Live-cell visualization of gasdermin D-driven pyroptotic cell death

Area of Science:

• Immunology
• Cell Biology
• Molecular Biology

Background:

• Pyroptosis is a critical inflammatory cell death pathway involved in host defense against pathogens.
• Gasdermin D (GSDMD) is the key effector protein that forms pores in cell membranes during pyroptosis.
• Previous visualization studies were limited by the lack of endogenous GSDMD expression and necessary proteases in common cell lines.

Purpose of the Study:

• To develop novel GSDMD fusion proteins for live-cell imaging of pyroptosis.
• To investigate the molecular mechanisms of GSDMD cleavage, oligomerization, and pore formation.
• To enable real-time visualization of pyroptotic cell death and its associated morphological changes.

Main Methods:

• Mutagenesis and molecular modeling were used to strategically engineer GSDMD fusion proteins.
• Fusion proteins were tested for cleavage by caspases-1 and -11.
• Mutations were introduced to disrupt the GSDMD autoinhibitory interface.
• Inflammasome-dependent pyroptotic cell death was induced by various stimuli.

Main Results:

• Novel GSDMD fusion proteins were successfully created, allowing for visualization of native pyroptosis.
• Fusion proteins were confirmed to be cleaved by caspases-1 and -11 at Asp-276.
• Mutations disrupting the autoinhibitory interface led to GSDMD aggregation, supporting oligomerization.
• The engineered GSDMD fusions executed inflammasome-dependent pyroptosis and enabled real-time imaging of cell death.

Conclusions:

• New GSDMD fusion proteins provide valuable tools for studying pyroptosis.
• These tools facilitate the direct visualization of pyroptotic cell death in real time.
• The findings enhance the molecular understanding of GSDMD function and pyroptosis mechanisms.