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.

项目摘要/摘要渗透压的波动代表了细胞环境的关键挑战。解决方案的差异跨质膜的组成在降低水活性的方向上导致大量水运动，驾驶细胞收缩或肿胀。细胞通过OSMO调节机制动态响应这种应力旨在保持体积（补品）控制。根据细胞对机械应力的耐受性，适应状态只能部分纠正潜在的渗透失衡。结果，细胞内水的变化活性还扰动渗透敏感的相互作用，涉及分子水合的变化。渗透压力是由于暴露于非异构环境或增殖细胞中的代谢快速周转而引起的，而越来越多的人类疾病与持续的渗透压有关。渗透压是因此，生物医学研究许多领域的感兴趣参数。当前的技术不能直接访问细胞内部的渗透压。他们从功能或其他相关性（例如细胞体积，气体蔬菜，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，但不是特定于渗透灾害的，请限制其一般效用。直接访问细胞内渗透压将使研究人员能够建立评估渗透的标准度量响应并比较不同的细胞系统或应力条件。为了满足这个未满足的需求，这个建议旨在验证一种新的解决方案，以直接报告使用常见的细胞内渗透压成像和流式细胞仪仪。我们的方法基于渗透敏感的转录与渗透压不同的高亲和力DNA靶序列结合的因素。我们假设通过渗透敏感的转录因子在高亲和力DNA增强子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，我们将用作初始设计转录因子pu.1，其渗透敏感性是特征的。我们将1）构建荧光蛋白报道器系统，这些系统响应于渗透压。 2）我们将使用渗透损害的突变因素和校准活细胞中的渗透压读数。 3）为了最大化可寻址的生物范围，我们将概括我们的设计，以消除特定因子的转录机械的要求。 4）最后，我们将通过控制代谢报告基因营业额来将时间敏感的功能整合到传感器中。强调我们方法是一种模块化设计，它将接受多种替代转录因子，启动子和记者部分。此功能大大增强了风险管理。如果成功，这些创新将导致直接和非侵入性方法直接确定低和完整的延迟，速率和完整性细胞中所有生命王国的过度渗透压力反应。

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