Professor Roger Hardie

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 familyWith 28 mammalian isoforms, this one of the largest and most diverse ion channel families in the genome and a major focus of biomedical research.

Inositol lipid signalling

In all eyes, phototransduction is based on G-protein coupled signalling cascades (reviews: Yau & Hardie 2009; Hardie & 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 (Gu et al 2005; Liu et al 2008).

Other project areas

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).

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.

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 (Krause et al 2008).

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.

Recent Lab Members
Dr Che Hsiung-Liu, Research Associate
Dr Sabrina Asteriti Research Associate
Dr Murali Bollepalli Research Associate
Mai Morimoto  (Graduate Student)

Collaborators
Don Ready (Purdue USA) website
Marten Postma (Amsterdam, Netherlands) website
Mikko Juusola (Sheffield) website
Patrick Dolph (Dartmouth USA) website
Craig Montell (UC Santa Barbara)

Ralf Stanewsky (Uni-Munster)