A FRET biosensor for necroptosis uncovers two different modes of the release of DAMPs

Shin Murai,Sotaro Uemura,Taichiro Tomida,Hideo Yagita,Ryosuke Miura,Satomi Adachi-Akahane,Ryodai Shindo,Osamu Nakabayashi,John Silke,Yoshifumi Yamaguchi,Masayuki Miura,Hiroyasu Nakano,Yoshitaka Shirasaki,Joanne M Hildebrand,Mai Yamagishi,Mamoru Totsuka

doi:10.1038/s41467-018-06985-6

Abstract

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.

Highlights

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)[3] and mixed lineage kinase domain-like (MLKL)
To investigate the mechanisms underlying the release of danger-associated molecular pattern (DAMP) from necroptotic cells, we developed a fluorescence resonance energy transfer (FRET) biosensor termed SMART
To develop a FRET biosensor that monitors necroptosis, we focused on the conformational change of MLKL

Summary

Introduction

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)[3] and mixed lineage kinase domain-like (MLKL). SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Analysis of SCAT3 transgenic mice revealed that the removal of apoptotic cells plays a crucial role in neural tube closure during the development of murine embryos[16] Together, these results indicate that live cell imaging of caspase activation in vitro and in vivo increases our understanding of pyroptosis and apoptosis under physiological and pathological conditions. To investigate the mechanisms underlying the release of DAMPs from necroptotic cells, we developed a FRET biosensor termed SMART (a Sensor for MLKL Activation by RIPK3 based on FRET). SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis

Methods

Results

Conclusion