Phosphoinositide cycle in Drosophila

Photoreceptors transduce light into electrical signals by a series of biochemical steps, each involving specific protein molecules (e.g. visual pigments and enzymes). The end result of this "phototransduction cascade" is the activation of proteins known as "ion channels", in the lipid membrane surrounding the cell. Once activated, ion channels open to allow charged ions, such as sodium and calcium, into the cell, thereby generating electrical signals for transmission to the brain. Phototransduction can be particularly well studied in the fruitfly Drosophila because of the ease with which we can manipulate specific genes (and hence proteins) and because we can record the activity from their photoreceptors with a range of high precision techniques. The molecules involved are not unique to fly photoreceptors and closely related molecules are found in cells throughout our own bodies. One such molecule is the so-called TRP channel. In flies, this is the channel activated during phototransduction; in mammals, TRP channels are essential for a wide range of vital processes such as hormonal responses, regulation of blood pressure, taste, smell, and sensations of pain, hot and cold.

The particular cascade used by the fly photoreceptor to activate the TRP channels is called the phospho-inositide (PI) cycle. This is one of the most widely used biochemical cascades in living cells. In humans it is found in almost every cell in the body and is responsible for a wide range and hormonal responses, such as those involved in regulating blood pressure as well as in communication between neurons in the brain and various senses, such as taste. Activation of this cascade involves the breakdown of an important lipid molecule found in all cell membranes known as PIP2. To maintain operation it is essential that PIP2 is continually resynthesised. This takes place via a complex cycle involving multiple steps and at least 5 distinct intermediates. If any of these steps is compromised, not only does the cascade cease working, but the cells affected can die or become cancerous. In order to study this cycle in living cells, researchers have developed fluorescently labelled molecules which bind to each intermediate which can then be viewed with special microscopes. Normally such experiments are performed under artificial conditions in cultured cells which may differ in their behaviour to cells in living tissue.

In our research we will use genetic techniques to express a range of such fluorescently labelled sensors in fly photoreceptors. By exploiting some unique optical features of the fly's eye this will enable us to image and measure their fluorescence in the completely intact living animal. This will allow us to follow the fate of the various intermediates in response to physiological stimulation, which in the eye can be precisely controlled by light. Additionally by making mutations in candidate genes, we will also be able to identify the genes responsible for regulating each step of the PI cycle. This approach will allow us to build up a comprehensive and quantitative picture of the functioning of this important and ubiquitous biochemical cycle in the intact animal. The knowledge we gain from these studies will not only further our 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.

Drosophila phototransduction is mediated by a phosphoinositide (PI) signalling cascade involving rhodopsin and a Gq protein which activates PLC. Hydrolysis of PIP2 results in activation of TRP channels which mediate the electrical response to light as well as Ca influx which regulates many components of the cascade. Following hydrolysis, PIP2 must be replenished by a multi-enzymatic pathway (PI cycle). Appropriate turnover of this cycle is essential for photoreceptor performance. Although extreme conditions (e.g. mutants or defects in Ca dependent regulation) are known where the cycle fails, there is virtually no information on how this cycle operates under physiological conditions; which steps are rate limiting, and genes encoding certain key components (PI kinase and PIP kinase) have yet to be identified.

We propose to address these questions by making the first in vivo measurement of the turnover of not only PIP2, but also several other components of the PI cycle, including PI(4)P, phosphatidyl inositol, diacyl glycerol, phosphatidic acid and PIP3. To achieve this we will generate transgenic flies expressing GFP-tagged sensors that bind specifically to each intermediate. By exploiting the optics of the compound eye we will quantify their dynamic behaviour (breakdown and resynthesis) in completely intact animals under physiological conditions using precisely calibrated illumination. Results will confirmed and complemented by mass spectrometric lipidomic analysis and electrophysiology. We will explore the regulation of the cycle (e.g by light and/or Ca) and identify/confirm genes responsible for each step of the cascade using mutants and/or RNAi of candidate genes. Our results will be incorporated into a detailed, realistic computational model of the PI cycle.

The immediate impact of the work in this proposal is primarily scientific; nevertheless, it should be of interest to several groups of potential beneficiaries outside academia.

Pharmaceutical and biotech industry: Drosophila is a key model organism in biomedical research, and over 70% of genes implicated in human disease have homologues in Drosophila. The fly retina is one of the most intensively studied systems making major contributions to development, hereditary disease, neural signalling and G-protein coupled signalling. Amongst these, my (BBSRC funded) discovery of the first TRP channel (now recognised as the 2nd largest ion channel family in our genome, and increasingly implicated in a broad spectrum of hereditary and non-inherited disease) continues to have a huge worldwide impact on clinically oriented research in both academia and the pharmaceutical industry. Like many of the 28 human TRP isoforms, the Drosophila TRP channels are regulated by the phospholipase C cascade arguably the most widespread and important of the G-protein coupling cascades. The phosphinositide cycle - the focus of this proposal - is an integral and essential feature of this ubiquitous signaling system and conserved from flies to humans. Uniquely, our proposal will monitor the turnover of this cycle in vivo, quantitatively and in real time. The methods we will develop are not only powerful but will also lend themselves to moderately high throughput screening in either genetic or drug-based screens.

Staff and students: Staff and students engaged in the research will receive multidisciplinary training in neuroscience, imaging, electrophysiology, molecular biology, and genetics. Previous post-docs/students have become successful not only in academia, but also in a broad range of employment (incl., government, education and industry). As well as post-docs and PhD students, we engage medical and basic science undergraduate students in lab-based projects related to ongoing research, for whom the experience can be inspirational. I routinely welcome short-term (up to one year) pre- and post-doctoral visitors (e.g. from Russia, USA, India, Japan, China, Singapore, and several European countries) to learn specific skills. I expect to host at least 1 per year during the course of the grant

General Public:
We expect the general public to be interested in several aspects of our work, e.g:
i) The sensory capabilities of insects, which have suprising differences to our own (eg flies can detect movement up to 10x faster than humans and can see UV and polarised light)
ii) Related TRP channels in humans are responsible for a variety of sensory perceptions - particularly sensations of hot and cold, pain and many tastes (chilli, menthol, horseradish, garlic, oregano, thyme) and play many other vital roles in the body
iii) The contributions Drosophila, as a model genetic organism, can make to our understanding of human biology and disease.

At a more advanced level, our research is widely considered as the definitive account of phototransduction in microvillar photoreceptors: it is taught at both 2nd and 3rd year level at our University and widely taught in relevant University courses internationally.