Mechanism of membrane pore formation by human gasdermin‐D

• Published in: The EMBO journal Vol. 37; no. 14
• Main Authors: Mulvihill, Estefania, Sborgi, Lorenzo, Mari, Stefania A, Pfreundschuh, Moritz, Hiller, Sebastian, Müller, Daniel J
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 13.07.2018; Springer Nature B.V; John Wiley and Sons Inc
• Subjects: Assembling; Assembly; Atomic force microscopy; Caspase; Caspases - metabolism; Cell death; Cell Membrane - metabolism; Cholesterol; EMBO07; EMBO19; gasdermin‐D pore assembly; Humans; Inflammation; Insertion; Inserts; Lipid membranes; Liposomes - metabolism; Membranes; Metals; Microscopy, Atomic Force; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Oligomerization; Oligomers; Pore formation; Pores; Porosity; Protein Multimerization; Protein Transport; Proteins; Pyroptosis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Time-Lapse Imaging; time‐lapse high‐resolution atomic force microscopy; transmission electron microscopy; time‐lapse high‐resolution atomic force microscopy; transmission electron microscopy; cell death; gasdermin‐D pore assembly; inflammation
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.15252/embj.201798321

Gasdermin‐D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N‐terminal domain (GSDMD Nterm ) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMD Nterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high‐resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMD Nterm inserts and assembles in membranes. We observe GSDMD Nterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMD Nterm assembles arc‐, slit‐, and ring‐shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase‐1, caspase‐4, or caspase‐5. Using time‐lapse AFM, we monitor how GSDMD Nterm assembles into arc‐shaped oligomers that can transform into larger slit‐shaped and finally into stable ring‐shaped oligomers. Our observations translate into a mechanistic model of GSDMD Nterm transmembrane pore assembly, which is likely shared within the gasdermin protein family. Synopsis Gasdermin‐D, which mediates pyroptosis in human and murine cells, is directly observed inserting into lipid membranes and assembling arc‐, slit‐ and ring‐shaped oligomers. The observations translate into a mechanistic model of gasdermin‐D assembling transmembrane lytic pores. High‐resolution time‐lapse imaging of gasdermin‐D pore formation. Gasdermin‐D assembles arc‐, slit‐ and ring‐shaped oligomers. Arc‐ and slit‐shaped pores transform into stable ring‐shaped pores. Phosphatidylinositide (PI(4,5)P2) increases gasdermin‐D pore formation. Cholesterol reduces gasdermin‐D pore formation. Graphical Abstract High‐resolution atomic force microscopy shows how a pyroptosis‐mediating gasdermin protein inserts into lipid membranes and assembles arc‐, slit‐ and ring‐shaped oligomers.