Lockyer_whole_thesis.pdf (2.98 MB)
Development of optogenetic approaches to selectively modulate G protein signalling
thesisposted on 2023-05-27, 23:57 authored by Lockyer, JL
G protein coupled receptors, or GPCRs, transduce signals from neuromodulators and permit dynamic regulation of neuronal circuits. Gradually, we have come to understand the processes underlying neural function and the molecular underpinnings of higher cognitive function. However, as we continue to push the limits of our understanding, so too do we push the technical demands required to reach the next breakthrough. Consequently, the need for a more diverse repertoire of tools for neuronal manipulation is continuously increasing. GPCRs - classified according to G protein association: G˜í¬±q, G˜í¬±s or G˜í¬±i-linked - are fundamentally associated with neuromodulation, as opposed to direct neuronal excitation and inhibition. Due to complexity, an ever-expanding molecular toolbox is needed for the artificial manipulation of these signalling cascades. Despite the large array of chemical and optogenetic-based tools already available; a selective, light-induced inhibitor of endogenous G protein signalling is yet to be described. Thus, this study designs and validates such a tool, with a focus on the selective inhibition of endogenous G˜í¬±q-linked GPCR signalling. The optogenetic tool described in this study utilised the protein domain of RGS2, a member of the regulator of G protein signalling (RGS) family. RGS2 acts as a GTPase-accelerating protein, or GAP, selectively inactivating the G˜í¬±q G protein associated with agonist bound GPCRs. It was proposed that optogenetic activation of this protein would allow for a selective, light-dependent tool that would inactivate endogenous G˜í¬±q-linked GPCR signalling. This study describes the design and validation of Photo-Induced G protein Modulator ‚Äö- Inhibitor G˜í¬±q, or PIGM-Iq. RGS2 functions at the membrane to inhibit G protein signalling. To control its activity and reduce possible interactions with other signalling pathways, the membrane binding domain, as well as a domain thought in interact with the G˜í¬±s signalling pathway, were removed. As expected, the resulting cytosolic variant of RGS2 was unable to inhibit G˜í¬±q-linked signalling in mammalian HEK293A cells. To allow for light-induced translocation of this protein to the membrane, the truncated RGS2 (˜ívÆRGS2) was fused to the photoreceptor, cryptochrome 2 (CRY2), with its binding partner, CIBN, localised at the membrane. Blue light illumination was shown to control ˜ívÆRGS2 activity via re-localisation of the ˜ívÆRGS2 from the cytosol to the membrane. Blue light illumination of cells expressing PIGM-Iq robustly reduced agonist induced G˜í¬±q-linked calcium efflux. Inhibition was absent in the three control conditions tested: 1) in cells not illuminated with light, 2) in cells expressing only one component of the system, and 3) in cells expressing a light-insensitive variant of the tool. The selectivity of the tool towards other GPCR signalling cascades was investigated. Both wild type RGS2 and PIGM-Iq did not possess affinity towards either the G˜í¬±i or G˜í¬±s-linked signalling pathways in mammalian HEK293A cell culture. It was therefore concluded that the tool selectively interacted with the endogenous G˜í¬±q G proteins. To validate the effectiveness of the tool in vivo, the mobility of the nematode, C. elegans, and courtship conditioning in the male fruit fly, D. melanogaster, were used as assays. In C. elegans, a single epoch of blue light delivered to transgenic animals produced a decrease in movement that was rescued after 20 minutes of dark habituation. This is consistent with the known phenotype of the G˜í¬±q mutant strains egl-30(ad803) and egl-30(ad806) in C. elegans. This movement deficit could be reinstated upon further illumination, highlighting both the sensitivity and reversibility of the tool. In D. melanogaster, the octopamine receptor, OAMB, is necessary for courtship learning and is hypothesised to signal through G˜í¬±q. Blue light illumination of expressing flies during courtship training subsequently produced a defect in courtship learning, consistent with the hypothesised function of G˜í¬±q-linked signalling in this behaviour. As this study presents a method to produce RGS-based G protein inhibitors, future studies will focus on the design and validation of a G˜í¬±i inhibitor tool. A selective G˜í¬±i inhibitor is currently being tested. Together, these tools will provide a valuable addition to the current methods for the modulation of GPCR signalling. Through the design and validation of an RGS2-based optogenetic tool, this study presents a reversible, light-induced inhibitor of endogenous G˜í¬±q-linked GPCR signalling that was previously absent from the molecular toolbox. This tool presents many advantages over current techniques used to manipulate GPCR signalling, including high spatiotemporal resolution, selectivity, and reversibility of inhibition, as well as the ability to be easily packaged for viral delivery. Moreover, its demonstrated ability to effectively function in both mammalian and invertebrate systems drastically increases the scope of its application for the investigation of G protein signalling and neuromodulation in a wide variety of fields.
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