Nanoscale organization of the endogenous ASC speck
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 ASC, denoted as '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 reach out into the periphery. We used a combination of anti-ASC antibodies as well as a much smaller nanobody for labeling and show that the larger complexes do not reliably label the dense core whereas the nanobody, which has a lower binding affinity, is less efficient in labeling the lower-density periphery. Imaging whole cells using dSTORM, furthermore, allowed us to sort the imaged structures into a quasi-temporal sequence suggesting that the endogenous ASC speck becomes mainly denser but not much larger during its formation.