Background

Regulated cell death is a common feature of multicellular organisms and plays a key role in development and tissue homeostasis and in protecting the host against malignant growth and various pathogens. Research over the last two decades has identified over 12 different forms of regulated cell death (Galluzzi et al., 2018); however, their study is often complicated by the complex crosstalk and interconnectivity between the different cell death pathways (Bedoui et al., 2020). Additionally, the consequences of different types of cell death in the tissue still remain insufficiently understood, which is at least partially due to the challenges of selectively targeting single cells in multicellular populations, as well as to the pleiotropic effects of commonly used natural cell death triggers both on the dying cells and their neighbors.

In recent years, multiple strategies have been developed to specifically ablate cells both in vitro and in living animals. However, some of these methods (such as laser ablation or photosensitization) still lack specificity regarding the type of cell death to be induced (Tirlapur et al., 2001; Qi et al., 2012), while others [such as chemically inducible dimerization of apoptotic or necroptotic effector proteins (Oberst et al., 2010; Wu et al., 2014)] suffer from a limited spatiotemporal control and require a delivery of soluble ligands, thus limiting their in vivo applications. To overcome these limitations and expand the scope of the tools available for programmed cell death induction, we recently developed a set of optogenetically activated cell death effectors (optoCDEs) (Shkarina et al., 2022), which enable selective induction of three major types of programmed cell death: apoptosis, pyroptosis, and necroptosis. These tools consist of three modules: 1) a photoactuator domain Cry2olig (Cry2 E490G), which responds to blue light by rapid homo-oligomerization, 2) an mCherry tag, which enables the detection of the cells expressing optoCDEs and estimation of the relative construct expression levels, and 3) an effector module (Figure 1A–1C). For opto-caspases, the effector module corresponds to the protease domain (p20 and p10 subunits) of corresponding caspases; the endogenous linkers and cleavage sites essential for the caspase activation are retained, while CARD (in caspase-1, -4, -5, -9, and -11) and DED (caspase-8) domains, responsible for the endogenous upper-level protein–protein interactions and homo-oligomerization, are removed. In opto-RIPK3, the design is similar, while the RHIM motif (responsible for the upstream interaction with the RIPK1) is mutated. In optoMLKL, the effector domain orientation in relation to Cry2olig and mCherry is reversed to keep the MLKL N-terminus (responsible for membrane binding and disruption) exposed. The considerations behind the construct design and testing of the different construct versions are described in more detail in the original paper (Shkarina et al., 2022). The blue light stimulation triggers the rapid activation and oligomerization of Cry2olig, which in turn results in the proximity-induced activation of effector domains and subsequent processing of downstream substrates, culminating in cell death. While Cry2olig alone responds to the blue light within seconds (Taslimi et al., 2014), the timing of cell death induction is defined by the kinetics of effector activation as well as availability and efficiency of the processing and activation of downstream substrates (such as apoptotic executioner caspases, necroptotic effector GSDMD, or pyroptotic effector MLKL); the first morphological features of cell death can usually be detected within minutes after the beginning of illumination.

Figure 1. Schematic representation of the optogenetically activated cell death effectors (optoCDE) system. (A) OptoCDEs are inactive and monomeric in the dark state but are activated within seconds upon blue light illumination, which induces oligomerization of the Cry2olig photoactuator domain, activation of downstream substrates, and induction of cell death. (B) General architecture of major cell death effectors and design of optoCDE constructs. Cell death effectors used in the study generally consist of an adaptor domain (CARD or DED for caspases and RHIM for RIPK3) and an effector domain bearing protease (caspases), kinase (RIPK3), or membrane-disrupting (MLKL) function. In the optoCDEs, the effector domain is retained, while the adaptor is replaced with the Cry2olig-mCherry photoactuator module (mCherry is used to visualize optoCDE expression and/or clustering). (C) Schematic representation of major types of optoCDE constructs used in the study. The detailed description and evaluation of the optoCDE tools is available in the original paper (Shkarina et al., 2022). Figure adapted from Shkarina et al. (2022).

The optoCDEs can be applied in vitro, as described in this protocol, as well as in vivo to selectively kill specific cells or cell populations in a highly controlled and specific manner (Shkarina et al., 2022). Additionally, the precise control over the illumination parameters, such as light intensity and duration, provides new means for cellular and mechanistic studies of these forms of cell death, as well as probing cell survival mechanisms that limit cellular damage downstream of these effectors.