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WILLIAM PAK
| Paul F. Oreffice Distinguished Professor of Biological Sciences LILY 3-219 494-8202 CV: Link |
We combine genetic, molecular, cell biological, physiological, and genomic techniques to investigate how signals are generated in response to light in photoreceptors and how these signals are transmitted to their postsynaptic target neurons. We use the fruit fly Drosophila to generate and utilize mutants that are impaired in a specific component of the process under investigation.
Genetic dissection of phototransduction: Phototransduction is the process by which light signals are converted to electrical signals in photoreceptors. The visual receptor molecule, rhodopsin, when light activated, interacts with a trimeric G protein, Gq, to activate a phospholipase Cβ (PLCβ) , to initiate a signaling cascade that results in the opening of two classes of light-activated channels, TRP and TRPL. Almost everything we know about this signaling cascade came from studies based on genetic approaches. The original genetic approach to phototransduction began in this laboratory over 40 years ago. We chose Drosophila because of its well-known genetics and well-developed eyes. We began chemically mutagenizing and isolating mutants that are defective in the electroretinogram (ERG), the light-evoked mass response of the eye. Over a period of 10 plus years, our laboratory isolated over 200 mutants falling into about 50 complementation groups. Many of the genes corresponding to these complementation groups, indeed, turned out to encode key components of the phototransduction machinery. We now know a great deal about the phototransduction cascade in Drosophila, though the knowledge is still incomplete. Not only do we know the identity of most of the key players in the process, but also many of the regulatory mechanisms involved. One of the most important early results to emerge from genetic studies of Drosophila phototransduction is to provide the first definitive evidence that, unlike vertebrate phototransduction, invertebrate phototransduction is based on inositol lipid-based signaling. Inositol lipid signaling had already been known to play a central role in controlling cellular Ca2+ levels in virtually every cell (Berridge et al., 2003). As a growing number of mutants affecting the Drosophila phototransduction cascade became identified and the cascade became understood in increasing detail, the Drosophila phototransduction cascade became the paradigm to explore inositol lipid signaling in depth. As Hardie (2003) stated, "With mutants available in all of these genes, the Drosophila compound eye represents a rich resource for genetic dissection of not only phototransduction but also more generally the ubiquitous inositol lipid signaling cascade." Many investigators contributed to the emergence of Drosophila phototransduction as a separate, vibrant and thriving discipline. These include not only those who were trained in this laboratory, but also many investigators who came into the field from various other laboratories. The infusion of the manpower and talent has transformed the field into what it is today. One of the most remarkable developments in this field has been the founding of a new superfamily of channels by the Drosophila phototransduction channel, TRP. This story began with the report of a spontaneously occurring mutant having a prematurely decaying ERG phenotype (Cosens and Manning, 1969). Soon after, our mutagenesis program generated 9 alleles of this mutant. Minke et al. (1975) carried out the first rigorous analysis of the mutant by intracellular recordings, noise analysis, and rhodopsin measurements. Intracellular recordings established definitively that the mutant defect resides within photoreceptors, whereupon these authors coined the term transient receptor potential (trp) to refer to these mutants and the gene identified by them. Minke went on to carry out several key studies elucidating TRP channel functions and laying the groundwork for search for mammalian homologs of TRP (Minke and Selinger, 1991; Hardie and Minke, 1992). The Drosophila trp gene was cloned by Montell and Rubin (1989) and Wong et al. (1989). Montell's group also was one of the two groups to isolate the first mammalian homologs of TRP (Wes et al., 1995; Zhu et al., 1995). There are now 28 TRP channels identified in mammals. They seem to constitute a major new superfamily of Ca2+ permeable ion channels, gated by signals other than voltage, responsible for regulation of Ca2+ homeostasis throughout the body throughout the animal phylogeny. Biological processes in which they have been implicated include: various sensory modalities, renal Ca2+/Mg2+ handling, smooth muscle tone, blood pressure regulation, growth cone guidance, etc. Not surprisingly, malfunctions of TRP channels have been implicated in a whole range of human diseases.
However, Drosophila phototransduction is still incompletely understood. Perhaps, the least understood aspects of this cascade are the mechanisms of activation and regulation of TRP/TRPL channels. Molecular signals to TRP/TRPL channels are not known. Previous workers reported on two potential candidate molecules of excitation, polyunsaturated acid (PUFA) (Chyb et al., 1999) and diacylglycerol (DAG) (Raghu et al., 2000). The first proposal was based largely on electrophysiological evidence and lacked genetic evidence; and the second proposal, though based on genetic evidence, could not distinguish between the effect of DAG and that of its metabolites, produced by the action of DAG lipase. Thus, the further progress on this problem seemed to hinge on the identification of the DAG lipase gene and isolation of mutations in the gene. Recently, we identified the first Drosophila DAG lipase gene expressed in photoreceptors, inaE, using mutants that had been isolated in the late '60's and the early '70's (Leung et al., 2008). An examination of a series of mutants of varying severity showed that the receptor potential was blocked to a varying degree in these mutants in an allele-dependent manner, suggesting that metabolites of DAG, rather than DAG, are the excitatory messenger. The DAG lipase identified is sn-1 specific. Its action on DAG generates 2-arachydonoyl glycerol (2-AG; a monoacylglycerol), which in turn is hydrolyzed to arachidonic acid (a PUFA) by monoacylglycerol lipase. Our current work is aimed at definitive identification of molecules of excitation to TRP and TRPL channels. This work is expected to provide insight into the activation mechanisms of mammalian TRP channels, most of which are also gated by inositol lipid signaling.
Genetic dissection of synaptic transmission: Although a great deal is now known about synaptic transmission, the precise in vivo roles of many of the molecules involved in it are still incompletely understood. Moreover, the roster of identified molecules is likely to be incomplete because the techniques used to identify them are heavily biased toward abundant ones. The use of genetic approach would bypass problems of this type because it is an unbiased approach with no a priori assumption about the protein product.
Among the mutants we isolated for use in genetic analysis of phototransduction are those that are impaired in synaptic transmission. These were identified and isolated in the mutant screen based on the ERG because the ERG contains contributions from photoreceptors as well as their target, postsynaptic neurons. We have identified synaptic transmission defective mutants falling into 8 complementation groups, of which four have been targeted for investigation in this first round. Functional analyses of synaptic transmission at the photoreceptor synapse are technically difficult but can be carried out very effectively at the larval neuromuscular junction. In addition, cloning genes that are identified only by chemically induced mutations is usually slow and laborious, but can be greatly facilitated by a novel microarray-based approach (Leung et al., 2008). Accordingly, we formed a collaboration with K. Broadie of Vanderbilt University and R. Doerge of Purdue Statistics Department aimed at cloning the genes corresponding to these complementation groups and analyzing the mutants at the larval neuromuscular junction. Cloning and the initial analysis of the first gene identified through this approach were completed about a year ago (Long et al., 2008). This gene, which was named fuseless (fusl), encodes a novel 8-pass transporter-like protein, which appears to regulate the assembly of the presynaptic Ca2+ channels, and thus is required for efficient synaptic vesicle exocytosis. Analysis of the second gene is currently well on the way. The second gene encodes a novel phosphatidylinositol 3-kinase (PI-3K). PI-3K has been implicated in vesicle priming and recycling previously (Richards et al., 2004; Meunier et al., 2005 ). However, its precise roles in synaptic transmission remain to be elucidated.
Education
A.B., summa cum laude, Boston University, 1955
Ph.D., Cornell University, 1960
Professional Faculty Research
(Molecular neurogenetics) Signal transduction; synaptic transmission.
Awards
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Other Activities
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