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The role of regulatory factors for primary endocytic vesicle formation during papillomavirus endocytosis

Das humane Papillomavirus Typ 16 ist die Hauptursache von Gebärmutterhalskrebs. Für die Aufnahme von HPV16 in Zellen wurde ein neuartiger Mechanismus der Endozytose identifiziert. Der neue Endozytoseweg ist unabhängig von Clathrin, Caveolin, Dynamin und Lipid Rafts. Die zelluläre Funktion ist bisher nicht bekannt, jedoch scheint er auch für die Aufnahme anderer Viren eine Rolle zu spielen. Die Arbeit strebt an die Regulation der einzelnen Schritte in der Entstehung des primären endozytischen Vesikels zu verstehen. Es zeigte sich, dass der Endozytoseweg ein allgemein verwendeter Mechanismus für die Aufnahme von Hochrisiko-Papillomaviren ist. Die Signale des EGF-Rezeptors initiierten die Entstehung der Vesikel. Proteine mit einer BAR-Domäne stabilisierten dann möglicherweise die Membrankrümmung. Die Reifung des Vesikels benötigte Signale von Serin-Threonin-Kinasen und Abl2. Der letzte Schritt der Vesikelabschnürung war angewiesen auf Aktinpolymerisierung, reguliert von Arp2/3 und WASH.

Titel: The role of regulatory factors for primary endocytic vesicle formation during papillomavirus endocytosis
Verfasser: Kühling, Lena GND
Gutachter: Schelhaas, Mario GND
Organisation: FB 13: Biologie
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 2015
Publikation in MIAMI: 23.10.2015
Datum der letzten Änderung: 23.10.2015
Schlagwörter: Papillomviren; HPV16; Endozytose; Aktin; Vesikelbildung; Signaltransduktion
Fachgebiete: Biowissenschaften; Biologie
Sprache: Englisch
Format: PDF-Dokument
video/mpeg
URN: urn:nbn:de:hbz:6-28209669742
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-28209669742
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Inhalt:
Table of content ....................................................................................................................... I
Table of figures ..................................................................................................................... IV
Summary ................................................................................................................................. 1
Zusammenfassung ................................................................................................................. 3
1 Introduction ..................................................................................................................... 5
1.1 Endocytosis ................................................................................................................. 5
1.1.1 Clathrin-mediated endocytosis as a model for an endocytic mechanism ........... 7
1.1.2 Clathrin-independent endocytic pathways .......................................................... 9
1.2 Virus entry .................................................................................................................. 14
1.2.1 The typical viral lifecycle ................................................................................... 15
1.2.2 Cell biology of virus entry .................................................................................. 16
1.2.3 Virus endocytosis .............................................................................................. 18
1.2.4 Papillomaviruses ............................................................................................... 20
1.2.5 Papillomavirus entry .......................................................................................... 23
1.3 Aim of this study ........................................................................................................ 27
2 Material and methods ................................................................................................... 29
2.1 Material ...................................................................................................................... 29
2.1.1 Cell lines ........................................................................................................... 29
2.1.2 Growth media .................................................................................................... 30
2.1.3 Specialized media ............................................................................................. 30
2.1.4 Cell culture reagents ......................................................................................... 31
2.1.5 Plasmids ........................................................................................................... 32
2.1.6 siRNA ................................................................................................................ 32
2.1.7 Virus production and labeling reagents ............................................................. 32
2.1.8 Virus stocks ....................................................................................................... 33
2.1.9 Inhibitors ........................................................................................................... 33
2.1.10 Antibodies ......................................................................................................... 34
2.1.11 Staining reagents .............................................................................................. 36
2.1.12 Buffers ............................................................................................................... 36
2.2 Methods ..................................................................................................................... 38
2.2.1 Cell culture ........................................................................................................ 38
2.2.2 Virus production and labeling ............................................................................ 40
2.2.3 Infection and internalization studies .................................................................. 42
2.2.4 Microscopy methods and quantification ............................................................ 45
2.2.5 Biochemical methods ........................................................................................ 47
3 Results ........................................................................................................................... 50
3.1 The novel endocytic pathway is used by other high risk papillomaviruses ................ 50
3.2 A targeted siRNA screen for cytoskeleton and regulatory proteins identified novel factors for HPV16 endocytosis ........................................................................................... 57
3.3 Regulatory factors in HPV16 endocytosis affect pit formation ................................... 62
3.4 Actin polymerization is required for scission .............................................................. 65
3.5 Internalization requires branched actin polymerization .............................................. 68
3.6 Different nucleation promoting factors are required in HPV16 endocytosis and macropinocytosis ................................................................................................................ 71
3.7 EGFR signaling is required throughout the protracted period of internalization ........ 76
3.8 Abl2 is required for endocytic vesicle maturation in HPV16 endocytosis .................. 81
4 Discussion ..................................................................................................................... 85
4.1 Model ......................................................................................................................... 85
4.2 Local EGFR activation is required to initiate pit formation ......................................... 86
4.3 BAR domain-containing proteins shape the membrane in HPV16 endocytosis ........ 90
4.4 Abl2 is a downstream target of EGFR required for pit maturation ............................. 93
4.5 Actin acts as a scission factor in HPV16 endocytosis ............................................... 95
4.6 Actin polymerization in HPV16 endocytosis requires WASH ..................................... 97
4.7 The novel endocytic pathway may be a common mechanism of uptake for high-risk papillomaviruses ................................................................................................................. 99
4.8 Concluding remarks ................................................................................................. 102
5 References .................................................................................................................. 103
6 Appendix ..................................................................................................................... 135
6.1 Infection data and cell numbers for siRNA screen ................................................... 135
6.2 Actin polymerization is required for scission ............................................................ 139
6.3 Establishment of the pHrodo internalization assay .................................................. 139
6.4 siRNA sequences and concentrations ..................................................................... 140
7 Abbreviations .............................................................................................................. 148
9 Publications and presentations ................................................................................ 153
10 Acknowledgments ...................................................................................................... 154