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A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells

Abstract

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.

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Fig. 1: Methanol fixation is optimal for the immunofluorescent detection of human MLKL.
Fig. 2: Better antibodies are needed for imaging human RIPK3.
Fig. 3: Three specific antibodies for imaging endogenous human RIPK1.
Fig. 4: A new monoclonal antibody to image endogenous mouse MLKL.
Fig. 5: Unlike human RIPK3, mouse RIPK3 is highly amenable to detection by immunofluorescence and immunoblotting.
Fig. 6: Three specific antibodies for imaging endogenous mouse RIPK1.
Fig. 7: Optimised antibody cocktails for visualising endogenous necroptotic signaling in fixed human and mouse cells.

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Acknowledgements

We thank the Walter and Eliza Hall Institute Monoclonal Antibody Facility for their assistance generating clones 3H1, 10C2, 7G2, 1H2, 5A6, 8G7 and 1H12; and Cathrine Hall for assistance generating Mlkl−/− Ripk1−/− MDF lines. We acknowledge the WEHI histology facility for hosting the Vectra Polaris Imaging System and Emma Pan for acquiring the accompanying data. We thank: the laboratory of Tracy Putoczki for use of their Qiagen TissueLyser II device; Kim Newton and Vishva Dixit for sharing Ripk3−/− mice; and Michelle Kelliher for the Ripk1−/− mice used to generate the Ripk1−/−Mlkl−/− strain.

Funding

We are grateful to the Australian National Health and Medical Research Council for fellowship (JMH, 1142669; EDH, 1159488; JMM, 1172929), grant (1124735, 1124737, 1105023 and 2002965), and infrastructure (IRIISS 9000653) support, with additional support from the CASS Foundation (ALS) and the Victorian Government Operational Infrastructure Support scheme. We acknowledge the support for AVJ from an Australian Research Training Program Scholarship.

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Investigation: ALS, KP, CF, JMH, LW, JR, AVJ, CRH, XG, SNY Supervision and Methodology: ALS, JMH, KLR, EDH and JMM; Conceptualization: ALS, EDH, and JMM; Writing – Original Draft: ALS and JMM; Writing – Review & Editing: all authors.

Corresponding authors

Correspondence to André L. Samson or James M. Murphy.

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ALS, CF, KP, JMH, SNY, and JMM contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma. The other authors declare no competing interests.

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All animal procedures were approved by and conducted in accordance with the Animal Ethics Committee of the Walter and Eliza Hall Institute, Australia.

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Samson, A.L., Fitzgibbon, C., Patel, K.M. et al. A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells. Cell Death Differ (2021). https://doi.org/10.1038/s41418-021-00742-x

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