Nanoscale Mapping Reveals Periodic Organization of Neutrophil Extracellular Trap Proteins

Neutrophils are essential cells of the innate immune system that release neutrophil extracellular traps (NETs) via NETosis. This specialized cell death pathway extrudes decondensed chromatin into the extracellular space. NET formation contributes to antimicrobial defense and coagulation, whereas dysregulated NETosis drives cancer, coagulopathy, and autoimmune diseases. Here we present a workflow combining optimized sample preparation, super-resolution imaging, and quantitative bioimage analysis to investigate the nanoscale organization of NETs. Our newly developed analysis tool, NanoNET, facilitates the NET protein binding pattern analysis. We identified specific NET proteins, including neutrophil elastase (NE) and proteinase 3 (PR3), that bind along DNA strands in a periodic fashion and are highly colocalized with nucleosomes, suggesting docking onto or around these structures. The workflow and analysis tools represent a significant methodological advance for studying protein distributions along NETs and filamentous structures in general. Understanding the NET filament organization is a critical step toward elucidating their formation and function.