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

Award Number: R21AI168983
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2023 ( Subtotal = $155,000 )
2023	2023	UNIVERSITY OF CALIFORNIA, MERCED FOUNDATION	5200 N LAKE RD	MERCED	CA	95343	MERCED	USA	Allergy and Infectious Diseases Research	000	2	4/20/2023	NON-COMPETING CONTINUATION	$155,000
														Subtotal = $155,000

Issue Date FY: 2022 ( Subtotal = $244,243 )
2022	2022	UNIVERSITY OF CALIFORNIA, MERCED	5200 N LAKE RD	MERCED	CA	95344	MERCED	USA	Allergy and Infectious Diseases Research	000	1	5/9/2022	NEW	$244,243
														Subtotal = $244,243

Grand Total All Awards = $399,243

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

Award Number: R21AI168983

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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