Yu Wei, Xiangyu Zi, Jia Zhai, Man Zhang, Jiaqi Li, Zhenglong Sun, Minzi Ju*, Xin Zhang* and Baoxing Shen*,
{"title":"利用黏度敏感荧光寿命探针探索内质网功能障碍对蛋白质相分离的影响","authors":"Yu Wei, Xiangyu Zi, Jia Zhai, Man Zhang, Jiaqi Li, Zhenglong Sun, Minzi Ju*, Xin Zhang* and Baoxing Shen*, ","doi":"10.1021/acs.analchem.5c0113110.1021/acs.analchem.5c01131","DOIUrl":null,"url":null,"abstract":"<p >Degenerative diseases are closely associated with protein phase transitions. Endoplasmic reticulum (ER), the primary site of protein synthesis, experiences homeostasis imbalance as the key trigger of the protein phase transition. Effective tools to monitor ER microenvironment changes are crucial for investigating protein phase behavior. In this work, we developed a set of viscosity-sensitive probes based on dicyanomethylene-4H-pyran (namely, VisDCM probes) with dual response of fluorescence intensity and fluorescence lifetime to local viscosity changes. Computational analysis demonstrated that fluorescence activation of VisDCM probes is due to the restricted accessible conical intersection mechanism under specific viscosity. Dual-color probes targeting the ER and protein of interest were designed. They revealed how ER stress regulates TDP-43 protein phase separation via Ca<sup>2+</sup> signaling. <i>In vitro</i> experiments exhibited that TDP-43 phase separation is Ca<sup>2+</sup>-dependent. Increased Ca<sup>2+</sup> promotes TDP-43 liquid–liquid phase separation and aggregation. Finally, fluorescence lifetime imaging was applied to map ERS-induced microenvironment changes. In summary, this work provides a novel toolbox to visualize protein phase transitions as well as highlights Ca<sup>2+</sup> role in TDP-43 phase separation and aggregation, offering insights and potential therapies for degenerative diseases.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"97 18","pages":"10038–10045 10038–10045"},"PeriodicalIF":6.7000,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Exploring Endoplasmic Reticulum Dysfunction on Protein Phase Separation Using Viscosity-Sensitive Fluorescent Lifetime Probe\",\"authors\":\"Yu Wei, Xiangyu Zi, Jia Zhai, Man Zhang, Jiaqi Li, Zhenglong Sun, Minzi Ju*, Xin Zhang* and Baoxing Shen*, \",\"doi\":\"10.1021/acs.analchem.5c0113110.1021/acs.analchem.5c01131\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >Degenerative diseases are closely associated with protein phase transitions. Endoplasmic reticulum (ER), the primary site of protein synthesis, experiences homeostasis imbalance as the key trigger of the protein phase transition. Effective tools to monitor ER microenvironment changes are crucial for investigating protein phase behavior. In this work, we developed a set of viscosity-sensitive probes based on dicyanomethylene-4H-pyran (namely, VisDCM probes) with dual response of fluorescence intensity and fluorescence lifetime to local viscosity changes. Computational analysis demonstrated that fluorescence activation of VisDCM probes is due to the restricted accessible conical intersection mechanism under specific viscosity. Dual-color probes targeting the ER and protein of interest were designed. They revealed how ER stress regulates TDP-43 protein phase separation via Ca<sup>2+</sup> signaling. <i>In vitro</i> experiments exhibited that TDP-43 phase separation is Ca<sup>2+</sup>-dependent. Increased Ca<sup>2+</sup> promotes TDP-43 liquid–liquid phase separation and aggregation. Finally, fluorescence lifetime imaging was applied to map ERS-induced microenvironment changes. In summary, this work provides a novel toolbox to visualize protein phase transitions as well as highlights Ca<sup>2+</sup> role in TDP-43 phase separation and aggregation, offering insights and potential therapies for degenerative diseases.</p>\",\"PeriodicalId\":27,\"journal\":{\"name\":\"Analytical Chemistry\",\"volume\":\"97 18\",\"pages\":\"10038–10045 10038–10045\"},\"PeriodicalIF\":6.7000,\"publicationDate\":\"2025-04-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical Chemistry\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acs.analchem.5c01131\",\"RegionNum\":1,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.analchem.5c01131","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
Exploring Endoplasmic Reticulum Dysfunction on Protein Phase Separation Using Viscosity-Sensitive Fluorescent Lifetime Probe
Degenerative diseases are closely associated with protein phase transitions. Endoplasmic reticulum (ER), the primary site of protein synthesis, experiences homeostasis imbalance as the key trigger of the protein phase transition. Effective tools to monitor ER microenvironment changes are crucial for investigating protein phase behavior. In this work, we developed a set of viscosity-sensitive probes based on dicyanomethylene-4H-pyran (namely, VisDCM probes) with dual response of fluorescence intensity and fluorescence lifetime to local viscosity changes. Computational analysis demonstrated that fluorescence activation of VisDCM probes is due to the restricted accessible conical intersection mechanism under specific viscosity. Dual-color probes targeting the ER and protein of interest were designed. They revealed how ER stress regulates TDP-43 protein phase separation via Ca2+ signaling. In vitro experiments exhibited that TDP-43 phase separation is Ca2+-dependent. Increased Ca2+ promotes TDP-43 liquid–liquid phase separation and aggregation. Finally, fluorescence lifetime imaging was applied to map ERS-induced microenvironment changes. In summary, this work provides a novel toolbox to visualize protein phase transitions as well as highlights Ca2+ role in TDP-43 phase separation and aggregation, offering insights and potential therapies for degenerative diseases.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.