Optogenetic activators of apoptosis, necroptosis, and pyroptosis

Abstract

Targeted and specific induction of cell death in an individual or groups of cells hold the potential for new insights into the response of tissues or organisms to different forms of death. Here, we report the development of optogenetically controlled cell death effectors (optoCDEs), a novel class of optogenetic tools that enables light-mediated induction of three types of programmed cell death (PCD)—apoptosis, pyroptosis, and necroptosis—using Arabidopsis thaliana photosensitive protein Cryptochrome-2. OptoCDEs enable a rapid and highly specific induction of PCD in human, mouse, and zebrafish cells and are suitable for a wide range of applications, such as sub-lethal cell death induction or precise elimination of single cells or cell populations in vitro and in vivo. As the proof-of-concept, we utilize optoCDEs to assess the differences in neighboring cell responses to apoptotic or necrotic PCD, revealing a new role for shingosine-1-phosphate signaling in regulating the efferocytosis of the apoptotic cell by epithelia.

The Opto-inflammasome in zebrafish: a tool to study cell and tissue responses to speck formation and cell death

“…We previously showed that periderm cells dying by opto-zfCaspase-8-induced apoptosis were also basally extruded from the periderm(Shkarina et al ., 2022). Thus, we analysed the behaviour of their actin cytoskeleton in larvae derived from a cross of Tg(Opto-caspase-8) with Tg(Krt4:Gal4_ UAS:mNG-UtrCH-UAS:Lyn-tagRFP) and found differences in the apical actin ridges behavior.…”

“…Apoptotic stimuli like geneticin have been shown to induce extrusion via sphingosine-1 phosphate (S1P) and its receptor, which is expressed in the surrounding cells. We have previously shown that S1P signaling does not seem to drive extrusion of pyroptotic cells in Caco-2 cell co-cultures (Shkarina et al, 2022), but so far it is unclear if S1P signaling or another mechanism activated downstream of the inflammasome is responsible for apical extrusion. Although the factors determining the direction of extrusion remain to be discovered, the different ratios of apical and basal extrusion of periderm cells in different zebrafish strains suggest a genetic component.…”

“…To induce specks and follow endogenous ASC we used Tg(HSE:asc-mKate2) and the endogenously tagged asc:asc-GFP line (Kuri et al ., 2017). To induce apoptotic cell death, we used Tg(HSE:mCherry-Cry2olig-caspase-8) (Shkarina et al ., 2022). For monitoring Ca 2+ signaling we used Tg(ß-actin:GCamp6) (Chen, Xia, Michael R Bruchas, et al ., 2017).…”

“…The Cry2olig-mCherry construct (plasmid #60032) was obtained from Addgene, and plasmids containing Asc, Asc(4xmut), Pyd, Nlr and Card published in (Kuri et al ., 2017) were used as cloning templates. The initial HSE:mCherry-Cy2olig ASC construct was cloned as described in (Shkarina et al ., 2022). Other constructs containing ASC variants (PYD and CARD) used the HSE:mCherry-Cy2olig-Asc as background and were modified by seamless cloning using the NEB Assembly Kit, after SmaI/EcoRV or XmaI/EcoRV digestion of the plasmid (replacing Asc).…”

“…We have previously developed optogenetic variants for human and zebrafish caspases which can be activated by light-induced oligomerization of Cry2olig, a photosensitive protein that undergoes rapid homo-oligomerization in response to blue light (Taslimi et al ., 2014; Shkarina et al ., 2022). This enabled us to selectively induce multiple forms of programmed cell death (PCD) in different cell types in vitro, organoids and in the live zebrafish larvae.…”