TY  - JOUR
A1  - Lohse, Christian
A1  - Bock, Andreas
A1  - Maiellaro, Isabella
A1  - Hannawacker, Annette
A1  - Schad, Lothar R.
A1  - Lohse, Martin J.
A1  - Bauer, Wolfgang R.
T1  - Experimental and mathematical analysis of cAMP nanodomains
JF  - PLoS ONE
N2  - In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells.
KW  - fluorescence resonance energy transfer
KW  - yellow fluorescent protein
KW  - radii
KW  - adenylyl cyclase signaling cascade
KW  - cell fusion
KW  - cytosol
KW  - isoproterenol
KW  - absorption
KW  - cyclic nucleotides such as cyclic adenosine monophosphate
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170972
VL  - 12
IS  - 4
ER  - 
TY  - JOUR
A1  - Maiellaro, Isabella
A1  - Lohse, Martin J.
A1  - Kitte, Robert J.
A1  - Calebiro, Davide
T1  - cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons
JF  - Cell Reports
N2  - The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. Here, we suggest a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, we demonstrate the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, we provide evidence that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons.
KW  - cAMP
KW  - synaptic plasticity
KW  - PDE
KW  - octopamine
KW  - FRET
KW  - active zone
KW  - dunce
KW  - GPCR
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-162324
VL  - 17
IS  - 5
ER  - 
TY  - JOUR
A1  - Schihada, Hannes
A1  - Vandenabeele, Sylvie
A1  - Zabel, Ulrike
A1  - Frank, Monika
A1  - Lohse, Martin J.
A1  - Maiellaro, Isabella
T1  - A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics
JF  - Communications Biology
N2  - G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the alpha(2 Lambda)-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning beta(2)-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands.
KW  - Fluorescence resonance energy transfer
KW  - G protein-coupled receptors
KW  - High-throughput screening
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228592
VL  - 1
IS  - 105
ER  -