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Cell-free formation, visualization and study of inflammasomes in real-time with optical tweezers and confocal fluorescence microscopy

使用光镊和共焦荧光显微镜实时观察炎症小体的无细胞形成、可视化和研究

基本信息

批准号：
10619602
负责人：
Eva de Alba Bastarrechea
金额：
$ 15.5万
依托单位：
UNIVERSITY OF CALIFORNIA, MERCED
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-09 至 2024-06-30
项目状态：
已结题

项目摘要

PROJECT SUMMARY/ABSTRACT. Inflammation is the primary response of the innate immune system to fight infection. Its dysregulation leads to chronic inflammation, a major cause of life-threatening diseases. The onset of inflammation depends on the intracellular assembly of multiprotein complexes known as inflammasomes, which form upon the presence of harmful substances by oligomerization of multiple copies of three proteins: - sensors that react upon danger signals derived from pathogens or damaged tissue; - procaspase-1 that activates inflammatory cytokines; - the adaptor ASC that functions as a molecular glue by connecting sensor and procaspase-1 molecules. Studying the molecular mechanisms that govern inflammasome assembly is challenging due to its dynamic nature and the strong tendency of inflammasome-related proteins to self- associate. These challenges limit our understanding of inflammasome assembly. Specifically, the AIM2-ASC inflammasome plays a critical role in the innate immune system against invading pathogens as the AIM2 sensor is capable of detecting foreign DNA. Despite extensive studies on the operating mode of the AIM2-ASC inflammasome, the interplay between AIM2, ASC and DNA in inflammasome formation is not fully understood. Particularly, the kinetic parameters involved in AIM2-ASC inflammasome assembly are not known and a knowledge gap exists regarding the molecular bases of AIM2 regulation. Filling these gaps will facilitate identifying molecular interventions for the manipulation of inflammasomes. Single-molecule techniques require sub-nanomolar protein concentrations thus enabling kinetic studies on inflammasome assembly before massive oligomerization occurs. In particular, optical tweezers can leverage AIM2-DNA binding and allow full mechanical control of the single DNA molecule. By combining optical tweezers, confocal fluorescence microscopy for real- time visualization and microfluidics for stepwise addition of inflammasome components, this proposal aims at forming and visualizing in a cell-free system and in real-time the AIM2-ASC inflammasome. Evidence is presented on the application of this strategy showing real-time assembly of AIM2 oligomers stochastically bound to a single DNA molecule tethered by two optically trapped beads. These promising results will be capitalized to determine the concerted roles of full-length ASC and AIM2 in inflammasome assembly (aim 1) and to establish the mechanism of action of a natural inhibitor of AIM2 on inflammasome regulation (aim 2). The overarching significance of the project is to obtain information on the mechanisms of inflammasome activation and regulation by overcoming current limitations using the proposed strategy: a) providing unknown information on the kinetics and assembly of the AIM2-ASC inflammasome; b) setting the grounds for the extension to other inflammasomes to obtain comprehensive relational data on inflammasome operating modes; c) testing inflammasome inhibitors to gain in-depth knowledge on their function at the molecular level; d) facilitating the design of molecular models of disease resulting from excessive inflammasome activation.

项目总结/抽象。炎症是先天免疫系统对抗的主要反应