A cellular osmotic pressure sensor

细胞渗透压传感器

基本信息

批准号：
10153828
负责人：
Gregory Man Kai Poon
金额：
$ 23.39万
依托单位：
GEORGIA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10153828
关键词：
Address Affinity Area Automobile Driving Behavior Binding Binding Sites Biochemical Biological Biomedical Research Cell Volumes Cell Wall Cell membrane Cell model Cells Communities Crowding DNA DNA Binding DNA Sequence Data Dependence Development Diffuse Elements Enhancers Environment Equipment Exposure to Future Gases Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Hydration status Image Impairment In Situ Investigation Knowledge Laboratories Laboratory Research Life Measurable Measurement Measures Mechanical Stress Mechanics Membrane Metabolic Modeling Molecular Organism Osmolalities Osmoregulation Osmotic Pressure Permeability Physical shape Physiological Plants Process Prokaryotic Cells Proliferating Property Proteins Publishing Reagent Recovery Regulation Reporter Reporter Genes Reporting Research Research Personnel Risk Management Stress Swelling System Technology Testing Time Transactivation Translating Variant Vesicle Water Water Movements Yeasts base biological adaptation to stress cell type cofactor cost design environmental stressor experimental study human disease in vivo innovation insight instrumentation interest miniaturize mutant novel novel strategies pressure sensor promoter ratiometric response sensor solute tool transcription factor

项目摘要

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.

项目概要/摘要渗透压的波动代表了细胞环境差异的关键挑战。跨质膜的成分导致大量水向水活度降低的方向运动，驱动细胞收缩或肿胀。细胞通过渗透调节机制动态地响应这种压力。旨在维持音量（强直）控制，具体取决于细胞对机械应力的耐受性。状态可能只能部分纠正潜在的渗透不平衡，从而导致细胞内水的变化。活性还会扰乱涉及分子水合作用变化的渗透敏感相互作用。压力是由于暴露于非等渗环境或增殖细胞的快速代谢更替而产生的，并且越来越多的人类疾病与持续的渗透压有关。因此，当前技术无法直接确定生物医学研究许多领域感兴趣的参数。他们从功能或其他相关因素（例如）推断渗透压。细胞体积、气泡、基因表达或大分子拥挤这些间接指标。特定于不同的细胞类型但不特定于渗透压紊乱，限制了它们的一般用途。细胞内渗透压将使研究人员能够建立评估渗透压的标准指标响应，并比较不同的细胞系统或压力条件，为了解决这一未满足的需求，这该提案旨在验证一种使用常见方法直接报告细胞内渗透压的新解决方案我们的方法基于渗透敏感转录。结合高亲和力和低亲和力 DNA 靶序列的因素，对渗透压有明显的依赖性。我们假设渗透敏感转录因子对报告基因的差异反式激活高亲和力和低亲和力 DNA 增强剂可以产生细胞内渗透压的直接比例读数。为了验证这个概念，我们将使用转录因子 PU.1 作为初始设计，其渗透敏感性我们将 1) 构建对不同的荧光蛋白有不同响应的荧光蛋白报告系统。 2）我们将使用渗透压受损的突变因子来验证其操作基础校准活细胞中的渗透压读数 3) 为了最大化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。通过控制代谢报告基因周转，将时间敏感功能集成到传感器中，这是我们的重点。方法是一种模块化设计，可接受多种替代转录因子、启动子和如果成功，这些创新将大大增强风险管理。直接和非侵入性方法，用于直接确定低级和低级的潜伏期、速率和完整性来自所有生命王国的细胞的高渗应激反应。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The Non-continuum Nature of Eukaryotic Transcriptional Regulation.

DOI：
10.1007/5584_2021_618
发表时间：
2022
期刊：
Advances in experimental medicine and biology
影响因子：
0
作者：
Poon GMK
通讯作者：
Poon GMK

Dissecting Dynamic and Hydration Contributions to Sequence-Dependent DNA Minor Groove Recognition.

剖析动态和水合对序列依赖性 DNA 小沟识别的贡献。

DOI：
10.1016/j.bpj.2020.08.013
发表时间：
2020
期刊：
Biophysical journal
影响因子：
3.4
作者：
Ha,VanLT;Erlitzki,Noa;Farahat,AbdelbassetA;Kumar,Arvind;Boykin,DavidW;Poon,GregoryMK
通讯作者：
Poon,GregoryMK

Dissecting Knowledge, Guessing, and Blunder in Multiple Choice Assessments

剖析多项选择评估中的知识、猜测和错误