| dc.description.abstracteng | In 2004, a new cell death program called neutrophil extracellular trap (NET) formation (also termed NETosis) was described, in which neutrophil granulocytes, stimulated by pathogens, formed web-like structures from their DNA and antimicrobial proteins, and then perished. Known as NETs, they can catch and immobilize pathogens like bacteria, viruses and fungi. The occurrence of NETs has also been observed in autoimmune diseases, malignant diseases and arteriosclerotic and thrombotic events such as myocardial infarction. As such, NETosis became increasingly important in the better understanding of the pathogenesis of these illnesses. Since it wasn’t clear, however, what mechanical and physical influences were leading to the release of NETs, one hypothesis was that the neutrophil cytoskeleton was playing a significant role. Thus, in this project, cytoskeletal components, including focal adhesions, tubulin and actin, were investigated by direct and indirect immunofluorescence and inhibition experiments. Using fluorescence microscopy, analysis of the data provided descriptive and quantitative results. Investigation of ezrin belonging to focal adhesions, showed the arrangement of this protein as a cap-like structure around the DNA during NETosis. Furthermore, it indicated structural parallels to F-actin. Examination of β-tubulin also presented a formation of a cap-like structure around the DNA, which was reproduced during life-cell staining experiments. While investigating α-tubulin, the cap-formation was not observed. Instead, α-tubulin seemed to mix with chromatin during NETosis, disassembling into small particles by the end of the process. However, α- and β-tubulin both showed spindle-like structures which lit up after around 90 minutes of NETosis. Inhibiting tubulin depolymerization with docetaxel led to no significant alteration of NETosis. Investigation of F-actin with phalloidin-staining showed an intense spreading of actin within the first 30 minutes. After that time, actin disassembled and was barely visible by the end of NETosis. Inhibiting actin polymerization with cytochalasin D showed a significant influence on the dynamics of NETosis, especially at the beginning of the process.
In conclusion, a key role during NETosis can be assigned to parts of the neutrophil cytoskeleton. Particularly F-actin appears to be crucial for maintaining cellular integrity and stability during the initial phase of NET formation. | de |