Molecular and cellular systems for visual pigment studies

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Visual pigment activation by light forms the first step in the phototransduction cascade within the photoreceptor cells of the eye. My research program investigates fundamental mechanisms of visual pigment function by studying the natural variation in vision genes. We study genetic variation in animals living in unusual light-limited environments, such as nocturnal snakes and diving whales, as well as human genetic variation in visual transduction genes. Our interdisciplinary approach combines evolutionary analyses with experimental studies of visual pigment function, in order to better understand the evolution of protein structure and function. We use evolutionary analyses to identify genetic adaptations that have resulted from natural experiments in tuning molecular function. These visual pigments are then transiently expressed in mammalian cell culture, purified, and functionally characterized using spectroscopic assays. Most recently, in order to work with more difficult visual pigment variants, we have found it increasingly necessary to construct stably transfected cell lines, and we have also expanded our research to include subcellular localization of visual pigment variants in cultured cells. For this RTI proposal, I am requesting new equipment that would expand our research capabilities, as well as updates and replacements for aging equipment essential to my research program. Specifically, I request an inverted fluorescent microscope that would be useful in constructing stably transfected cell lines, and would also allow my lab to rapidly screen large numbers of rhodopsin mutants expressed in cultured cells for more detailed analysis using confocal microscopy. I am also requesting replacements and upgrades of equipment crucial to our research program, much of which was purchased in 2003 when I arrived at the University of Toronto, including a refrigerated tabletop centrifuge, and CO2 incubator. Some of the requested equipment will be located in a new light-sealed tissue culture area specifically designed for our work with light-sensitive visual pigments that the University is currently in the process of renovating. This new facility, in conjunction with the equipment requested here, will allow us to be able to express visual pigments in cultured cells in the presence of the light-sensitive chromophore. This will allow us to express and assay new and previously inaccessible pigment variants, and to potentially rescue improperly folded mutants. The requested equipment is vital for the ongoing success of my research program, and will support and substantially enhance our research on the molecular evolution of visual pigment structure and function. This equipment will also be used by nearby faculty in my department (Tepass, Mitchell) who are working with mammalian cell culture, and require the use of a fluorescent inverted microscope.