title: Biophysical forces and intracellular restructuring during neutrophil extracellular trap formation
Creators: name: Holsapple, Jason
Publisher: name: University of Muenster, Institutional Repository MIAMI
Year: 2025
Subjects: subject: 570 Biology

description: Neutrophile extrazelluläre Fallen (NETs) sind Werkzeuge zur Infektionsbekämpfung, die aus dekondensiertem Chromatin, Histonen und antimikrobiellen Proteinen bestehen. Wenn sie dysreguliert sind, werden NETs mit Pathologien in Verbindung gebracht. Diese Regulierung ist nur unzureichend erforscht, so dass die Umstrukturierung der Zellzyklusmaschinerie während der NETose ein Ziel darstellt. Insbesondere wird Ki-67 wieder exprimiert. Durch Computermodellierung wurde Ki-67 als potenzielles Substrat für Peptidyl-Arginin-Deiminase 4 (PAD4) identifiziert. In primären menschlichen Neutrophilen wurden Ki-67 und PAD4 immunpräzipitiert und wiesen eine nanoskalige Nähe auf. Die NET-Bildung nahm in Depletionsmodellen zu, einschließlich Ki-67-Knockout-Mäusen, gehemmten menschlichen Neutrophilen und Knockdown von Zelllinien. Dies schloss eine erhöhte Citrullinierung von Histon H3 und Chromatin-Geschwindigkeit ein, was bedeutet, dass diese in Gegenwart von Ki-67 negativ reguliert werden.
description: Neutrophil extracellular traps (NETs) are tools to combat infection, composed of decondensed chromatin, histones, and antimicrobial proteins. When dysregulated, NETs are linked to pathologies. This regulation is poorly understood, and so the repurposing of cell cycle machinery during NETosis provides a target. Specifically, Ki-67 is re-expressed. Through computer modelling, Ki-67 was identified as a potential substrate for peptidyl arginine deiminase 4 (PAD4). In primary human neutrophils, Ki-67 and PAD4 immunoprecipitated and showed nanoscale proximity. NET formation increased in depletion models, including Ki-67 knockout mice, inhibited human neutrophils, and cell line knockdown. This included increased citrullination of histone H3 and chromatin velocity, thus that these are negatively regulated in the presence of Ki-67.
description: Declaration ..... viii 
Acknowledgements ..... ix 
Abbreviations ..... x
Summary ..... xii 
Zussamenfassung ..... xiii 
1 Introduction 
1.1 Neutrophil biology and the nuclear environment ..... 1 
1.2 Neutrophil extracellular trap (NET) formation ..... 3
1.3 Ki-67 and the repurposing of mitotic machinery during NETosis ..... 6
1.4 Regulation of NET formation ..... 14
1.5 PAD4 as a driver of NETosis ..... 16
1.6 Clinical relevance of dysregulated NETosis 20 1.7 Aims of this study ..... 22
2 Methods ..... 23
3 Results 
3.1 Ki-67 characterization during NET formation in primary neutrophils ..... 36
3.2 Theoretical suitability of Ki-67 as a novel nuclear PAD4 substrate ..... 50
3.3 Ki-67 citrullination, histone competition assay, and biophysical interactions with PAD4 through QCM-D ..... 56
3.4 Nanoscale interaction of Ki-67 and PAD4 in primary human neutrophils ..... 65
3.5 Ki-67 depletion in primary human neutrophils via translation inhibition ..... 68 
3.6 Genetic manipulation of Ki-67 through knockdown in the human HL-60 cell line and knockout in mouse models ..... 78 
3.7 Clinical relevance of Ki-67 during NET formation in disease models ..... 93
4 Discussion ..... 108
5 Conclusion ..... 115 
6 Appendix ..... 116
7 References ..... 122
8 Lebenslauf ..... 134