Molecular and cellular systems for visual pigment studies

由光引起的视觉色素激活形成眼睛的感光细胞内的光转导级联中的第一步。我的研究项目通过研究视觉基因的自然变异来研究视觉色素功能的基本机制。我们研究生活在不寻常的光限制环境中的动物的遗传变异，如夜行蛇和潜水鲸，以及人类视觉转导基因的遗传变异。我们的跨学科方法将进化分析与视觉色素功能的实验研究相结合，以更好地了解蛋白质结构和功能的进化。我们使用进化分析，以确定遗传适应，从自然实验中调整分子功能。这些视觉色素，然后瞬时表达在哺乳动物细胞培养，纯化，并使用光谱分析功能。最近，为了研究更困难的视色素变异体，我们发现越来越有必要构建稳定转染的细胞系，我们还扩大了我们的研究范围，包括视色素变异体在培养细胞中的亚细胞定位。对于RTI的这份提案，我要求提供新的设备，以扩大我们的研究能力，以及更新和更换对我的研究计划至关重要的老化设备。具体来说，我要求倒置荧光显微镜，这将是有用的，在构建稳定转染的细胞系，也将使我的实验室快速筛选大量的视紫红质突变体表达在培养细胞中更详细的分析，使用共聚焦显微镜。我还要求更换和升级对我们的研究计划至关重要的设备，其中大部分是在2003年我到达多伦多大学时购买的，包括一台冷冻台式离心机和二氧化碳培养箱。所要求的一些设备将位于一个新的光密封组织培养区，该区域是专门为我们的光敏视色素工作设计的，该大学目前正在翻新。这个新的设施，与这里要求的设备相结合，将使我们能够在光敏发色团存在的情况下，在培养的细胞中表达视觉色素。这将使我们能够表达和分析新的和以前无法获得的色素变体，并可能拯救不正确折叠的突变体。所要求的设备对我的研究计划的持续成功至关重要，并将支持和大大加强我们对视觉色素结构和功能的分子进化的研究。该设备也将由我系（Tepass，Mitchell）附近的教师使用，他们正在进行哺乳动物细胞培养，并需要使用荧光倒置显微镜。

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.