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Updated: Feb 6, 2026
Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis
Published on: August 7, 2018
Generation and Use of Chimeric RIP Kinase Molecules to Study Necroptosis
Diego A Rodriguez1, Douglas R Green2
1Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Abstract:
Necroptosis, a form of regulated necrosis, is triggered by a variety of signals that converge to activate receptor interacting protein kinase-3 (RIPK3), consequently promoting the direct phosphorylation and activation of the mixed lineage kinase like (MLKL) protein. Active MLKL executes necroptosis by disrupting the integrity of the plasma membrane. Stimuli that can induce necroptosis include ligation of death receptors (a subset of the TNFR family), toll-like receptors (in particular, TLR3 and TLR4), interferons, and the intracellular viral sensor, DAI/ZBP1, among others. To study the process in more detail, it is useful to have a means to directly activate RIPK3. Here we provide protocols and procedures to artificially induce necroptotic cell death by drug-induced forced dimerization of RIPK3. We also provide information on specific kinase inhibitors, procedures to monitor RIPK3 and MLKL activation, and real-time quantification of cell death.
Insights
Necroptosis, a regulated cell death pathway, is initiated by RIPK3 activation. This study introduces a method for drug-induced RIPK3 activation to study necroptosis and cell death.
Area of Science:
- Cellular Biology
- Immunology
- Biochemistry
Background:
- Necroptosis is a regulated form of necrosis.
- It is triggered by various signals activating RIPK3 and MLKL.
- Active MLKL disrupts plasma membrane integrity, causing cell death.
Purpose of the Study:
- To provide protocols for artificially inducing necroptosis.
- To enable detailed study of necroptosis by direct RIPK3 activation.
- To offer methods for monitoring RIPK3/MLKL activation and cell death.
Main Methods:
- Drug-induced forced dimerization of RIPK3.
- Monitoring RIPK3 and MLKL activation.
- Real-time quantification of cell death.
Main Results:
- Successful induction of necroptotic cell death via forced RIPK3 dimerization.
- Established protocols for monitoring key necroptosis pathway components.
- Quantified cell death in real-time.
Conclusions:
- Drug-induced RIPK3 activation provides a robust method to study necroptosis.
- The presented protocols facilitate research into necroptosis signaling.
- This approach aids in understanding regulated necrosis and developing therapeutic strategies.
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