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Camello-XR Enables Visualization and Optogenetic Control of Gq/11 Signals and Receptor Trafficking in GPCR-Specific Domains

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Camello-XR Enables Visualization and Optogenetic Control of Gq/11 Signals and Receptor Trafficking in GPCR-Specific Domains ARTICLE https://doi.org/10.1038/s42003-019-0292-y OPEN CaMello-XR enables visualization and optogenetic control of Gq/11 signals and receptor trafficking in GPCR-specific domains Dennis Eickelbeck1, Raziye Karapinar1, Alexander Jack 2, Sandra T. Suess1, Ruxandra Barzan3, Zohre Azimi3, 1234567890():,; Tatjana Surdin1, Michelle Grömmke1, Melanie D. Mark1, Klaus Gerwert4, Dirk Jancke3, Petra Wahle2, Katharina Spoida1 & Stefan Herlitze1 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 2+ CaMello (Ca -melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic- 2+ dependent Ca signals in 5-HT2A-R domains. These constructs consist of the light-activated 2+ Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca 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. 1 Department of General Zoology and Neurobiology, ND7/31, Ruhr-University Bochum, Universitätsstr. 150, D-44780 Bochum, Germany. 2 Developmental Neurobiology, ND6/72, Ruhr-University Bochum, Universitätsstr. 150, D-44780 Bochum, Germany. 3 Optical Imaging Group, Institut für Neuroinformatik, NB 2/27, Ruhr-University Bochum, Universitätsstr. 150, D-44780 Bochum, Germany. 4 Department of Biophysics, ND04/596, Ruhr-University Bochum, Universitätsstr. 150, D-44780 Bochum, Germany. Correspondence and requests for materials should be addressed to S.H. (email: [email protected]) COMMUNICATIONS BIOLOGY | (2019) 2:60 | https://doi.org/10.1038/s42003-019-0292-y | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-019-0292-y hanges in the intracellular Ca2+ concentration in neurons membrane-localized calcium sensing domain combined with a regulate various cellular processes including synaptic second spectrally shifted fluorescent tag and a receptor trafficking C fi transmitter release, gene transcription, and various forms signal, allowing for passive visualization of receptor traf cking of synaptic plasticity1. These Ca2+ signals are spatio-temporally and intracellular Ca2+ signals in receptor-specific domains, controlled in their amplitude and can occur as fast Ca2+ spikes or CaMello-XRs are engineered in a comparable manner, but are Ca2+ oscillations2. Many Ca2+ signaling molecules are assembled built around a light-activated GPCR as a backbone and thus allow into macromolecular complexes in specific subcellular micro- for additional optogenetic control of Gq/11 signals. While this domains, which function autonomously within highly specialized technique can most likely be applied to every GPCR coupling to 1 environments . An example for such a functional subcellular the Gq/11 pathway, we established the method using the 5-HT2A- microdomain is the assembly of voltage gated Ca2+ channels with R because of its importance for neuropsychiatric disorders and the transmitter release machinery at the presynaptic terminal3. the lack of understanding of how this GPCR modulates neuronal Transmitter-mediated increases in intracellular Ca2+ levels not activity and plasticity. only involve the fast gating of plasma membrane ion channels but also GPCRs, coupling to the Gq/11 pathway. Ion channels and GPCRs are activated by various transmitters such as glutamate, Results histamine, oxytocin or serotonin, where the Ca2+ signaling Visualization of GPCR trafficking and intracellular Ca2+ sig- components are often assembled into spatially separated signaling nals. In order to engineer a light-activated GPCR for the visua- complexes. lization of receptor trafficking and intracellular Ca2+ signals we For example, metabotropic mGluRs assemble into a macro- introduced the red fluorescent protein mCherry (mCh) into the molecular complex with IP3 receptors via the scaffolding protein III intracellular loop of the light-activated GPCR melanopsin Homer and co-purify with protein phosphatases and protein from mouse (mOpn4L or moMo). In addition, we fused kinase A (PKA)1,4.Ca2+ release from internal stores in neurons is GCaMP6m19 to the CT of moMo. We call this construct CaMello regulated via the activation of phospholipase C (PLC), hydrolysis (Ca2+-melanopsin-local-sensor, Fig. 1a). Expression of CaMello of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylgly- in HEK tsA201 cells and illumination of the cell with red light cerol (DAG) and inositol 1,4,5-trisphosphate (IP3) and activation reveals the localization of the GPCR at the plasma membrane by of IP3 receptors located at the endoplasmic reticulum (ER), which the intracellular mCh tag. Activation of CaMello by blue light leads to the release of Ca2+ from the ER5. The opening of small leads to an immediate increase in intracellular Ca2+ signal 2+ 2+ groups of IP3 receptors induces a puff of Ca . Summation of detected by the C-terminally localized GCaMP6m. The Ca several of these puffs can elicit an intracellular Ca2+ wave3. signal is sustained during receptor activation20 (Fig. 1b, Supple- Depending on which isoforms of the different signaling compo- mentary Movie 1) and correlates with the stable fluorescent mCh nents are activated different forms of Ca2+ signals are induced in signal at the membrane (Fig. 1c) suggesting that CaMello is not neurons. For instance, activation of mGluR1 in neurons produces internalized during Gq/11 activation. a single Ca2+ transient, whereas mGluR5 generates an oscillatory In former studies we showed that the CT of GPCRs such as the 2+ 1,6 Ca wave . 5-HT1A-R, 5-HT1B-R, and 5-HT2C-R is sufficient for localization fi 21–24 5-HTRs coupling to the Gq/11 pathway such as 5-HT2A/C-Rs of the GPCR into the receptor-speci c domain in neurons . are abundantly expressed in the brain and are molecular targets Thus, in order to analyze GPCR-specificGq/11 signals in the brain 7 for atypical antipsychotic drugs and most hallucinogens .5- we tagged CaMello with the CT of the 5-HT2A-R at the C- HT2A-Rs are expressed on apical dendrites of cortical pyramidal terminal end and analyzed if the 5-HT2A-CT could change GPCR fi 2+ neurons and cerebellar Purkinje cells. 5-HT2A-Rs colocalize with traf cking and Gq/11 induced Ca signals. This construct is PSD95 and MUPP1 (multi-PDZ domain protein 1) in apical called CaMello-5HT2A. The 5-HT2A-R is a Gq/11 coupled GPCR, dendrites, dendritic shafts and spines8. The targeting and sub- which is expressed throughout the brain, involved in various cellular localization of 5-HT2A-Rs involve a PDZ binding domain psychiatric disorders such as schizophrenia, anxiety, and depres- 9,10 in the C-terminus (CT) of the 5-HT2A-R . Activation of 5- sion, activated by hallucinogens and a drug target for various 25 HT2A has been associated with changes in spine and dendritic anxiolytics, antidepressants, and antipsychotics . As detected by morphology8, changes in BDNF levels in the hippocampus and the mCh tag our engineered GPCR is localized to the membrane neocortex11 and normally results in an increased neuronal and to intracellular vesicle like structures. Blue light-mediated 12,13 activity . It has been suggested that increased activity of 5- activation of CaMello-5HT2A leads to a fast rise in intracellular 2+ HT2A-Rs might be responsible for some of the psychotic symp- Ca levels, which declines to a steady state level at about 50% of toms in schizophrenia14 and that atypical antipsychotic agents the maximal response amplitude (Fig. 1b, Supplementary may antagonize the hyperactivity and membrane targeting of 5- Movie 2). The decline in response amplitude during receptor 15 fl HT2A-Rs . In addition, depending on the agonist and cell-type 5- activation correlates with a decrease in the uorescent mCh signal 2+ HT2A-Rs not only stimulate the Gq-PLC pathway, but also other at the plasma membrane (Fig. 1c). The decline in mCh and Ca 16–18 pathways such as the G12/13-PLA2 and Gi/o-Src pathway . signals during light activation of CaMello-5HT2A are blocked by These observations suggest that alterations in 5-HT2A-R traf- Dynasore (Fig. 1b, c), an inhibitor of dynamin-dependent ficking and G protein signaling contribute to the development endocytosis26,27. Five-minute light stimulation of CaMello- and manifestation of neuropsychiatric disorders. 5HT2A leads to an increase of cytoplasmic (vesicle) localization Thus, decoding Ca2+ signals in GPCR-specific microdomains relative to transmembrane localization, which is recovered after is important for understanding the functions of GPCRs in their 2 h (Fig. 1d). The increase in cytoplasmic vesicle localization is native environment. It is also important to understand how these blocked by Dynasore (Fig. 1d). In addition, the 5-HT2A-R and fi signals are shaped by GPCR traf cking and internalization, how CaMello-5HT2A colocalize with early (Rab5) and late (Rab7) they contribute to neuronal excitation and plasticity and how endosome markers, but not with the Golgi marker GALT. The these signals are altered
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