albicans ( G) colored boxes around images correspond to color coded cellular events in the graphs. Double-arrow-headed lines indicate presence of ionomycin ( A, C, and E) or phagocytosed C. Time (min) relative to plasma membrane microvesicle (MV) shedding noted. albicans, MOI of 2, G– J, dTomato shown in blue), and imaged by DIC and confocal microscopy at the coverslip–cell interface (Z = 0 μm) and in the cell center (Z = +3 μm) at 1- to 2-min intervals for 4 h. Mouse (Ms A, B, I, and J) or human (Hm E– J) PMNs or dHL-60 cells ( C, D, I, and J) stained with far-red SiR-DNA were stimulated with ionomycin (4 μM, A– F, I, and J) or dTomato-expressing C. NETosis proceeds by vesiculation, DNA decondensation, DNA release from the nucleus into the cytoplasm, and extracellular DNA expulsion in mouse and human blood neutrophils and dHL-60 cells. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.Ĭopyright © 2020 the Author(s). Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Inhibition of actin disassembly blocked NET release. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. ![]() We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. ![]() Eukaryotes are fundamentally diff… Nuclear Energy, Nuclear power is any method of doing work that makes use of nuclear fission or nuclear fusion reactions.Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation.
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