Nanoscale organization of the endogenous ASC speck

Abstract

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to the formation of a supramolecular assembly of the inflammasome adaptor protein ASC, commonly referred to as the "ASC speck". Different models of the overall structure of the ASC speck, as well as the entire NLRP3 inflammasome, have been reported in the literature. While many experiments involve overexpression or in vitro reconstitution of recombinant ASC, the cytoplasmic endogenous ASC speck remains difficult to study due to its relatively small size and structural variability. Here, we use a combination of fluorescence imaging techniques including dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT) to visualize the endogenous ASC speck following NLRP3 inflammasome activation. We observe that the complex varies in diameter between [~]800 and 1000 nm and is composed of a dense core from which filaments extend into the periphery. We used a combination of anti-ASC antibodies as well as much smaller nanobodies for labeling, and find that the larger antibodies reliably label the lower-density periphery whereas the nanobody, which has a lower binding affinity, is more efficient in labeling the dense core. Imaging whole cells using dSTORM, furthermore, allowed us to sort the imaged structures into a pseudo-time sequence suggesting that the endogenous ASC speck becomes denser but not much larger during its formation. The reported results add an important piece of information towards a comprehensive understanding of the supramolecular structure of the endogenous inflammasome complex. SignificanceThe assembly of ASC into a supramolecular complex referred to as the ASC speck is a critical step in the NLRP3 inflammasome response. Despite significance research, the organization and formation of the endogenous ASC speck remain elusive. We used a combination of differently sized ASC labels with super-resolution microscopy to address the organization of the ASC speck at the nanoscale. Our results allow inference of the supramolecular structure of the unperturbed, endogenous speck and characterize how it changes during ASC recruitment. We find that the complex varies in size and is composed of a dense core from which filaments extend into the periphery. Notably, our results further indicate that during inflammasome formation, ASC specks become denser but only slightly larger.