Abstract
NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization.
One-Sentence Summary Electron tomography of frozen cells reveals the ultrastructure of ASC specks and gasdermin D pores in adjacent mitochondria.
Competing Interest Statement
C.E.B are Y.M. are consultants for Related Sciences LLC and have profits interests in Danger Bio LLC. CEB is on the SAB of NodThera and Lightcast.
Footnotes
Main and supplementary text revised. Figures 1, 2, 4, 5, S3, S4, S5, S6 and S7 updated. Movies S2 and S4 updated. Data S1 and S4 updated.
https://github.com/jboulanger/imagej-macro/tree/main/FRAP_Measure
