Abstract
Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.
Highlights
Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens
Released NETs were visualized by stimulated emission depletion (STED) nanoscopy to reveal the architecture of hydrated NETs below the resolution limit of normal fluorescence microscopy (Supplementary Fig. 2)
We provide a comprehensive and unique picture of the complex biophysical aspects that govern the different phases of NETosis using an interdisciplinary approach and innovative imaging methods
Summary
We imaged chromatin and cell membranes of human neutrophils stimulated by 100 nM PMA (Fig. 1a, b, Supplementary Movies 1, 2). To address the question of how chromatin exits the nucleus, we labeled lamin B1, a constitutive component of the nuclear envelope that surrounded all individual lobuli of the nucleus (Fig. 2c, Supplementary Fig. 5). Over time, those lobuli merged and became less distinctive, indicating profound changes in the separation between chromatin. Several rupture events of this layer were discernable
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