Molecular and cellular systems for visual pigment studies

用于视色素研究的分子和细胞系统

• 批准号: RTI-2016-00643
• 负责人: Chang, Belinda
• 金额: $ 2.63万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=591510
• 关键词: Molecular; cellular; systems; visual; pigment

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.

光激活视觉色素是眼睛感光细胞内光转导级联反应的第一步。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。我们研究了生活在异常光线受限环境中的动物的遗传变异，如夜间活动的蛇和潜水鲸，以及人类视觉传导基因的遗传变异。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。我们使用进化分析来确定在调节分子功能的自然实验中产生的遗传适应。然后，这些视觉色素在哺乳动物细胞培养中瞬时表达，纯化，并使用光谱分析进行功能表征。最近，为了处理更困难的视觉色素变体，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，包括在培养细胞中视觉色素变体的亚细胞定位。对于这项RTI提案，我要求新的设备来扩展我们的研究能力，以及更新和更换对我的研究计划至关重要的老化设备。具体地说，我要求一台倒置荧光显微镜，它将有助于构建稳定转染的细胞系，并使我的实验室能够快速筛选大量在培养细胞中表达的视紫红质突变，以便使用共聚焦显微镜进行更详细的分析。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，包括一台冷藏桌面离心机和二氧化碳孵化器。所要求的一些设备将设在一个新的光封组织培养区，专门为我们使用光敏视觉颜料工作而设计，大学目前正在进行翻新。这一新设施与这里所需的设备相结合，将使我们能够在光敏发色团存在的情况下，在培养细胞中表达视觉色素。这将使我们能够表达和检测新的和以前无法获得的色素变体，并有可能挽救不正确折叠的突变。所要求的设备对我的研究计划的持续成功至关重要，将支持并大大加强我们对视觉色素结构和功能的分子进化的研究。我所在系附近的教员(泰帕斯、米切尔)也将使用这种设备，他们正在研究哺乳动物细胞培养，并且需要使用荧光倒置显微镜。