Spatiotemporal control of necroptotic cell death and plasma membrane recruitment using engineered MLKL domains

Area of Science:

• Cell Biology
• Molecular Biology
• Immunology

Background:

• Necroptosis is a programmed cell death pathway crucial for antiviral defense and implicated in diseases like cancer and diabetes.
• The precise mechanisms of necroptosis and protective cellular factors remain incompletely understood.
• Mixed lineage kinase domain-like (MLKL) protein oligomerization is a key event triggering plasma membrane rupture during necroptosis.

Purpose of the Study:

• To engineer an optogenetic tool for precise spatiotemporal control of necroptosis.
• To utilize this tool for chemical genetic screening to identify necroptosis inhibitors.
• To develop a novel optogenetic tool for modulating protein function at the plasma membrane.

Main Methods:

• Engineered a light-activated MLKL variant that induces rapid cell death upon light exposure.
• Utilized the tool for spatial and temporal control of necroptosis in cellular models.
• Performed chemical genetic screens to identify compounds and pathways modulating MLKL-induced cell death.
• Re-engineered MLKL to create a non-lethal, light-recruitable membrane-binding protein.

Main Results:

• Demonstrated precise spatial and temporal control over MLKL-induced necroptosis using light.
• Identified novel chemicals and pathways that protect cells from necroptosis.
• Successfully developed a light-inducible membrane recruitment tool for protein function modulation.
• Enabled the study of signaling responses in non-dying bystander cells.

Conclusions:

• The light-activated MLKL tool provides unprecedented control over necroptosis, facilitating mechanistic studies.
• This optogenetic approach is effective for high-throughput screening of necroptosis regulators.
• The developed tools offer new avenues for investigating cell death pathways and developing therapeutic strategies for related diseases.