CaMello-XR enables visualization and optogenetic control of Gq/11 signals and receptor trafficking in GPCR-specific domains

Abstract

The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.

Signaling in the primary cilium through the lens of the Hedgehog pathway

“…Additional HH components regulate GLI transport and processing, mediating pathway activation. Kinesin family member 7 (KIF7) is responsible for moving GLI2 and GLI3 to the ciliary tip by IFT (Endoh-Yamagami et al, 2009;Haycraft et al, 2005;Liem et al, 2009;S. Y. Tay et al, 2005).…”

“…Similarly, by adapting biosensors to the ciliary environment, the lipid composition of the membrane, Ca 2+ flux and cAMP dynamics can be followed in vivo (Delling et al, 2016;Jiang, Falcone, Curci, & Hofer, 2019;Mukherjee et al, 2016;Su et al, 2013;Yuan et al, 2015). Optogenetic versions of such constructs can manipulate these same aspects of ciliary biology (Eickelbeck et al, 2019;Jansen et al, 2015). Finally, as the processes that regulate ciliary localization and signaling are better understood, in vivo gene editing techniques will enable researchers to target precise mutations that disrupt specific aspects of these processes, in contrast to removing cilia all together (Gigante et al, 2019).…”

Expression of serotonin 1A and 2A receptors in molecular- and projection-defined neurons of the mouse insular cortex

“…Guinea pig polyclonal anti-NeuN (1:500; 266004, SYSY) [35], mouse monoclonal anti-GAD67 (1:500; MAB5406, Millipore) [36][37][38], rabbit polyclonal anti-5-HT1A receptor (1:500; ADI-905-741, Enzo) [39] and rabbit polyclonal anti-5-HT2A receptor (1:250; ab66049, Abcam) [40][41][42][43][44] were used as primary antibodies. The primary antibodies used in this study were validated and widely used in previous publications [35][36][37][38][39][40][41][42][43][44]. Alexa Fluor 488 goat anti-rabbit IgG (A11034, Fisher Scientific), Alexa Fluor 555 goat antiguinea pig IgG (A21435, Fisher Scientific) and Alexa Fluor 647 goat anti-mouse IgG (A21236, Fisher Scientific) were used as secondary antibodies.…”

Reverse optogenetics of G protein signaling by zebrafish non-visual opsin Opn7b for synchronization of neuronal networks

“…AAV virus production. Recombinant AAV stocks of serotype 9 were produced via the triple-transfection method 53 and purified using chloroform (described in 54 ). In short, HEK293T cells were transfected with the vector of interest, the serotype plasmid and helper plasmid using polyethylenimine.…”