Mechanisms of inactivation in Drosophila phototransduction

Lead Research Organisation: University of Cambridge
Department Name: Physiology Development and Neuroscience

Abstract

Photoreceptors respond to light by converting it into electrical signals. This process of 'phototransduction' involves a cascade of biochemical steps, each of which involves one or more specific protein molecules (e.g. visual pigments and catalytic enzymes). The end result is the activation of specialised proteins known as 'ion channels', embedded in the membrane surrounding the cell. Once activated, ion channels allow charged ions, such as sodium and calcium, into the cell, thereby generating electrical signals that are transmitted along nerves to the brain. Fly photoreceptors are remarkable in being able to generate responses 10-100 x more rapidly than equivalent photoreceptors in vertebrate eyes thereby representing the fastest biochemical signalling cascade of this sort in the animal kingdom (one of the main reasons why it is so hard to swat a fly!). Phototransduction can be particularly well studied in the fruitfly Drosophila for several reasons. Firstly, we now know its entire genetic code, and can manipulate its genes so that individual genes (and hence proteins) can be altered, deleted or introduced into the fly. Secondly, we can isolate fly photoreceptors and record their electrical signals with extreme precision using a technique known as 'patch-clamp'. Thirdly, our laboratory has developed specialized and sophisticated physiological tools that allow us to monitor the rates of individual molecular steps in phototransduction in living, responding cells. In order to respond quickly and reliably photoreceptors must not only activate in response to light, but must also be able to terminate their activity when the light goes off. Although equally important for photoreceptor performance, the molecular steps involved in inactivation are poorly understood. In this research programme we will combine our biochemical and physiological approaches with genetic manipulation of different molecular components of the phototransduction cascade to provide a detailed understanding of how these inactivation mechanisms are controlled and co-ordinated to generate the remarkable performance of these photoreceptors. The molecules involved in generating the fly's response to light are not unique to fly photoreceptors. Even in humans, molecules closely related to those we are studying are found throughout the body. They play important roles in a wide range of processes such as all manner of hormonal responses, regulation of blood pressure, taste and smell, and sensations of pain, hot and cold. The knowledge we gain from these studies will not only give us a detailed understanding of how photoreceptors see but, because the basic underlying biochemical mechanisms are so widely found, will provide new insight into many other, often clinically important processes in the body.

Technical Summary

The fly phototransduction cascade represents the fastest known G-protein coupled signalling system. Its performance is remarkable, generating large quantum bumps in response to single photons with kinetics 10-100x faster than in vertebrate rods. High temporal resolution requires not only fast activation but also rapid inactivation. Inactivation steps include quenching of active metarhodopsin (M*) by arrestin, termination of G-protein and PLC activity, metabolism of second messenger (diacylglycerol), closure of the channels, and resynthesis of PIP2. However, the contribution of these steps to response kinetics remains largely unexplored as does the question of whether and how they are regulated (eg by Ca2+). Rapid response termination also requires myosin III (NINAC), but how NINAC functions in phototransduction has remained largely mysterious. Recently we have developed a method (instantaneous photoinactivation of M* by photoreisomerisation) which allows us to measure the response deactivation time course of different steps of the cascade. Our preliminary results using this technique showed that M* deactivation by arrestin is strongly Ca2+ dependent, and implicated NINAC in this process. Objectives: i) To determine the mechanism underlying NINAC's role in Ca2+ dependent M* deactivation. ii) To investigate the molecular basis of two further separable phenotypes of ninaC mutants, (the occurrence of spontaneous quantum bump-like events in the dark and an increase in quantum bump duration) iii) To determine the contribution of specific deactivation steps to overall response deactivation. kinetics iv) To investigate the Ca2+ dependence of specific inactivation steps by using the Na/Ca exchanger to quantitatively control cytosolic Ca2+ whilst measuring the response deactivation time-course of different steps of the cascade.

Publications

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Abou Tayoun AN (2011) The Drosophila SK channel (dSK) contributes to photoreceptor performance by mediating sensitivity control at the first visual network. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Fain GL (2010) Phototransduction and the evolution of photoreceptors. in Current biology : CB

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Hardie R (2012) Phototransduction mechanisms in Drosophila microvillar photoreceptors in Wiley Interdisciplinary Reviews: Membrane Transport and Signaling

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Hardie RC (2015) Phototransduction in Drosophila. in Current opinion in neurobiology

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Hardie RC (2012) Regulation of arrestin translocation by Ca2+ and myosin III in Drosophila photoreceptors. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Hardie RC (2014) Photosensitive TRPs. in Handbook of experimental pharmacology

 
Description 1. Discovery that arrestin (the protein that binds to inactivate the active state of the visual pigment - metharhodopsin) translocates reversibly between the phototransduction compartment (rhabdomere) and cell body on a timescale of seconds. Overturning an influential model, we showed that translocation is mediated by diffusion between two light-regulated sinks: metarhodopsin in the rhabdomere, and myosin III (NINAC) in the cell body, and that this process is regulated by Ca2+ influx.

2. Absolute sensitivity is ultimately limited by "dark noise" (spontaneous activation of the phototransduction cascade). We quantified this, showed that this is mediated by ion channels (TRP) activated by spontaneous G-protein activation independently of rhodopsin and measured its dependence on Ca2+. We identified a novel protein (retinophilin) that suppresses dark noise and found that noise suppression also required DAG kinase and the interaction of the C-terminal of NINAC with the scaffolding protein INAD.

3. The exact mechanism of phototransduction in invertebrate photoreceptors, such as those of the fly is still not understood and represents one of the major outstanding questions in sensory physiology. Our work in this grant suggested a radical a new hypothesis for the mechanism of phototransduction: we showed that phospholipase C (PLC) rapidly acidifies the rhabdomere and that the light-sensitive channels are activated in a combinatorial manner by protons and PIP2 depletion.
Exploitation Route Our work on arrestin translocation introduced in vivo live imaging of GFP tagged constructs in the fly's eye: a methodology that may find braod applicability

Our discovery that TRP channels may be gated by a combination of PIP2 depletion and protons released by PLC represents a new paradigm in cell signalling
Sectors Education,Pharmaceuticals and Medical Biotechnology

URL http://www.pdn.cam.ac.uk/staff/hardie/index.html
 
Description This research is of a fundamental curiosity and hypothesis driven nature. It has been published in high impact journals but has no immediate commercial application. As world-leading research into animal vision, some of the results have been integrated into University courses in UK and abroad.
First Year Of Impact 2009
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

 
Description European Commission
Amount € 183,455 (EUR)
Funding ID 658818 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 11/2015 
End 11/2017
 
Title Dynamic In vivo monitoring of various protein/intermediates 
Description We have generated range of GFP-tagged constructs for monitoring a variety of cellular processes (including protein translocation, phospholipid metabolism and pH) in real time with millisecond resolution. By targetting these to the Drosophila eye, measurements can be made in completely intact animals over extended periods as well as in acutely dissociated cells 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2012 
Provided To Others? Yes  
Impact We have already published a number of papers using this technology in high impact journals (eg Neuron). 
 
Description Arrestin interactions and translocation 
Organisation Purdue University
Country United States 
Sector Academic/University 
PI Contribution Our contribution included intellectual input, electrophysiology, imaging and molecular biology. I was corresponding or joint corresponding authors on three major publications arising from this collaboration. Post-docs funded on the grants (Huang and Liu) worked on these projects
Collaborator Contribution Intellectual (project design and development) Immunocytochemistry Generation of transgenic flies In vivo confocal imaging of GFP tagged probes
Impact Liu CH, Satoh AK, Postma M, Huang J, Ready DF, Hardie RC (2008) Ca2+-dependent metarhodopsin inactivation mediated by Calmodulin and NINAC myosin III. Neuron 59:778-789. Satoh AK, Xia H, Yan L, Liu CH, Hardie RC, Ready DF (2010) Arrestin translocation is stoichiometric to rhodopsin isomerization and accelerated by phototransduction in Drosophila photoreceptors. Neuron 67:997-1008. Sengupta S, Barber TR, Xia H, Ready DF, Hardie RC (2013) Depletion of PtdIns(4,5)P2 underlies retinal degeneration in Drosophila trp mutants. J Cell Sci 126:1247-1259. plus presentations (invited) at various meetings
Start Year 2006
 
Description Atomic Force Microscopy 
Organisation University of Cambridge
Department Department of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual (designed project, wrote paper) Electrophysiology
Collaborator Contribution Atomic force microscope measurements of photoreceptor contractions
Impact publs. include Hardie RC, Franze K (2012) Photomechanical responses in Drosophila photoreceptors. Science 338:260-263. Randall AS, Liu CH, Chu B, Zhang Q, Dongre SA, Juusola M, Franze K, Wakelam MJ, Hardie RC (2015) Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids. J Neurosci 35:2731-2746. plus invited presentations at several meetings and coverage in popular scientific press and websites
Start Year 2011
 
Description INAD complex 
Organisation University of Hong Kong
Country Hong Kong 
Sector Academic/University 
PI Contribution generation of mutants in INAD scaffolding protein (transgenic flies) and electrophysiology. Post-doc on grant (Liu) worked this
Collaborator Contribution Structural analysis (crystallography and NMR) of INAD
Impact Liu W, Wen W, Wei Z, Yu J, Ye F, Liu CH, Hardie RC, Zhang M (2011) The INAD scaffold is a dynamic, redox-regulated modulator of signaling in the Drosophila eye. Cell 145:1088-1101.
Start Year 2010
 
Description Publicity following publication of Science article (Hardie and Franze 2012) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact I publicised the results of our Science article (Hardie & Franze 2012 "Photomechanical responses in Drosophila photoreceptors" Science 338,260) on the University Research News website and gave several interviews to various media outlets by phone and in person - including New Scientist, and BBC's "Naked Scientist) .

The work was reported in perspectives/commentaries in many Scientific journals (incl. New Scientist, Nat Neuroscience, Science, Current Biology, J Gen Physiology , J Exp Biology BBSRC research news, Faculty 1000 and many popular science websites.)
Year(s) Of Engagement Activity 2012
URL http://www.cam.ac.uk/research/news/surprising-solution-to-fly-eye-mystery
 
Description Radio interview for BBC World Service 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Visited and interviewd by presenter of BBC World Service "Crowd Science" program. Broadcast Aug 31 2017 and extended information included in a feature on the BBC "Science & Environment" website
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/news/science-environment-41284065