Cryo-electron tomography of ASC signalling sites in pyroptotic cells

BioRxiv : the Preprint Server for Biology
Y. LiuYorgo Modis

Abstract

Inflammasomes induce cell death in response to infection, chemical entities and cell damage. Human genetics and animal models have identified the NLRP3 inflammasome as a gatekeeper of caspase-1-dependent pyroptosis. NLRP3 activation induces polymerization of ASC into a single, micron-scale perinuclear punctum, where caspase-1 is activated. These puncta have yet to be imaged at sufficient resolution to resolve their ultrastructure. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 puncta and mitochondrial membrane remodelling within NLRP3-activated cells. The puncta are composed primarily of branched ASC filaments. The N-terminal pyrin domain forms an 8-10 nm-diameter tubular core, which is decorated by the flexibly linked C-terminal caspase recruitment domain. The variable filament density allows ribosomes and trans-Golgi-like vesicles to permeate the network. We propose this provides structural integrity while allowing macromolecules and vesicles to diffuse in or bind with the necessary density, timing and localization.

Methods Mentioned

BETA
fluorescence microscopy
light microscopy
electron microscopy
electron tomography
ELISAs
NMR
Fluorescence recovery after photobleaching
Flow cytometry
ELISA
confocal microscopy

Key Resources (RRID) Mentioned

AB_2783550
AB_631746
AB_2490440
AB_2799736
AB_955792
AB_416684
AB_477584
AB_1522533
AB_2207533
AB_2534102

Software Mentioned

Fiji
UCSF Chimera
SubTOM
MATLAB
Leica
IMOD
Flowjo10
ImageJ
GraphPad Prism
Leica LAS X

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