Single-Molecule Fluorescence Reveals the Oligomerization and Folding Steps Driving the Prion-like Behavior of ASC

J Mol Biol. 2018 Feb 16;430(4):491-508. doi: 10.1016/j.jmb.2017.12.013. Epub 2017 Dec 27.


Single-molecule fluorescence has the unique ability to quantify small oligomers and track conformational changes at a single-protein level. Here we tackled one of the most extreme protein behaviors, found recently in an inflammation pathway. Upon danger recognition in the cytosol, NLRP3 recruits its signaling adaptor, ASC. ASC start polymerizing in a prion-like manner and the system goes in "overdrive" by producing a single micron-sized "speck." By precisely controlling protein expression levels in an in vitro translation system, we could trigger the polymerization of ASC and mimic formation of specks in the absence of inflammasome nucleators. We utilized single-molecule spectroscopy to fully characterize prion-like behaviors and self-propagation of ASC fibrils. We next used our controlled system to monitor the conformational changes of ASC upon fibrillation. Indeed, ASC consists of a PYD and CARD domains, separated by a flexible linker. Individually, both domains have been found to form fibrils, but the structure of the polymers formed by the full-length ASC proteins remains elusive. For the first time, using single-molecule Förster resonance energy transfer, we studied the relative positions of the CARD and PYD domains of full-length ASC. An unexpectedly large conformational change occurred upon ASC fibrillation, suggesting that the CARD domain folds back onto the PYD domain. However, contradicting current models, the "prion-like" conformer was not initiated by binding of ASC to the NLRP3 platform. Rather, using a new method, hybrid between Photon Counting Histogram and Number and Brightness analysis, we showed that NLRP3 forms hexamers with self-binding affinities around 300nM. Overall our data suggest a new mechanism, where NLRP3 can initiate ASC polymerization simply by increasing the local concentration of ASC above a supercritical level.

Keywords: inflammasome; prion-like polymerization; single-molecule spectroscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CARD Signaling Adaptor Proteins / chemistry*
  • Fluorescence
  • Fluorescence Resonance Energy Transfer / methods*
  • Humans
  • Models, Molecular
  • NLR Family, Pyrin Domain-Containing 3 Protein / chemistry
  • Prions / chemistry
  • Protein Aggregates
  • Protein Conformation
  • Protein Domains
  • Protein Folding*
  • Protein Multimerization
  • Single Molecule Imaging / methods*


  • CARD Signaling Adaptor Proteins
  • NLR Family, Pyrin Domain-Containing 3 Protein
  • PYCARD protein, human
  • Prions
  • Protein Aggregates