Curated Optogenetic Publication Database

Abstract: G protein-coupled receptors (GPCRs) are efficient Guanine nucleotide exchange factors (GEFs) and exchange GDP to GTP on the Gα subunit of G protein heterotrimers in response to various extracellular stimuli, including neurotransmitters and light. GPCRs primarily broadcast signals through activated G proteins, GαGTP, and free Gβγ and are major disease drivers. Evidence shows that the ambient low threshold signaling required for cells is likely supplemented by signaling regulators such as non-GPCR GEFs and Guanine nucleotide Dissociation Inhibitors (GDIs). Activators of G protein Signaling 3 (AGS3) are recognized as a GDI involved in multiple health and disease-related processes. Nevertheless, understanding of AGS3 is limited, and no significant information is available on its structure-function relationship or signaling regulation in living cells. Here, we employed in silico structure-guided engineering of a novel optogenetic GDI, based on the AGS3’s G protein regulatory (GPR) motif, to understand its GDI activity and induce standalone Gβγ signaling in living cells on optical command. Our results demonstrate that plasma membrane recruitment of OptoGDI efficiently releases Gβγ, and its subcellular targeting generated localized PIP3 and triggered macrophage migration. Therefore, we propose OptoGDI as a powerful tool for optically dissecting GDI-mediated signaling pathways and triggering GPCR-independent Gβγ signaling in cells and in vivo.

Abstract: 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 PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ 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 PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ 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-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Abstract: Chemokine-induced directional cell migration is a universal cellular mechanism and plays crucial roles in numerous biological processes, including embryonic development, immune system function, and tissue remodeling and regeneration. During the migration of a stationary cell, the cell polarizes, forms lamellipodia at the leading edge (LE), and triggers the concurrent retraction of the trailing edge (TE). During cell migration governed by inhibitory G protein (Gi)-coupled receptors (GPCRs), G protein βγ (Gβγ) subunits control the LE signaling. Interestingly, TE retraction has been linked to the activation of the small GTPase Ras homolog family member A (RhoA) by the Gα12/13 pathway. However, it is not clear how the activation of Gi-coupled GPCRs at the LE orchestrates the TE retraction in RAW264.7 macrophages. Here, using an optogenetic approach involving an opsin to activate the Gi pathway in defined subcellular regions of RAW cells, we show that in addition to their LE activities, free Gβγ subunits also govern TE retraction by operating two independent, yet synchronized, pathways. The first pathway involves RhoA activation, which prevents dephosphorylation of the myosin light chain, allowing actomyosin contractility to proceed. The second pathway activates phospholipase Cβ and induces myosin light chain phosphorylation to enhance actomyosin contractility through increasing cytosolic calcium. We further show that both of these pathways are essential, and inhibition of either one is sufficient to abolish the Gi-coupled GPCR-governed TE retraction and subsequent migration of RAW cells.