Premium
A small optogenetic tool for spatiotemporal control of intracellular cAMP
Author(s) -
O'Banion Colin Padraic,
Lawrence David S
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.566.1
Subject(s) - optogenetics , microbiology and biotechnology , signal transduction , protein kinase a , cyclic adenosine monophosphate , intracellular , cell signaling , biology , phosphorylation , chemistry , biochemistry , neuroscience , receptor
Cellular signaling is highly compartmentalized in both time and space as exemplified by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway. PKA and associated signaling proteins are sequestered to specific subcellular compartments by A‐kinase anchoring proteins to generate distinct signaling microenvironments. These signaling nodes provide spatial specificity to PKA so that this otherwise ubiquitous signaling pathway is only activated in the right location and at the right time. Although many tools are available to manipulate cellular signaling on a global scale, it is difficult to control intracellular signaling with any degree of spatiotemporal resolution. In order to overcome this challenge, we have generated a light‐activated adenylate cyclase (bPAC) targeted to the plasma membrane, nucleus, outer mitochondrial membrane, inner mitochondrial membrane, and mitochondrial matrix as an optogenetic tool which provides high spatial and temporal resolution over the cAMP/PKA pathway. We used molecular engineering approaches in conjunction with biochemical and cell biology assays such as western blotting and fluorescent microscopy to show that bPAC activation results in compartment specific PKA phosphorylation events upon stimulation with light, and that activity is localized to discrete locations within the cell using mitochondria as a model for compartmentalized signaling.