Detection of Gasdermin Activation and Lytic Cell Death During Pyroptosis and Apoptosis

Methods Mol Biol. 2022:2523:209-237. doi: 10.1007/978-1-0716-2449-4_14.

Abstract

Cytosolic pattern recognition receptors trigger pyroptosis by detection of danger- or pathogen-associated molecular patterns. These receptors initiate the assembly of inflammasomes, multimeric protein complexes that drive caspase-1 activation. Active caspase-1 cleaves the proinflammatory cytokines IL-1β and IL-18 and the pore-forming protein gasdermin-D (GSDMD) thereby liberating its N-terminal domain. The GSDMD N-termini form multimeric pores at the plasma membrane that allow leakage of intracellular content and ultimately trigger a type of cell death called "pyroptosis." Emerging studies have revealed that GSDMD is also processed by apoptotic caspases-8/-3/-7. In this chapter, we aim to describe methods to monitor lytic cell death and to distinguish between GSDMD processing events and the GSDMD fragments that are generated after pyroptosis or apoptosis induction. We also illustrate the difference between GSDMD pore formation, and final cell lysis, and how this affects to the release of intracellular content. Finally, we show that the activation of another pore-forming protein, gasdermin-E, does not exclusively translate into lytic cell death in bone marrow-derived macrophages.

Keywords: Apoptosis; Cell death; Gasdermin; IL-1β quantification; Immunoblotting; Inflammasomes; LDH detection; Pyroptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Caspase 1 / metabolism
  • Inflammasomes / metabolism
  • Intracellular Signaling Peptides and Proteins* / metabolism
  • Neoplasm Proteins / metabolism
  • Phosphate-Binding Proteins / metabolism
  • Pyroptosis*

Substances

  • Inflammasomes
  • Intracellular Signaling Peptides and Proteins
  • Neoplasm Proteins
  • Phosphate-Binding Proteins
  • Caspase 1