Imaging sub-plasma membrane cAMP dynamics with fluorescent translocation reporters

Methods in Molecular Biology
Anders Tengholm, Olof Idevall-Hagren

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 imagi...Continue Reading

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.