WILLIAM PAKPaul F. Oreffice Distinguished Professor of Biological Sciences
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 specific components 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) and 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 in the mid-60’s (review: Pak, 2010). We chose Drosophila for this purpose and 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 15 plus years, our laboratory isolated over 200 mutants falling into about 50 complementation groups (genes). 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 cellular signaling in virtually every cell (Berridge & Irvine, 1989). 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 in depth, not only phototransduction, but also the ubiquitous inositol lipid signaling in general. In addition, because this was the first sensory transduction mechanism to be subjected to a forward genetic analysis, many of the proteins identified turned out to be novel proteins or novel isoforms of identified proteins. Thus, investigations of these proteins often resulted in the creation of new subdisciplines of study. Many investigators from all over the world contributed to the emergence of Drosophila phototransduction as a vibrant and thriving discipline.
Although there are many examples, 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 by Cosens and Manning (1969) that a spontaneously occurring mutant displayed a prematurely decaying ERG phenotype (Cosens and Manning, 1969). Soon after, our mutagenesis program generated 9 alleles of this mutant. Minke, Wu, and Pak (1975), by means of intracellular recordings, established definitively that the mutant defect resides within photoreceptors and 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 the subsequent search for mammalian homologs of TRP (Suss et al., 1989; Minke and Selinger, 1991; Rom-Glas et al., 1992). The Drosophila trp gene was cloned by Montell and Rubin (1989) and Wong et al. (1989). The first definitive evidence that trp encodes a channel was obtained by Hardie and Minke (1992). Wes et al. (1995) and Zhu et al. (1995) isolated the first mammalian homologs of TRP. 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 the regulation of Ca2+ homeostasis throughout the body and throughout the animal phylogeny. Biological processes in which they have been implicated include: various sensory modalities, renal Ca2+/Mg2+ handling, smooth muscle tone and 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. At least five different hypotheses of TRP/TRPL activation have been proposed, none of which has found universal acceptance. Light-induced hydorolysis of phosphotidylinositol 4,5-bisphosphate (PIP2) by PLC results in the production of H+, inositol 1,4,5-trisphosphate (IP3) and diacylglylclerol (DAG) accompanied by a decrease in the PIP2 level. A long-standing and still viable hypothesis is that a metabolic product of DAG, polyunsaturated fatty acid (PUFA), is critical in TRP channel excitation (Chyb et al., 1999). Very recently, it has been proposed that a light–induced local acidification by H+ production combined with a decrease in the PIP2 level results in excitation (Huang et al., 2010). However, this hypothesis too has met with criticisms (see: Lev et al., 2012; Montell, 2012). A few years ago, we identified the first Drosophila DAG lipase gene expressed in photoreceptors, inaE, using mutants that had been isolated decades earlier (Leung et al., 2008). Mutations in this gene drastically impairs the receptor potential, suggesting that metabolites of DAG, such as PUFA, might, indeed, be important in TRP excitation. However, the inaE-encoded DAG lipase is sn-1 specific. Its action on DAG releases 2-monoacyl glycerol (2-MAG), which in turn must be hydrolyzed through the action of a MAG lipase to produce a PUFA. Little is known about MAG lipases in Drosophila. We have taken a bioinformatic approach to identify potential MAG lipases expressed in photoreceptors. However definitive results on the identity of molecules of excitation have not yet been obtained.
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 a forward 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 initially targeted mutants falling into 4 complementation groups. 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 microarray-based approach (Leung et al., 2008). Accordingly, we formed a collaboration with K. Brodie of Vanderbilt University and R. Doerge of Purdue Statistics Department to carry out this work and some initial results have been published (Long et al., 2008; 2010). Our goal, however, is to analyze additional mutants in many different genes with the hope of discovering novel principles of synaptic transmission that may not have been apparent in traditional studies or ones based on a small number of genes.
A.B., summa cum laude, Boston University, 1955
Ph.D., Cornell University, 1960
Professional Faculty Research
(Molecular neurogenetics) Signal transduction; synaptic transmission.
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