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Updated: Nov 4, 2025
Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis
Published on: August 7, 2018
Time-Lapse Imaging of Necroptosis and DAMP Release at Single-Cell Resolution
Shin Murai1, Yoshitaka Shirasaki2, Hiroyasu Nakano3
1Department of Biochemistry, Toho University School of Medicine, Ota-ku, Tokyo, Japan.
Abstract:
Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). Necroptotic cells release a variety of cellular and nuclear factors, referred to as danger-associated molecular patterns (DAMPs). We recently developed a förster resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation based on FRET). SMART comprises a fragment of MLKL, and it monitors necroptosis, but not apoptosis or necrosis. We performed live-cell imaging for secretion activity (LCI-S) to observe the release of high-mobility group box 1 (HMGB1) from necroptotic cells at single-cell resolution. Moreover, we combined SMART and LCI-S imaging techniques and found two different modes of HMGB1 release from necroptotic cells. Thus, SMART and LCI-S are valuable tools for investigating intimate cross talk between necroptosis and DAMP release at single-cell resolution.
Insights
Researchers developed a novel FRET biosensor (SMART) to monitor necroptosis and combined it with imaging to reveal distinct modes of DAMP release, offering new insights into cell death signaling.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Necroptosis is a programmed form of necrosis crucial in immunity and disease.
- Necroptosis involves RIPK3 and MLKL, leading to the release of danger-associated molecular patterns (DAMPs).
- Understanding DAMP release mechanisms during necroptosis is vital for therapeutic development.
Purpose of the Study:
- To develop and validate a Förster Resonance Energy Transfer (FRET) biosensor for monitoring MLKL activation during necroptosis.
- To investigate the dynamics and modes of DAMP release, specifically HMGB1, from necroptotic cells.
- To establish a single-cell resolution method for studying necroptosis and DAMP secretion.
Main Methods:
- Development of SMART (a sensor for MLKL activation based on FRET) biosensor.
- Live-cell imaging for secretion activity (LCI-S) to track HMGB1 release.
- Combined SMART and LCI-S imaging for simultaneous monitoring of necroptosis and DAMP release.
Main Results:
- The SMART biosensor specifically detects necroptosis, distinguishing it from apoptosis and necrosis.
- Two distinct modes of high-mobility group box 1 (HMGB1) release were identified from necroptotic cells.
- Single-cell resolution imaging revealed the kinetics and patterns of DAMP secretion.
Conclusions:
- SMART is a reliable tool for studying necroptosis.
- The combination of SMART and LCI-S provides unprecedented resolution for analyzing necroptosis-associated DAMP release.
- These findings enhance our understanding of the interplay between necroptosis and immune signaling.
