# Cell-free formation, visualization and study of inflammasomes in real-time with optical tweezers and confocal fluorescence microscopy > **NIH NIH R21** · UNIVERSITY OF CALIFORNIA, MERCED · 2023 · $155,000 ## Abstract 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 g... ## Key facts - **NIH application ID:** 10619602 - **Project number:** 5R21AI168983-02 - **Recipient organization:** UNIVERSITY OF CALIFORNIA, MERCED - **Principal Investigator:** Eva de Alba Bastarrechea - **Activity code:** R21 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2023 - **Award amount:** $155,000 - **Award type:** 5 - **Project period:** 2022-05-09 → 2024-06-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10619602 ## Citation > US National Institutes of Health, RePORTER application 10619602, Cell-free formation, visualization and study of inflammasomes in real-time with optical tweezers and confocal fluorescence microscopy (5R21AI168983-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10619602. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*