: 10.1007/978-1-4939-2537-7_7ISI: 000369927200008PubMedID: 25783879ISBN: 9781493925377ISBN: 9781493925360OAI: oai:DiVA.org:uu-300477DiVA: diva2:951478
Available from: 2016-08-09 Created: 2016-08-09 Last updated: 2016-09-01Bibliographically approved

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Tengholm, AndersIdevall-Hagren, Olof
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