Design of Genetically Encoded Ca2+ Indicators for in Vivo Application

描述(由申请人提供)：遗传编码的钙传感器在体内解剖复杂的生理方面有很大的希望。在哺乳动物中实时进行分子尺度测量，并根据基因规范确定特定谱系的信号事件的能力，为确定构成正常器官功能的复杂细胞-细胞通讯以及伴随疾病和疾病标志的功能障碍提供了前所未有的实验力量。许多实验室已经开发出循环排列的EGFP-钙调蛋白/M13融合蛋白来了解体内几个复杂的生物学过程，这些工具已经开始为心脏发育、心脏修复和内皮细胞信号转导提供一个新的窗口。虽然这些研究证明了使用遗传编码的钙离子指示剂在哺乳动物分子水平上进行实时活体成像的可行性，但当前分子的局限性阻碍了对其的全面开发。这些限制包括不太理想的信号/噪声特性、非线性的钙离子响应、有效探针的有限光谱范围以及它们的非比率性质。总体目标是通过确定钙依赖荧光的结构基础，开发能够量化体内钙信号的多波长指示剂，以及创建红移的能够在体内研究细胞间信号的GECI，来开发改进的遗传编码的钙传感器(GECI)。这项工作代表着迈克尔·科特利科夫博士、霍尔格·桑德曼博士和沃伦·齐普费尔博士实验室之间正在进行的合作的延伸。科特利科夫博士在GECI的设计和功能方面拥有丰富的经验，霍尔格·桑德曼博士是蛋白质结构和功能方面的专家，沃伦·齐普费尔博士是一名具有荧光光物理专长的生物物理学家。这些研究将解决当前分子的几个重大局限性，并扩大此类分子将有用的研究范围。重点将放在开发的传感器的体内性能的优化上，这取决于它们在转基因小鼠中的表达。与公共卫生相关：该项目将生产可在体外细胞系统和动物体内使用的新分子，以确定疾病或器官修复背景下的细胞功能。该提议将产生新的蛋白质，将在分子水平上跟踪细胞的功能。

DESCRIPTION (provided by applicant): Genetically encoded Ca2+ sensors hold great promise for the dissection of complex physiology in vivo. The ability to make molecular scale measurements in real time in mammals, and to determine lineage-specific signaling events by genetic specification, provides unprecedented experimental power to determine the complex cell-cell communications that underlie normal organ function, and the dysfunction that attends and is the hallmark of disease. A number of laboratories have developed circularly permutated EGFP-Calmodulin/M13 fusion proteins to understand several complex biological processes in vivo, and these tools have begun to provide a novel window on heart development, heart repair, and endothelial cell signaling. While these studies demonstrate the feasibility of real-time, in vivo imaging at the molecular scale in mammals using genetically encoded Ca2+ indicators, limitations of current molecules prevent their comprehensive exploitation. These limitations include less than optimal signal/noise characteristics, a nonlinear Ca2+ response, the limited spectral range of effective probes, and their non-ratiometric nature. The overall goal is to develop improved genetically encoded Ca2+ sensors (GECIs) through the determination of the structural basis of Ca2+ -dependent fluorescence, the development of multiwavelength indicators that provide the ability to quantify Ca2+ signals in vivo, and the creation of red- shifted GECIs that enable studies of cell-cell signaling in vivo. The effort represents an extension of an ongoing collaboration between the laboratories of Dr. Michael Kotlikoff, who has significant experience with the design and function of GECIs, Dr. Holger Sondermann, who is an expert in protein structure and function, and Dr. Warren Zipfel, a biophysicist with expertise in fluorescence photophysics. These studies will address several significant limitations of current molecules and extend the range of studies for which such molecules will be useful. Emphasis will be placed on the optimization of developed sensors for in vivo performance, as determined by their expression in transgenic mice. PUBLIC HEALTH RELEVANCE: This project will produce novel molecules that can be used in vitro in cell systems and in vivo in animals to determine cellular function in the context of disease or organ repair. The proposal will produce novel proteins that will track the function of cells at the molecular level.