Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method

Area of Science:

• Immunology
• Cell Biology
• Innate Immune Response

Background:

• Inflammatory programmed cell death, including PANoptosis, is crucial for removing infected or damaged cells during innate immune responses.
• PANoptosis involves PANoptosomes, complex protein structures integrating elements from pyroptosis, apoptosis, and necroptosis.
• While PANoptosis is implicated in infections, cancers, and cytokine storms, PANoptosomes remain poorly characterized at the single-cell level.

Purpose of the Study:

• To optimize immunofluorescence and expansion microscopy techniques for visualizing PANoptosome assembly in individual cells.
• To validate antibodies for key PANoptosome components: ASC, CASP8, and RIPK3.
• To confirm PANoptosome formation across various innate immune cell death-inducing conditions.

Main Methods:

• Validated antibodies for endogenous mouse ASC, CASP8, and RIPK3 using knockout cells.
• Assessed PANoptosome formation by monitoring colocalization of ASC with CASP8 and/or RIPK3 via immunofluorescence.
• Employed expansion microscopy with validated antibodies to image the molecular organization within PANoptosomes.

Main Results:

• Successfully identified and validated antibodies for staining endogenous ASC, CASP8, and RIPK3.
• Demonstrated PANoptosome assembly by observing the colocalization of ASC with CASP8 and/or RIPK3 in response to cell death triggers.
• Visualized the nanoscale organization of ASC, CASP8, and RIPK3 within the PANoptosome using expansion microscopy.

Conclusions:

• Developed and optimized robust immunofluorescence and expansion microscopy protocols for studying PANoptosomes.
• Confirmed PANoptosome assembly at the single-cell level, providing critical insights into this innate immune pathway.
• Established a foundation for deeper molecular understanding of PANoptosomes and PANoptosis, facilitating future therapeutic targeting.