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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.
- NSF-PAR ID:
- 10510660
- Publisher / Repository:
- Current Protocols
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 4
- Issue:
- 4
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein‐protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light‐activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light‐tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC.
This article was corrected on 25 July 2022. See the end of the full text for details.
Basic Protocol 1 : Genetic engineering strategy for the generation of modular light‐activated protein dimerization unitsSupport Protocol 1 : Molecular cloningBasic Protocol 2 : Cell culture and transfectionSupport Protocol 2 : Production of dark containers for optogenetic samplesBasic Protocol 3 : Confocal microscopy and light‐dependent activation of the dimerization systemAlternate Protocol 1 : Protein recruitment to intracellular compartmentsAlternate Protocol 2 : Induction of organelles’ membrane tetheringAlternate Protocol 3 : Optogenetic reconstitution of protein functionBasic Protocol 4 : Image analysisSupport Protocol 3 : Analysis of apparent on‐ and off‐kineticsSupport Protocol 4 : Analysis of changes in organelle overlap over time -
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Basic Protocol 1 : PD‐1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cellsBasic Protocol 2 : Plate‐based ligand binding assay to study PD‐1 function in human T cellsSupport Protocol 1 : T cell proliferation assay in the presence of PD‐1 ligationBasic Protocol 3 : In vitro APC/T cell co‐culture system to evaluate therapeutic interventions targeting the PD‐1/PD‐L1 axisSupport Protocol 2 : Microscopy‐based approach to evaluate the consequences of PD‐1 ligation on immune synapse formationBasic Protocol 4 : Tetramer‐based approach to study PD‐1/PD‐L1 interactions