Nanoscale organization of the endogenous ASC speck

Ivo M Glück; Grusha Primal Mathias; Sebastian Strauss; Virgile Rat; Irene Gialdini; Thomas Sebastian Ebert; Che Stafford; Ganesh Agam; Suliana Manley; Veit Hornung; Ralf Jungmann; Christian Sieben; Don C Lamb

doi:10.1016/j.isci.2023.108382

Nanoscale organization of the endogenous ASC speck

iScience. 2023 Nov 2;26(12):108382. doi: 10.1016/j.isci.2023.108382. eCollection 2023 Dec 15.

Authors

Ivo M Glück^{1

2}, Grusha Primal Mathias¹, Sebastian Strauss^{3

4}, Virgile Rat^{1

2}, Irene Gialdini^{1

2}, Thomas Sebastian Ebert⁵, Che Stafford⁵, Ganesh Agam^{1

2}, Suliana Manley⁶, Veit Hornung^{4

5}, Ralf Jungmann^{3

4}, Christian Sieben⁶, Don C Lamb^{1

2}

Affiliations

¹ Department of Chemistry, Ludwig Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany.
² Center for Nano Science (CENS), Ludwig Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany.
³ Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
⁴ Max Planck Institute of Biochemistry, Martinsried, Germany.
⁵ Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany.
⁶ Laboratory of Experimental Biophysics, École Polytechnique Fédérale de Lausanne, BSP 427 (Cubotron UNIL), Rte de la Sorge, CH-1015 Lausanne, Switzerland.

Abstract

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to formation of the ASC speck, a supramolecular assembly of the inflammasome adaptor protein ASC. Different models, based on ASC overexpression, have been proposed for the structure of the ASC speck. Using dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT), we visualized the ASC speck structure following NLRP3 inflammasome activation using endogenous ASC expression. A complete structure was only obtainable by labeling with both anti-ASC antibodies and nanobodies. The complex varies in diameter between ∼800 and 1000 nm, and is composed of a dense core with emerging filaments. Dual-color confocal fluorescence microscopy indicated that the ASC speck does not colocalize with the microtubule-organizing center at late time points after Nigericin stimulation. From super-resolution images of whole cells, the ASC specks were sorted into a pseudo-time sequence indicating that they become denser but not larger during formation.

Keywords: Biotechnology; Cell biology; Immunology.