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Development of novel methods that monitor necroptosis and the release of DAMPs at the single cell resolution.

ABSTRACT: Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). While danger-associated molecular pattern (DAMP)s are released from dead cells and involved in various pathological conditions, the mechanisms underlying regulation of the release of DAMPs are not fully understood. Apoptosis and pyroptosis can be detected by several types of sensors such as Forster resonance energy transfer (FRET) biosensors, termed SCAT1 (a sensor for caspase 1 activation based on FRET) and SCAT3, respectively. These sensors have provided better understanding of pyroptosis and apoptosis in vitro and in vivo. However, there have been no biosensors to monitor necroptosis. Development of a FRET biosensor that monitors necroptosis and generation of transgenic mice expressing such FRET biosensor might be useful to understand the mechanisms underlying the execution of necroptosis and also the consequences of necroptosis in vivo. In our recent study (Nat Commun, 9(1):4457), we developed a FRET biosensor for necroptosis, 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. Moreover, we recently developed a platform called Live-Cell Imaging for Secretion activity (LCI-S) to monitor protein secretion at the single cell level. This platform has enabled us to monitor the release of HMGB1 (High Mobility Group Box 1), one of the DAMPs, at the single cell level and reveals two different modes of the release of HMGB1 from necroptotic cells.

SUBMITTER: Nakano H

PROVIDER: S-EPMC6551708 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Development of novel methods that monitor necroptosis and the release of DAMPs at the single cell resolution.

Nakano Hiroyasu H Murai Shin S Yamaguchi Yoshifumi Y Shirasaki Yoshitaka Y Nakabayashi Osamu O Yamazaki Soh S

Cell stress 20190122 2

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). While danger-associated molecular pattern (DAMP)s are released from dead cells and involved in various pathological conditions, the mechanisms underlying regulation of the release of DAMPs are not fully understood. Apoptosis and pyroptosis can be detected by several types of sensors such as Forster resonance energy transfer (FRET) biosensors ...[more]

PMID: 31225501

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Project description:Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila melanogaster embryos spanning three landmark embryonic stages: 2-4 h after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises around 6,000 multipotent cells; 6-8 h after egg laying (predominantly stage 10-11), to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying (predominantly stage 13), when each of the embryo's more than 20,000 cells are undergoing terminal differentiation. Our results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. We identify 30,075 distal regulatory elements that exhibit tissue-specific accessibility. We validated the germ-layer specificity of a subset of these predicted enhancers in transgenic embryos, achieving an accuracy of 90%. Overall, our results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types.

Dataset Information

Development of novel methods that monitor necroptosis and the release of DAMPs at the single cell resolution.

Publications

Development of novel methods that monitor necroptosis and the release of DAMPs at the single cell resolution.

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