Dataset Information

Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.

ABSTRACT: Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.

SUBMITTER: O'Banion CP

PROVIDER: S-EPMC6800631 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.

O'Banion Colin P CP Vickerman Brianna M BM Haar Lauren L Lawrence David S DS

Cell chemical biology 20190725 10

Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is ...[more]

PMID: 31353320

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Project description:Adenosine 3',5'-cyclic monophosphate (cAMP) is a key second messenger that activates several signal transduction pathways in eukaryotic cells. Alteration of basal levels of cAMP is known to activate protein kinases, regulate phosphodiesterases and modulate the activity of ion channels such as Hyper polarization-activated cyclic nucleotide gated channels (HCN). Recent advances in optogenetics have resulted in the availability of novel genetically encoded molecules with the capability to alter cytoplasmic profiles of cAMP with unprecedented spatial and temporal precision. Using single molecule based super-resolution microscopy and different optogenetic modulators of cellular cAMP in both live and fixed cells, we illustrate a novel paradigm to report alteration in nanoscale confinement of ectopically expressed HCN channels. We characterized the efficacy of cAMP generation using ensemble photoactivation of different optogenetic modulators. Then we demonstrate that local modulation of cAMP alters the exchange of membrane bound HCN channels with its nanoenvironment. Additionally, using high density single particle tracking in combination with both acute and chronic optogenetic elevation of cAMP in the cytoplasm, we show that HCN channels are confined to sub 100 nm sized functional domains on the plasma membrane. The nanoscale properties of these domains along with the exchange kinetics of HCN channels in and out of these molecular zones are altered upon temporal changes in the cytoplasmic cAMP. Using HCN2 point mutants and a truncated construct of HCN2 with altered sensitivity to cAMP, we confirmed these alterations in lateral organization of HCN2 to be specific to cAMP binding. Thus, combining these advanced non-invasive paradigms, we report a cAMP dependent ensemble and single particle behavior of HCN channels mediated by its cyclic nucleotide binding domain, opening innovative ways to dissect biochemical pathways at the nanoscale and real-time in living cells.

Dataset Information

Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.

Publications

Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.

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