Genetically encoded sensors shed light on zinc homeostasis

描述(申请人提供)：据我们所知，过渡金属离子对生命至关重要。所有蛋白质中有30%含有金属离子辅因子，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发挥着重要作用。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深远的影响，并与一系列病理疾病相关，如阿尔茨海默病、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，确定细胞使用金属作为信号媒介的条件，并阐明金属失衡是如何导致疾病和退化的。目前的建议侧重于锌离子，因为有新的证据表明，细胞内可以产生瞬时的锌离子信号，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，在过渡金属离子中，锌离子是独一无二的，因为它集中在细胞亚群中的分泌小泡中，在细胞功能中扮演着特殊的、但不明确的角色。锌离子在这些细胞中的干扰具有毁灭性的后果，这突显了需要更深入地了解锌离子的生理作用以及锌离子干扰细胞过程的方式。我们目前对细胞内锌离子动态平衡的理解是有限的，因为缺乏合适的工具来研究高空间分辨率的锌离子分布。为了满足这一需求，我们建议开发一系列全面的锌离子荧光传感器，这些传感器可以通过基因编码，即明确地针对细胞的不同细胞器和亚域。这些传感器将定位于内质网、高尔基体和线粒体，以成像活细胞中锌的分布和转位。我们假设细胞含有不稳定的锌池，可以对细胞信号和压力做出反应，并且细胞细胞器在调节这些锌信号方面起着关键作用。我们的工作有三个具体目标：(1)通过系统研究自然存在的锌离子结合结构域和传感器文库的微流控筛选，开发可遗传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；以及(3)通过细胞信号(如一氧化氮)和细胞压力(如氧化还原失稳)来确定锌离子的来源和汇。与公共健康相关：对活细胞中金属离子定位和移位的定量成像将改变我们目前对金属动态平衡的认识，提供对细胞基本工作原理的洞察，并揭示当金属调控出错时被扰乱的细胞过程。活细胞内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，反过来也将改变金属离子如何影响细胞功能的理解。由于金属失衡和调节失调与多种疾病有关，如阿尔茨海默病、癌症和糖尿病，金属动态平衡对人类健康具有深远的影响。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。

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.