Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

Summary

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

Abstract

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Introduction

Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) are central regulators of necroptotic cell death^1,2. Necroptosis is a lytic and inflammatory form of regulated cell death involved in antiviral immunity and autoinflammation. Necroptosis of virus-infected cells immediately shuts down virus replication. Cell lysis following necroptosis induction also releases damage-associated molecular patterns, which stimulate antiviral immunity^3,4. Necroptosis is initiated by the activation of RIPK3 following RI....

Protocol

1. Preparation of biotinylated tyramide

1. Prepare biotinylated tyramide starting from biotin-tyramide. To make a 10 mM stock solution, dissolve 3.6 mg of biotin-tyramide in 1 mL of DMSO. Store the dissolved product in aliquots at −20 °C to preserve the quality.

2. Maintaining HT-29 cells in culture

NOTE: ZBP1-expressing HT-29 were generated by transduction with a lentivector²⁷ encoding human ZBP1.

1. Keep ZBP1-expressing HT-29 cells in McCoy's 5A medium supplemented with L-glutamine, sodium-pyruvate, and 10% fetal b....

Results

The immunofluorescent detection of MLKL phosphorylation and especially RIPK3 phosphorylation in human cells is technically challenging²⁶. We here present an improved staining protocol for human p-RIPK3 (S227) and p-MLKL (S358) upon the activation of ZBP1. The protocol includes a TSA step to improve the detection limit and sensitivity of the fluorescent signals. To validate the method, a side-by-side comparison of the TSA-mediated immunofluorescence with standard indirect fluorescent staining of bo.......

Discussion

This immunofluorescent staining protocol describes the use of tyramide signal amplification (TSA) to increase the sensitivity for signaling events of the human necroptotic signaling pathway that are difficult to detect, including the phosphorylation of RIPK3 and MLKL²⁶. The inclusion of a TSA step significantly improves the detection threshold of p-RIPK3 (S227) and p-MLKL (S358) and increases the sensitivity of p-MLKL (S358) straining. TSA revealed a p-RIPK3 (S227) signal already present in the mo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies
Anti-rabbit HRP	Agilent Technologies Belgium	K4002	Envision+ System-HRP Labelled Polymer anti-rabbit
Goat anti-mouse DyLight 633	Thermofisher	35513	Secundary antibody
HSV-1 ICP0	Santa Cruz	sc-53070	Mouse anti-ICP0(HSV-1) antibody
IAV-PR8 mouse serum	In house production	xx	Mouse anti-IAV-PR8 polyclonal antibody
pMLKL	Abcam	ab187091	Rabbit anti-MLKL-phospho S358 antibody
pRIPK3	Abcam	ab209384	Rabbit anti-RIPK3-phospho S227 antibody
Fluorophores
DAPI	Thermofisher	D21490	To visualise the nucleus of the cells
Streptavidin coupled to Alexa Fluor 568	Thermofisher	S11226	To visulalise biotin molecules
Compounds
30% H2O2	Sigma	H1009	Oxidising substrate, necessary for HRP activity
4% PFA	SANBIO	AR1068	To fix/crosslink the cells
Biotinyl-tyramide	R&D Systems	6241	To amplify signal, HRP substrate
BV-6	Selleckchem	S7597	BV6 IAP Inhibitor
			For cell culture: to detach the cells
			8.0 g/L NaCl
			0.4 g/L disodium salt of EDTA
EDTA 0.04%	In house formulation		1.1 g/L Na2HPO4
			0.2 g/L NaH2PO4
			0.2 g/L KCl
			0.2 g/L Glucose
Fetal Bovine serum	TICO	FBS EU XXX	For cell culture, maintaining cell culture; lot number: 90439
GSK'840	Aobious	AOB0917	RIPK3 kinase inhibitor
L-Glutamine	Sigma-Aldrich	G7513	For cell culture, maintaining cell culture
MAXblock	Active Motif	15252	Blocking solution
PBS	Gibco	10444402
Sodium pyruvate	Sigma-Aldrich	S8636	For cell culture, maintaining cell culture
TNF-α	In house production	-	Signaling molecule, able to trigger cell death in combination with BV6 and zVAD
Triton X-100	Sigma Aldrich	T8787-50ML	To permeabilise the cells
Trypan blue	Merck	11732	For cell counting, used as live/dead marker at 0,1%
Trypsine	Sigma-Aldrich	T4424	For cell culture: to detach the cells
zVAD	Bachem	BACE4026865.0005	Z-Val-Ala-DL-Asp-fluoromethylketone
Material
µ-Slide 8 well high glass bottom	iBidi	80807	To culture the cells
Cotton Preping Balls-size medium	Electron Microscopy Sciences	71001-10	To clean the objectives
Immersol 518 F / 30 °C	ZEISS	444970-9000-000	To visualise the sample at high magnifications
Lens Cleaner	ZEISS	000000-0105-200	To clean the objectives
LSM880 Fast Airyscan confocal microscope			To visualise the sample
Software
Excel	Office	xx	To process the data
Prism 9	Graphpad	xx	To analyse the data- statistical testing and graph generation
Volocity 6.3	Volocity	xx	To perform quantifications
Zen black	ZEISS	xx	To aquire and process images
Zen blue	ZEISS	xx	To visualise images
Viruses
HSV-1 (mutRHIM) F strain	produced by  Dr. Jiahuai Han	in house replication	HSV-1 as a trigger for necroptosis; RHIM core domain of UL39/ICP6 is mutated (VQCG>AAAA)
HSV-1 (WT) F strain	Produced by Dr. Jiahuai Han	in house replication	HSV-1 (WT) as a negative control for necroptosis induction (ICP6 inhibition)
IAV PR8	in house stock	in house replication	IAV as a trigger for necroptosis