In-silico predicted mouse melanopsins with blue spectral shifts deliver efficient subcellular signaling.

Melanopsin is a photopigment belonging to the G Protein-Coupled Receptor (GPCR) family expressed in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) and responsible for a variety of processes. The bistability and, thus, the possibility to function under low retinal availability would make melanopsin a powerful optogenetic tool. Here, we aim to utilize mouse melanopsin to trigger macrophage migration by its subcellular optical activation with localized blue light, while simultaneously imaging the migration with red light. To reduce melanopsin's red light sensitivity, we employ a combination of in silico structure prediction and automated quantum mechanics/molecular mechanics modeling to predict minimally invasive mutations to shift its absorption spectrum towards the shorter wavelength region of the visible spectrum without compromising the signaling efficiency. The results demonstrate that it is possible to achieve melanopsin mutants that resist red light-induced activation but are activated by blue light and display properties indicating preserved bistability. Using the A333T mutant, we show that the blue light-induced subcellular melanopsin activation triggers localized PIP3 generation and macrophage migration, which we imaged using red light, demonstrating the optogenetic utility of minimally engineered melanopsins.

Spatiotemporal Optical Control of Gαq-PLCß Interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Spatiotemporal optical control of Gαq-PLCß interactions.

Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Molecular regulation of PLCß signaling.

Phospholipase C (PLC) enzymes convert the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG regulate numerous downstream pathways, eliciting diverse and profound cellular changes and physiological responses. In the six PLC subfamilies in higher eukaryotes, PLCß is intensively studied due to its prominent role in regulating crucial cellular events underlying many processes including cardiovascular and neuronal signaling, and associated pathological conditions. In addition to GαqGTP, Gßγ generated upon G protein heterotrimer dissociation also regulates PLCß activity. Here, we not only review how Gßγ directly activates PLCß, and also extensively modulates Gαq-mediated PLCß activity, but also provide a structure-function overview of PLC family members. Given that Gαq and PLCß are oncogenes, and Gßγ shows unique cell-tissue-organ specific expression profiles, Gγ subtype-dependent signaling efficacies, and distinct subcellular activities, this review proposes that Gßγ is a major regulator of Gαq-dependent and independent PLCß signaling.

The spatial distribution of GPCR and Gßγ activity across a cell dictates PIP3 dynamics.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various diseases, including cancer, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.