Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Summary

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.

Abstract

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

Introduction

Identifying genetic components of necroptosis has facilitated the use of animal models to test the implication of necroptosis in physiology and disease^1,2,3,4,5. Knockout of RIPK3 or MLKL in mice had minimal implication in development and adult homeostasis suggesting that necroptosis is not essential for life^3....

Protocol

1. Cloning and Cell Line Generation

1. PCR amplify the NBB region, corresponding to amino acid residues 1-140 (NBB₁₄₀), from human MLKL cDNA for in frame standard restriction enzyme-based cloning with the oligomerization domain 2x FK506 binding protein (2xFKBP or 2xFV) and Venus fluorescent protein into the Doxycycline (Dox)-inducible retroviral vector pRetroX-TRE3G to obtain NBB₁₄₀-2xFV-Venus (Table 1, Figures 1A-B).
2. Immortalize primary mlkl^-/- or ripk3^-/- mlkl^-/- mouse embryonic fibroblasts (MEFs) obtained from the respective mice available in the Green ....

Results

Visualizing regulated necroptosis execution in live cells has been possible through inducible expression of a minimal truncated MLKL construct, NBB₁₄₀-2xFV-Venus. This construct maintains the ability to induce plasma membrane permeabilization and is activated through Dim-induced oligomerization of the FKBP cassette (2xFV). We observe and quantify necroptosis by live-cell microscopy imaging, monitoring kinetically (every 5 min) the uptake of a cell impermeable green fluorescence.......

Discussion

We provide protocols for techniques that we combined to implicated MLKL as the putative executioner of plasma membrane rupture²⁴. In addition to deciphering the regulatory network that regulates MLKL-mediated necroptosis, these techniques can be used independently to characterize other suitable biological systems. Practically speaking, these techniques are medium- to low-throughput discovery tools.

We have routinely used live-cell imaging of NBB₁₄₀-2xFV-Venus.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cloning and cell line generation
pRetroX-TRE3G	Clontech	631188
Tet-On transactivator plasmid			Llambi et al., 2016
Mouse Embryonic Fibroblasts (MEFs) mlkl-/-			Dillon et al., 2014
Blasticidin S Hydrochloride	Thermo Fisher Scientific	BP2647100 CAS#3513-03-9
Cell death quantification and live-cell microscopy
Doxycycline	Clontech	631311 CAS# 24390-14-5
B/B Homodimerizer AP20187	Takara	635059 CAS# 195514-80-8
SYTOX Green	Thermo Fisher Scientific	S7020
Syto16	Thermo Fisher Scientific	S7578
NMR
15 N Ammonium Chloride	Cambridge Isotope Laboratories	NLM-467-10 CAS# 12125-02-9
Deuterated DTT	Cambridge Isotope Laboratories	DLM-2622-1
Deuterium Oxide	Sigma Aldrich	617385-1 CAS# 7789-20-0
n-Dodecyl-β-D-Maltopyranoside	Anatrace	D310 CAS# 69227-93-6
L-α-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt)	Avanti Polar Lipids	840046X CAS# 383907-42-4
1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (18:0 PI)	Avanti Polar Lipids	850143 CAS# 849412-67-5
1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium salt) (18:1)	Avanti Polar Lipids	850149 CAS# 799268-53-4
Specialized Equipment
IncuCyte FLR or ZOOM	Essen BioScience, Inc.		Live-cell microscopy imaging
Helios NanoLab 660 DualBeam	Thermo Fisher Scientific		Electron microscope
Software
IncuCyte 2011A Rev2 v20111.3.4288 (FLR)	Essen BioScience, Inc.	http://www.essenbioscience.com	Imaging analysis
FEI MAPS	Thermo Fisher Scientific	https://www.fei.com/software/maps/	EM analysis
TopSpin v3.2	Bruker BioSpin	http://www.bruker.com	NMR data collection
CARA v1.9.1.7		http://cara.nmr.ch/	NMR data analysis
Slidebook	3i (Intelligent Imaging Innovations)	https://www.intelligent-imaging.com/slidebook	Confocal microscopy