The development of optogenetic tools for investigations into mammalian circadian organisation

  • Ling Zhuang

Student thesis: Phd

Abstract

Many biological oscillators are perpetuated by an autonomous molecular clockwork that generates rhythmic expression of clock genes. Whilst the periods of these endogenous rhythms deviate from 24h, daily cues in the natural environment synchronise all internal rhythms precisely to 24h. Molecular signals, such as the principle intracellular messenger cAMP, are amplified by the Galphas signalling pathway upon activation of GPCRs and regulate a diverse array of physiological processes, one of which is entrainment of the mammalian circadian clock. cAMP conveys environmental cues to the molecular oscillatory network by interacting with the autonomous feedback loop of genes and proteins, thereby resetting their rhythmic expression. Many pharmacological studies have shown that the cAMP signalling cascade is relevant to the entrainment of the mammalian clock. However, due to inherent complications arising from lack of target and subtype specificity, pharmacological agents are known to elicit confounding side-effects in in vivo models. Indeed, there is still ambiguity about the relevance of physiological cAMP signalling in regulating circadian dynamics.Opsin photopigments offer a powerful and drug free platform to initiate signalling cascades under temporally controlled parameters. The Galphas-coupled Carybdea rostonii opsin (JellyOp) robustly activates cAMP signalling in functional studies, and is thus suitable for addressing the contributions of cAMP to circadian regulation. Furthermore a Galphas-decoupled opsin variant (F139A JellyOp) was engineered which, despite being deficient in Galphas interactions, retained the ability to modulate the MAPK pathway in fibroblasts, comparably to JellyOp. Both the wildtype and F139A JellyOp photopigments were therefore employed for investigating contributions of GPCR triggered cAMP dependent pathways to regulating the mammalian circadian clock over background signals.Upon stimulating wildtype or F139A JellyOp photopigments with equivalent irradiances and durations of ultraviolet and infra-red filtered light, both Galphas dependent and independent signalling impacted on the phasing of the fibroblast clock respectively. However, the two JellyOp signalling profiles produced divergent phase response curves in the fibroblast clock, which reflected inherent differences in the phase-responsiveness of the clock to cAMP dependent and independent cascades. A strong relationship was reported between the magnitude of a JellyOp triggered circadian response and the duration of photostimulation, a correlation that was been previously described though in vivo studies of photo-entrainment. These studies have confirmed that mammalian circadian dynamics are responsive to temporally controlled cAMP signalling in a phase and duration dependent manner. Thus, JellyOp and F139A JellyOp are suitable opsin photopigments for investigating the influences of Galphas dependent and independent mechanisms on mammalian clock dynamics.
Date of Award1 Aug 2014
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorRobert Lucas (Supervisor) & Paul Bishop (Supervisor)

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