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Roger Hardie FRS

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Professor of Cellular Neuroscience
Tel: 01223 339771 Fax: +44 (0)1223 333840 E-mail: rch14@cam.ac.uk

Phototransduction, TRP channels, lipid and Calcium signalling in Drosophila
Our lab studies the cellular and molecular machinery underlying phototransduction - the mechanisms by which photoreceptors respond and adapt to light. We investigate this in the fruitfly Drosophila, using powerful molecular genetic and in vivo physiological approaches. A major aim of our research is to understand the mechanisms of activation and regulation of the light-sensitive channels. In Drosophila, we discovered these are encoded by the trp gene (Hardie & Minke 1992), which became the prototypical member of the intensively studied TRP ion channel family. With 28 mammalian isoforms, this one of the largest and most diverse ion channel families in the genome and a major focus of biomedical research.

PhD students. I will be pleased to consider enquiries from prospective PhD students. Please contact me by e-mail in the first instance.

Inositol lipid signalling In all eyes, phototransduction is based on G-protein coupled signalling cascades (reviews: Yau & Hardie 2009Hardie & Raghu 2001).  Drosophila  uses one of the most widespread versions – the inositol lipid cascade, characterised by the effector enzyme phospholipase C (PLC). This generates the second messengers, inositol trisphosphate (InsP3) and diacylglycerol (DAG) by hydrolysis of the minor membrane phospholipid, PIP2. Drosophila phototransduction represents an important and influential genetic model for this ubiquitous signalling cascade.  As in many cell types throughout the body, this PLC dependent pathway leads to activation of TRP channels, but the exact mechanism has long remained mysterious. We have recently found evidence for an unexpected, and novel solution to this enigma: namely the channels may be combinatorially activated by the reduction of PIP2 and protons, which are also released by the PLC reaction (Huang et al 2010). Fascinatingly, the effect of PIP2  reduction may be mediated mechanically (Hardie & Franze 2012):  hydrolysing this minor membrane lipid, effectively increases membrane tension, resulting in visible contractions of the photoreceptors in response to light (see movie

Calcium  signalling  Fly photoreceptors respond sensitively to single photons; do so ~10 times more rapidly than vertebrate rods and can also light adapt over more than a million-fold range of light intensities. Ca2+ influx via the light-sensitive TRP channels is critical for this performance. We have identified several Ca2+ dependent targets mediating both positive and negative feedback, and are investigating details of the underlying molecular mechanisms (e.g. Gu et al 2005; Liu et al 2008).

Other project areas

(i)             Arrestin translocation. Several key proteins of the transduction cascade undergo massive intracellular movements in response to light and dark-adaptation. By imaging GFP-tagged arrestin we are analyzing the dynamics and mechanism of arrestin translocation in vivo (Satoh et al 2010).

(ii)           Molecular mechanisms of retinal degeneration. Mutations in many elements of the transduction cascade lead to retinal degeneration via pathways that are often conserved between flies and humans. We are analysing mechanisms underlying degenerative phenotypes resulting from calcium and lipid dyshomeostasis.

(iii)          Voltage gated potassium channels. The final voltage response of the photoreceptors is fine-tuned by the activity of a variety of voltage-gated channels, which are subject to second-messenger mediated modulation (e.g. Krause et al 2008).

(iv)          Ion channels at the photoreceptor synapse. We discovered that the photoreceptor neurotransmitter in flies and other arthropods is histamine, which directly gates a novel class of ligand (histamine) gated ion channel.

Techniques  Classical and molecular genetic approaches are complemented by sophisticated electro- and opto-physiological tools, including single channel and whole-cell patch-clamp from dissociated photoreceptors, imaging of fluorescent indicators & genetically targeted reporters and flash photolysis of caged compounds. Both in vivo and heterologous expression systems are used. Confocal and electron microscopy are both available as are standard molecular biological techniques.

Lab Members
Dr Che Hsiung-Liu, Research Associate
Dr Murali Bollepalli Research Associate
Mai Morimoto  (Graduate Student)
Alex Randall (Graduate Student)

Collaborators
Don Ready (Purdue USA) website
Marten Postma (Amsterdam, Netherlands) website
Mikko Juusola (Sheffield) website
Patrick Dolph (Dartmouth USA) website
Nansi Colley (Wisconsin USA) website
Padinjat Raghu (Banglaore India) website
Joseph O’Tousa (Notre Dame USA) website

Main sources of funding: BBSRC

Selected publications: (and a full list)

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 ref

Hardie RC, Franze K (2012) Photomechanical responses in Drosophila photoreceptors. Science 338:260-263ref

 Song Z, Postma M, Billings SA, Coca D, Hardie RC, Juusola M (2012) Stochastic, adaptive sampling of information by microvilli in fly photoreceptors. Curr Biol 22:1371-1380 ref

Hardie RC (2012) Phototransduction mechanisms in Drosophila microvillar photoreceptors. WIRES Membr Transp Signal 1:WIREs Membr Transp Signal 2012, 2011:2162–2187. doi: 2010.1002/wmts.2020

Huang, J., Liu, C.H., Hughes, S.A., Postma, M., Schwiening, C.J., and Hardie, R.C. (2010). Activation of TRP Channels by Protons and Phosphoinositide Depletion in Drosophila Photoreceptors. Curr Biol 20, 189-197. ref

Satoh, A.K., Xia, H., Yan, L., Liu, C.H., Hardie, R.C., and Ready, D.F. (2010). Arrestin translocation is stoichiometric to rhodopsin isomerization and accelerated by phototransduction in Drosophila photoreceptors. Neuron 67, 997-1008. ref

Yau, K.W., and Hardie, R.C. (2009). Phototransduction motifs and variations. Cell 139, 246-264. ref

Liu, C.H., Satoh, A.K., Postma, M., Huang, J., Ready, D.F., and Hardie, R.C. (2008). Ca2+-dependent metarhodopsin inactivation mediated by Calmodulin and NINAC myosin III. Neuron 59, 778-789.ref

Krause, Y., Krause, S., Huang, J., Liu, C.H., Hardie, R.C., and Weckstrom, M. (2008). Light-Dependent Modulation of Shab Channels via Phosphoinositide Depletion in Drosophila Photoreceptors. Neuron 59, 596-607. ref

Liu, C. H., Wang, T., Postma, M., Obukhov, A. G., Montell, C., and Hardie, R. C. (2007). In Vivo Identification and Manipulation of the Ca2+ Selectivity Filter in the Drosophila Transient Receptor Potential Channel. J Neurosci. 27, 604-615.ref

Gu, Y, Oberwinkler, J, Postma, M., Hardie, R.C. (2005) Mechanisms of light adaptation in Drosophila photoreceptors. Curr Biol 15,1228-1234 ref

Hardie, R.C. & Raghu, P. (2001) Visual transduction in Drosophila. Nature 41:186-193  ref

Hardie RC (2001) Phototransduction in Drosophila melanogaster. J Exp Biol 204:3403-3409. ref Ukrainian translation

Image

Above : Drosophila phototransduction cascade. Inset Cross section of a Drosophila rhabdomere composed of tightly packed microvilli. (1) Photoisomerization of rhodopsin to metarhodopsin (Rh M, encoded by ninaE gene) activates Gq (2) Gq activates phospholipase C (PLC), generating InsP3 and DAG from PIP2. DAG may release polyunsaturated fatty acids (PUFAs) via action of DAG lipase; (3) Two classes of light-sensitive channels (trp and trpl genes) are activated by an unknown mechanism. Several components of the cascade are coordinated into a signalling complex by the scaffolding protein, INAD. (4) At the base of the microvilli there is a system of submicrovillar cisternae (SMC). Although these may represent Ca2+ stores endowed with InsP3 receptors, they may play a more important role in phosphoinositide turnover (5).

rh1

Above: Molecular model of the TRP channel pore based on crystal structure of KcsA. By site-directed mutagenesis we identified aspartate 621 (D621) as the critical acidic residue conferring the high Ca2+ selectivity of the channel (PCa:PNa ≈ 50:1). In transgenic flies where the aspartate was neutralised (TRPD621G) the channels became impermeable to Ca2+, allowing only currents mediated by monovalent cations. In these photoreceptors all Ca2+ dependent amplification, kinetics and adaptation were eliminated ((Liu et al 2007)

rh2

Above : Dissociated Drosophila ommatidia. The light absorbing rhabdomere has been labelled by a genetically targeted GFP-tagged ion channel protein (Kir2.1). As well as visualizing the rhabdomere, this ion channel is specifically activated by PIP2 and can be used as an electrophysiological biosensor to monitor PIP2 levels in real time in vivo).

a movie

Above: Movie of contracting ommatidium . Movie showing dissociated live ommatidum contracting in response to brief light flash.  See also articles on University research news pages and BBSRC business news