Spatial and temporal control of cell behavior with a genetically-encoded photoswi

用基因编码的照片对细胞行为进行空间和时间控制

• 批准号: 7903492
• 负责人: Michael A Glotzer
• 金额: $ 30.36万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7903492
• 关键词: Adaptor Signaling Protein; Affinity; Biochemical; Biological; Biological Process; Biology; Cells; Complex; Development; Dimerization; Generic Drugs; Green Fluorescent Proteins; Heterodimerization; Life; Light; Location; Methods; Molecular Analysis; Pathway interactions; Proteins; Regulatory Pathway; Resolution; System; Technology; Time; biological research; cell behavior; cell type; cofactor; flexibility; interest; protein activation; protein function; public health relevance; response; spatiotemporal

DESCRIPTION (provided by applicant): Many biological processes occur at specific times and subcellular locations. Although technologies exist for descriptive analysis of molecular changes in living cells with high spatiotemporal resolution, only limited methods exist that permit experimental control of protein function in living cells. We propose to fulfill this unmet need with a flexible, genetically encoded system that uses light to locally induce the association of two proteins. Such complex formation is a widespread mechanism for protein activation in biology. Our proposed system consists of two adaptor proteins that have the capacity to heterodimerize with high affinity. A light sensing domain fused to one of the two partner proteins will inhibit heterodimerization in the dark state. Absorbance of light will trigger a reversible conformational change that will relieve this inhibition and allow dimerization. These adaptor proteins will be fused to proteins that trigger a biological response when colocalized. Hence, heterodimerization of the adaptor proteins will cause the fusion partners to associate with each other and activate the pathway of interest. Thus, we envisage a general system that will afford the ability to activate arbitrary biological pathways with high temporal and spatial resolution. Importantly, our strategy is genetically encoded and applicable to most cell types. These are the two features that have enabled the widespread use of green fluorescent protein (GFP) and we anticipate that our strategy will likewise be of broad utility and significantly impact biological research. PUBLIC HEALTH RELEVANCE: This project proposes a generic strategy to spatially and temporally control the activation of many proteins and regulatory pathways using light. This strategy is genetically encoded and does not require exogenous cofactors, therefore it will be suitable for use in a wide range of cellular and developmental contexts as well as biochemical studies.

描述（由申请人提供）：许多生物学过程发生在特定的时间和亚细胞位置。虽然存在高时空分辨率的活细胞中分子变化的描述性分析技术，但仅存在有限的方法允许实验控制活细胞中的蛋白质功能。我们建议用一种灵活的遗传编码系统来满足这一未满足的需求，该系统利用光来局部诱导两种蛋白质的结合。这种复合物的形成是生物学中蛋白质活化的普遍机制。我们提出的系统由两个衔接蛋白，具有高亲和力异二聚化的能力。与两个伴侣蛋白之一融合的光敏结构域将抑制黑暗状态下的异源二聚化。光的吸收将触发可逆的构象变化，这将缓解这种抑制并允许二聚化。这些接头蛋白将融合到蛋白质，当共定位时触发生物反应。因此，接头蛋白的异源二聚化将导致融合伴侣彼此缔合并激活感兴趣的途径。因此，我们设想一个通用的系统，将提供激活任意的生物途径的能力，具有高的时间和空间分辨率。重要的是，我们的策略是基因编码的，适用于大多数细胞类型。这两个特征使得绿色荧光蛋白（GFP）的广泛应用成为可能，我们预计我们的策略也将具有广泛的实用性，并对生物学研究产生重大影响。公共卫生相关性：该项目提出了一种通用策略，利用光在空间和时间上控制许多蛋白质和调节途径的激活。这种策略是遗传编码的，不需要外源辅因子，因此它将适用于广泛的细胞和发育背景以及生化研究。