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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
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This content will become publicly available on July 16, 2026
Investigation of CD36 interactome provides insights into multimolecular complexes necessary for anti-angiogenic signalling
Abstract Signal transduction is a fundamental process that enables cells to adapt to external cues and organize adequate responses including survival, death, growth, and homeostasis. A key mechanism modulating signal transduction relies on the formation of multimolecular complexes optimized for specificity, modularity and signal amplification. The scavenger receptor CD36, which binds diverse ligands in different cellular contexts, illustrates this principle. To uncover the nature of CD36 multimolecular complexes, we employed a proximity biotinylation labeling approach on human endothelial cells, where CD36 binds to thrombospondin-1 (TSP-1) to initiate a signaling cascade promoting programmed cell death. Using biotin capture and mass spectrometry protein identification, we uncovered a list of proteins in the vicinity of CD36. This list of candidates was refined by proximity ligation assays. The relationship between key CD36 interacting molecules, in particular active integrin beta-1 (ITGB1) and CD9, was further decoded by conditional colocalization analysis, providing support for their association within a tri-molecular complex. The implication of selected candidates in the signaling function of CD36 was further evaluated using shRNA knockdown, revealing that active ITGB1 is essential for Fyn activation downstream of CD36, with the tetraspanin playing a connecting role between CD36 and active ITGB1. Our approach to investigating CD36 complexes emphasizes the complexity and fundamental role of protein-protein interactions and coordination in the context of transmembrane signal transduction.
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- Award ID(s):
- 2114417
- PAR ID:
- 10635256
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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