Genetically encoded sensors shed light on zinc homeostasis

描述（由申请人提供）：众所周知，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助因子，这些蛋白质在呼吸、氧气运输和储存、细胞分裂和迁移以及基因转录等基本过程中起着重要作用。矛盾的是，这些必需金属也是有毒的，因此细胞必须严格控制金属的积累、运输、分配和出口。毫不奇怪，金属失衡在细胞和机体水平上都有着深远的影响，并与许多病理状况有关，如阿尔茨海默病、神经变性、糖尿病、前列腺癌、威尔逊病和门克斯病。我们研究的长期目标是确定细胞平衡金属离子的机制，确定细胞使用金属作为信号剂的条件，并阐明金属失衡如何导致疾病和变性。目前的研究重点是Zn2+，因为有新的证据表明，瞬态Zn2+信号可以在细胞内产生，这代表了金属离子如何影响细胞功能的一个令人兴奋的新范式。此外，Zn2+在过渡金属离子中是独一无二的，因为它被浓缩到细胞亚群的分泌囊泡中，在细胞功能中起着专门的作用，但作用尚不明确。在这些细胞中破坏Zn2+具有毁灭性的后果，强调需要更深入地了解Zn2+的生理作用以及Zn2+破坏细胞过程的方式。我们目前对细胞Zn2+稳态的理解受到缺乏适当的工具来询问高空间分辨率的Zn2+分布的限制。我们建议通过开发一个全面的荧光Zn2+传感器家族来解决这一需求，这些传感器可以遗传编码，即明确针对不同的细胞器和细胞的亚结构域。这些传感器将定位于内质网、高尔基体和线粒体，以成像活细胞中Zn2+的分布和易位。我们假设细胞中含有不稳定的锌池，可以被动员起来响应细胞信号和应激，细胞器在调节这些锌信号中起关键作用。我们提出的工作有三个具体目标：(1)通过系统研究自然存在的Zn2+结合域和传感器文库的微流控筛选，开发遗传可编码的荧光锌传感器；(2)传感器的生物物理特性和原位验证；(3)在细胞信号（如一氧化氮）和细胞应激（如氧化还原不稳定）的动员下，确定Zn2+的来源和汇。公共卫生相关性：活细胞中金属离子定位和易位的定量成像将改变我们目前对金属稳态的认识，提供对细胞基本工作的洞察，并揭示当金属调节出错时受到干扰的细胞过程。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可用性的理解，反过来，金属离子如何影响细胞功能。由于金属失衡和失调与多种疾病有关，如阿尔茨海默病、癌症和糖尿病，因此金属稳态对人类健康具有深远的影响。了解生物体控制金属离子的详细机制将突出潜在的干预途径，并可能最终导致靶向治疗。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.