Genetically encoded sensors shed light on zinc homeostasis

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， 这些蛋白质在呼吸、氧运输和 储存、细胞分裂和迁移以及基因转录。巧合的是，这些必需金属也是 因此，细胞必须严格调节金属的积累、运输、分配和输出。不 令人惊讶的是，金属不平衡在细胞和生物体水平上都有深远的影响， 与许多病理状况相关，如阿尔茨海默病，神经变性，糖尿病， 前列腺癌和肝豆状核变性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 代理商，并阐明如何金属失衡导致疾病和退化。现时的建议 关注Zn 2+，因为有新的证据表明，瞬时Zn 2+信号可以在细胞内产生， 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，Zn 2+是唯一的 在过渡金属离子，因为它是集中到分泌囊泡在一个子集的细胞，它发挥了 专门的，但在细胞功能中定义不清的作用。这些细胞中的Zn 2+的破坏具有破坏性 结果，强调需要更深入地了解Zn 2+的生理作用以及 Zn 2+破坏细胞过程的手段。我们目前对细胞锌稳态的理解是 受限于缺乏适当的工具来以高空间分辨率询问Zn 2+分布。我们提出 通过开发一个全面的荧光Zn 2+传感器家族来解决这一需求， 在一些实施方案中，所述靶向分子是编码的，即明确靶向细胞的不同细胞器和亚结构域。这些传感器将 定位于ER、高尔基体和线粒体以成像活细胞中的Zn 2+分布和易位。我们 假设细胞含有不稳定的锌池，可以响应细胞信号而动员， 细胞器在调节这些锌信号中起着关键作用。我们建议的工作 本论文的主要目的有三：（1）系统研究锌离子荧光传感器的基因编码， 天然存在的Zn 2+结合结构域的研究和传感器库的微流体筛选;（2） 传感器的生物物理表征和原位验证;以及（3）确定Zn 2+的源和汇 通过细胞信号如一氧化氮和细胞应激如氧化还原失稳而动员。 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的 金属稳态的知识，提供深入了解细胞的基本运作，并揭示 当金属调节出错时，细胞过程会受到干扰。活细胞中过渡金属离子的定量成像将改变我们对细胞 调节金属离子的可用性，以及金属离子如何影响细胞功能。因为金属 失衡和失调与多种疾病，如阿尔茨海默病， 癌症和糖尿病，金属稳态对人类健康有着深远的影响。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。

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.