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Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation
Benoit Arnould1, Adeline Martz1, Pauline Belzanne2
1Laboratoire de Chémo-Biologie Synthétique et Thérapeutique (CBST) UMR 7199, équipe Ingénierie Canaux Ioniques, Centre National de la Recherche Scientifique, Université de Strasbourg, Faculté de Pharmacie, Illkirch, France.
Abstract:
ATP-gated purinergic P2X7 receptors are crucial ion channels involved in inflammation. They sense abnormal ATP release during stress or injury and are considered promising clinical targets for therapeutic intervention. However, despite their predominant expression in immune cells such as microglia, there is limited information on P2X7 membrane expression and regulation during inflammation at the single-molecule level, necessitating new labeling approaches to visualize P2X7 in native cells. Here, we present X7-uP, an unbiased, affinity-guided P2X7 chemical labeling reagent that selectively and covalently biotinylates endogenous P2X7 in BV2 cells, a murine microglial cell line, allowing subsequent labeling with streptavidin-Alexa 647 tailored for super-resolution imaging. We uncovered a nanoscale microglial P2X7 redistribution mechanism where evenly spaced individual receptors in quiescent cells undergo upregulation and clustering in response to the pro-inflammatory agent lipopolysaccharide and ATP, leading to synergistic interleukin-1β release. Our method thus offers a new approach to revealing endogenous P2X7 expression at the single-molecule level.
Insights
New chemical probe X7-uP visualizes P2X7 receptors in microglia. This reveals how P2X7 channels cluster during inflammation, impacting interleukin-1β release and offering therapeutic insights.
Area of Science:
- Immunology
- Cell Biology
- Neuroscience
Background:
- ATP-gated P2X7 receptors are key in inflammation and potential drug targets.
- Microglial P2X7 expression and regulation at the single-molecule level during inflammation are poorly understood.
- Novel labeling methods are needed to visualize P2X7 in native immune cells.
Purpose of the Study:
- To develop a chemical probe for visualizing endogenous P2X7 receptors in microglia.
- To investigate the nanoscale distribution and regulation of P2X7 during inflammation.
- To understand the role of P2X7 clustering in interleukin-1β release.
Main Methods:
- Development of X7-uP, an affinity-guided chemical labeling reagent for P2X7.
- Selective biotinylation of endogenous P2X7 in BV2 microglial cells.
- Super-resolution imaging using streptavidin-Alexa 647 after X7-uP labeling.
Main Results:
- X7-uP selectively labels endogenous P2X7 in microglial cells.
- Pro-inflammatory stimuli (lipopolysaccharide and ATP) induce P2X7 upregulation and nanoscale clustering.
- P2X7 redistribution correlates with enhanced interleukin-1β release.
Conclusions:
- X7-uP provides a novel method for single-molecule visualization of P2X7 in native cells.
- Inflammation triggers P2X7 redistribution and clustering in microglia, impacting inflammatory signaling.
- Understanding P2X7 dynamics offers new avenues for anti-inflammatory therapies.
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