Gene Delivery and Analysis of Optogenetic Induction of Lytic Cell Death

Abstract

Necroptosis is a form of inflammatory lytic cell death involving active cytokine production and plasma membrane rupture. Progression of necroptosis is tightly regulated in time and space, and its signaling outcomes can shape the local inflammatory environment of cells and tissues. Pharmacological induction of necroptosis is well established, but the diffusive nature of chemical death inducers makes it challenging to study cell-cell communication precisely during necroptosis. Receptor-interacting protein kinase 3, or RIPK3, is a crucial signaling component of necroptosis, acting as a crucial signaling node for both canonical and non-canonical necroptosis. RIPK3 oligomerization is crucial to the formation of the necrosome, which regulates plasma membrane rupture and cytokine production. Commonly used necroptosis inducers can activate multiple downstream signaling pathways, confounding the signaling outcomes of RIPK3-mediated necroptosis. Opsin-free optogenetic techniques may provide an alternative strategy to address this issue. Optogenetics uses light-sensitive protein-protein interaction to modulate cell signaling. Compared to chemical-based approaches, optogenetic strategies allow for spatiotemporal modulation of signal transduction in live cells and animals. We developed an optogenetic system that allows for ligand-free optical control of RIPK3 oligomerization and necroptosis. This article describes the sample preparation, experimental setup, and optimization required to achieve robust optogenetic induction of RIPK3-mediated necroptosis in colorectal HT-29 cells. We expect that this optogenetic system could provide valuable insights into the dynamic nature of lytic cell death. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of lentivirus encoding the optogenetic RIPK3 system Support Protocol: Quantification of the titer of lentivirus Basic Protocol 2: Culturing, chemical transfection, and lentivirus transduction of HT-29 cells Basic Protocol 3: Optimization of optogenetic stimulation conditions Basic Protocol 4: Time-stamped live-cell imaging of HT-29 lytic cell death Basic Protocol 5: Quantification of HT-29 lytic cell death.

Keywords: RIPK3; cell signaling; lytic cell death; necroptosis; optogenetic.

Figure 1.. HEK293T cells show sickened morphology during lentivirus production.

A. Non-transfected HEK293T cells show stretched cell morphology. B. When transfected with the transfer plasmid, psPAX2, pMD2.G, cells show aggregation and shrinkage, a good sign for potent lentivirus production.

Figure 2.. Lentiviral transduction provides a more effective gene delivery rate than chemical transfection.

A. HEK293T cells were treated with a complete media supplemented with polybrene (8 μg/mL). Right: HEK293T cells were treated with a complete media supplemented with polybrene (8 μg/mL) and the optogenetic RIPK3 lentivirus after concentrating the original virus-containing media 10 times. C. HT-29 cells were transiently transfected with non-viral optogenetic RIPK3 plasmid, and the images were taken 24 h post-transfection. D. HT-29 cells were transduced with lentivirus supplemented with polybrene (8 μg/mL), and images were taken 96 h post-treatment. Scale bar 50 μm.

Figure 3.. Setting for spinning down tissue culture plate.

The tissue culture plate is wrapped before spinning to keep humidity and CO₂ in the wells.

Figure 4.. Hardware and software for acquiring optogenetic induction of RIPK3-mediated necroptosis.

A. Side view of the microscope equipped with an environment-control chamber that tunes the temperature and CO2 concentration for long-term live-cell imaging. A1. Sample holder and a plate with cultured cells. B. Rear view of the microscope highlighting the tubes and CO₂ regulator. B1. Fluorescence intensity manager (FIM) and the liquid guide for light delivery. C. Software interface for data acquisition.

Figure 5.. Time-stamped images of optogenetic induction of RIPK3-mediated lytic cell death.

A. HT-29 cells were transduced with lentivirus encoding the optogenetic RIPK3 system and cultured in an environment chamber. Blue light stimulation causes RIPK3-containing necrosomes and membrane rupture. SytoxGreen stain was used to stain lytic cells. A pulse of 95 ms blue light stimulation was applied every five minutes when cell morphology and staining were also monitored. Representative images of a cluster of cells undergoing necroptosis were shown. Cell death proceeds through a burst and flattening of cell membranes concurrently stained by SytoxGreen. B. Time-dependent cell death in dark and light conditions.

Figure 6.. Representative images of HT-29 cells 20 hours after blue light stimulation under each condition.

La-RIPK3-transfected cells (top) undergo blue-light-inducible lytic cell death, which is confirmed by SytoxGreen staining. mCherry-CRY2clust (no RIPK3, middle) and Wild-type (no transfection, bottom) cells are not stained, indicating intact membranes.