A cellular osmotic pressure sensor

项目总结/摘要 渗透压的波动代表了细胞环境的关键挑战。解决方案的差异 组合物穿过质膜导致大量水在降低水活性的方向上移动， 驱动细胞收缩或膨胀。细胞动态地响应这种压力与非调节机制 旨在保持音量（紧张）控制。根据细胞对机械应力的耐受性， 国家只能部分纠正潜在的渗透压失衡。结果，细胞内水的变化 活性还干扰涉及分子水合变化的敏感相互作用。渗透 应激产生于暴露于非等渗环境或增殖细胞中的快速代谢周转， 越来越多的人类疾病与持续的渗透压有关。渗透压 因此是生物医学研究的许多领域感兴趣的参数。目前的技术不能直接 获取细胞内的渗透压。他们从功能性或其他相关因素，如 细胞体积、气泡、基因表达或大分子拥挤。这些间接指标， 特别是对于不同的细胞类型，但不特异于渗透干扰，限制了它们的一般用途。直接访问 将使研究人员能够建立一个标准的指标，用于评估渗透压 反应，并比较不同的细胞系统或压力条件。为了满足这一未满足的需求， 一项提案旨在验证一种新的解决方案，以直接报告细胞内渗透压， 成像和流式细胞仪。我们的方法是基于免疫敏感的转录 这些因子结合高亲和力和低亲和力DNA靶序列，对渗透压具有明显的依赖性。 我们推测，在转录水平上，敏感的转录因子对报告基因的差异反式激活作用可能是由于转录水平的不同而引起的。 高亲和力和低亲和力DNA增强子可以产生细胞内渗透压的直接比率读数。 为了验证这一概念，我们将使用转录因子PU.1作为初始设计，其渗透敏感性 是有特点的。我们将1）构建荧光蛋白报告系统， 渗透压2)我们将使用免疫受损的突变因子来验证它们的操作基础， 校准活细胞中的渗透压读数。3)为了最大限度地扩大生物体的可寻址范围，我们将 推广我们的设计，以消除对因子特异性转录机制的要求。4)最后我们将 通过控制代谢报告子转换将时间敏感特征集成到传感器中。我们的重点是 方法是一种模块化设计，将接受广泛的替代转录因子，启动子， 报告分子部分。这一特点大大加强了风险管理。如果成功，这些创新将导致一个 直接和非侵入性的方法，用于直接确定潜伏期，速率和完整性的低， 高渗应激反应的细胞从所有王国的生活。

PROJECT SUMMARY/ABSTRACT Fluctuations in osmotic pressure represent a critical challenge of the cellular environment. Differences in solution composition across plasma membranes cause bulk water movement in the direction of decreasing water activity, driving cell shrinkage or swelling. Cells dynamically respond to such stresses with osmo-regulatory mechanisms aimed at maintaining volume (tonic) control. Depending on the cell’s tolerance for mechanical stress, the adapted state may only partially correct the underlying osmotic imbalance. As a result, variations in intracellular water activity also perturb osmotically sensitive interactions that involve changes in molecular hydration. Osmotic stress arises from exposure to non-isotonic environments or rapid metabolic turnover in proliferating cells, and an increasing number of human diseases are connected to persistent osmotic stress. Osmotic pressure is therefore a parameter of interest to many areas of biomedical research. Current technologies cannot directly access osmotic pressure inside the cell. They infer osmotic pressure from functional or other correlates such as cell volume, gas vesicles, gene expression or macromolecular crowding. These indirect metrics, which are particular to different cell types but not specific to osmotic disturbances, limit their general utility. Direct access to intracellular osmotic pressure would enable investigators to establish a standard metric for evaluating osmotic responses, and compare different cellular systems or stress conditions. To address this unmet need, this proposal is aimed at validating a novel solution to directly report intracellular osmotic pressure using common imaging and flow cytometric instrumentation. Our approach is based on osmotically sensitive transcription factors, which bind high- and low-affinity DNA target sequences with distinct dependence on osmotic pressure. We postulate that differential transactivation of reporter genes by osmotically sensitive transcription factors at high- and low-affinity DNA enhancers could yield a direct ratiometric readout of the intracellular osmotic pressure. To validate this concept, we will use as initial design the transcription factor PU.1, whose osmotic sensitivities are characterized. We will 1) construct fluorescent protein reporter systems that are differentially responsive to osmotic pressure. 2) We will validate their operational basis using osmotically impaired mutant factors and calibrate the osmotic pressure readout in live cells. 3) To maximize the addressable range of organisms, we will generalize our design to remove the requirement for factor-specific transcriptional machinery. 4) Finally, we will integrate a time-sensitive feature into the sensor by controlling metabolic reporter turnover. An emphasis in our approach is a modular design that will accept a wide range of alternate transcription factors, promoters, and reporter moieties. This feature greatly enhances risk management. If successful, these innovations will lead to a direct and non-invasive approach for directly determining the latency, rate, and completeness of hypo- and hyperosmotic stress response by cells from all kingdoms of life.