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 results in the extrusion of decondensed chromatin into the extracellular space. NET formation participates in antimicrobial defense and coagulation but dysregulated NETosis can contribute to neoplasticity, coagulopathy, and autoimmune disease. 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, facilitated the automated identification of DNA filaments and the analyses of NET binding proteins along those filaments through auto- and cross-correlation. We identify specific NET proteins, including neutrophil elastase (NE) and proteinase 3 (PR3), that are bound along DNA strands in a periodic fashion that mirrored nucleosomes. Cross-correlation of NE and PR3 with nucleosomes revealed that these proteins were highly colocalized suggesting that they dock onto or around nucleosomes. The presented workflow and analysis tools represent a significant methodological advance for studying protein distributions along NETs and along filamentous structures in general. Understanding how NET filaments are organized is a critical step toward elucidating both their formation and function.