Genetically encoded sensors shed light on zinc homeostasis

基因编码传感器揭示了锌稳态

• 批准号: 7921863
• 负责人: Amy E Palmer
• 金额: $ 3.04万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921863
• 关键词: Address; Affect; Affinity; Alzheimer' s Disease; Base Sequence; Binding; Buffers; Calibration; Cell division; Cell physiology; Cells; Cellular Stress; Cellular biology; Classification; Cytoplasm; Development; Diabetes Mellitus; Disease; Ensure; Epithelial Cells; Equilibrium; Family; Fingers; Fluorescence Resonance Energy Transfer; Genetic Transcription; Goals; Golgi Apparatus; Health; Hela Cells; Hippocampus (Brain); Homeostasis; Human; Human Cell Line; Image; In Situ; In Vitro; Intervention; Investigation; Ions; Kinetics; Knowledge; Lead; Libraries; Life; Light; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Mammalian Cell; Measurement; Measures; Menkes Kinky Hair Syndrome; Metallothionein; Metals; Microfluidics; Mitochondria; Monitor; Movement; Nerve Degeneration; Neurons; Nitric Oxide; Nucleic acid sequencing; Organelles; Organism; Oxidation-Reduction; Peptide Signal Sequences; Peptides; Physiological; Play; Process; Property; Prostate; Proteins; Regulation; Research; Resolution; Respiration; Rest; Role; Screening procedure; Secretory Cell; Secretory Vesicles; Series; Signal Transduction; Source; Specificity; Stress; Technology; Tetracyclines; Transfection; Transgenic Organisms; Transition Elements; Translations; Validation; Vesicle; Work; Zinc; base; cellular imaging; cofactor; design; human disease; improved; insight; migration; oxygen transport; plasmid DNA; promoter; prototype; response; sensor; tool

Transition metal ions are critical to life as we know it. 30% of all proteins contain a metal ion cofactor and these proteins play essential roles in fundamental processes such as respiration, oxygen transport and storage, cell division and migration, and gene transcription. Paradoxically, these essential metals are also toxic and therefore cells must tightly regulate metal accumulation, transport, distribution and export. Not surprisingly, metal imbalance has profound implications at both the cellular and organismal level and is correlated with a host of pathological conditions such as Alzheimer's disease, neurodegeneration, diabetes, prostate cancer, and Wilson's and Menkes disease. The long term goals of our research are to identify the mechanisms by which cells balance metal ions, to define conditions under which cells use metals as signaling agents, and to elucidate how metal imbalance leads to disease and degeneration. The current proposal focuses on Zn2+ as there is emerging evidence that transient Zn2+ signals can be generated within the cell, representing an exciting new paradigm in how metal ions influence cellular function. Moreover, Zn2+ is unique among transition metal ions as it is concentrated into secretory vesicles in a sub-set of cells where it plays a specialized, but poorly defined role in cellular function. Disruption of Zn2+ in these cells has devastating consequences, highlighting the need for a deeper understanding of the physiological role of Zn2+ as well as the means by which Zn2+ disrupts cellular processes. Our current understanding of cellular Zn2+ homeostasis is limited by the lack of appropriate tools to interrogate Zn2+ distribution with high spatial resolution. We propose to address this need by developing a comprehensive family of fluorescent Zn2+ sensors that can be genetically encoded, i.e. explicitly targeted to distinct organelles and sub-domains of the cell. These sensors will be localized to the ER, Golgi, and mitochondria to image Zn2+ distribution and translocation in living cells. We hypothesize that cells contain labile pools of zinc that can be mobilized in response to cellular signals and stresses, and that cellular organelles play a critical role in modulating these zinc signals. Our proposed work has 3 specific aims: (1) Development of genetically encodable fluorescent zinc sensors by systematic investigation of naturally occurring Zn2+ binding domains and microfluidic screening of sensor libraries; (2) Biophysical characterization and in situ validation of sensors; and (3) Identify sources of and sinks for Zn2+ upon mobilization by cellular signals such as nitric oxide, and cellular stresses such as redox destabilization. Quantitative imaging of metal ion localization and translocation in living cells would transform our current knowledge of metal homeostasis, providing insight into the fundamental workings of the cell, and shedding light on cellular processes that are perturbed when metal regulation goes awry. Quantitative imaging of transition metal ions in living cells will transform our understanding of how cells regulate metal ion availability, and conversely how metal ions influence cellular function. Because metal imbalance and dysregulation have been correlated with a wide variety of diseases, such as Alzheimers, cancer, and diabetes, metal homeostasis has profound implications for human health. Understanding the detailed mechanisms by which organisms control metal ions will highlight potential avenues for intervention, and could ultimately lead to targeted therapies.

正如我们所知，过渡金属离子对生命至关重要。30%的蛋白质含有金属离子辅因子和 这些蛋白质在基本过程中发挥着重要作用，如呼吸、氧气运输和 储存、细胞分裂和迁移以及基因转录。矛盾的是，这些必需金属也是 因此，细胞必须严格控制金属的积累、运输、分配和出口。不 令人惊讶的是，金属失衡在细胞和组织水平上都有深远的影响，而且 与阿尔茨海默病、神经退行性变、糖尿病、 前列腺癌，以及威尔逊和门克斯病。我们研究的长期目标是确定 细胞平衡金属离子的机制，以确定细胞使用金属作为信号的条件 并阐明金属失衡是如何导致疾病和退化的。目前的提案 集中在锌离子上，因为有新的证据表明可以在电池内产生瞬时锌离子信号， 代表了金属离子如何影响细胞功能的令人兴奋的新范例。此外，锌离子是独一无二的 在过渡金属离子中，因为它被浓缩到细胞亚群中的分泌小泡中，在那里它扮演着 在细胞功能中专门的，但定义不明确的角色。这些细胞中锌离子的破坏具有破坏性 结果，突出了需要更深入地了解锌的生理作用以及 锌离子扰乱细胞过程的方式。我们目前对细胞内锌稳态的理解是 由于缺乏适当的工具来询问高空间分辨率的锌离子分布，这一点受到限制。我们建议 为了满足这一需求，我们开发了一系列可遗传的锌离子荧光传感器 编码的，即明确地针对细胞的不同细胞器和亚域。这些传感器将是 定位于内质网、高尔基体和线粒体，以显示活细胞中锌离子的分布和转位。我们 假设细胞含有不稳定的锌池，可以根据细胞信号和 细胞细胞器在调节这些锌信号方面起着关键作用。我们建议的工作 有3个具体目标：(1)利用系统的方法开发可遗传编码的荧光锌传感器 天然锌离子结合区的研究和传感器文库的微流控筛选 传感器的生物物理表征和现场验证；以及(3)确定锌离子的源和汇 通过一氧化氮等细胞信号和氧化还原失稳等细胞应激进行动员。 对活细胞中金属离子定位和移位的定量成像将改变我们的电流 金属动态平衡的知识，提供对细胞的基本工作原理的洞察，并提供启示 当金属调控出现问题时，细胞过程就会受到干扰。活细胞中过渡金属离子的定量成像将改变我们对细胞如何 调节金属离子的可用性，反之，金属离子如何影响细胞功能。因为金属 失衡和调节失调与多种疾病有关，例如阿尔茨海默氏症， 癌症、糖尿病、金属动态平衡对人类健康有着深远的影响。了解 生物控制金属离子的详细机制将突出潜在的干预途径， 并最终可能导致靶向治疗。