The role of MEL-28 in nuclear pore complex formation [Elektronische Ressource] / Rudolf Walczak

Walczak

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences




























presented by

Diplom-Biochem.: Rudolf Walczak

born in: Hamburg, Germany

Oral examination: ...........................







The role of MEL-28 in
nuclear pore complex formation










PhD student:

Rudolf Walczak




Group leader:

Prof. Dr. Iain W. Mattaj











Referees:

Dr. Jan Ellenberg

Prof. Dr. Elmar Schiebel






INAUGURAL - DISSERTATION

zur
Erlangung der Doktorwürde
der
Naturwissenschaftlich-Mathematischen Gesamtfakultät
der
Ruprecht – Karls – Universität
Heidelberg


























vorgelegt von

Diplom-Biochem.: Rudolf Walczak

aus: Hamburg, Deutschland

Tag der mündlichen Prüfung: ...........................







Die Rolle von MEL-28 bei der
Bildung des Kernporenkomplexes










Doktorand:

Rudolf Walczak




Betreuer:

Prof. Dr. Iain W. Mattaj











Gutachter:

Dr. Jan Ellenberg

Prof. Dr. Elmar Schiebel



"The greatest shortcoming of the human race is our
inability to understand the exponential function."

(A. A. Bartlett) 6
Table of contents

Short summary............................................................................................................9

Zusammenfassung ...................................................................................................11

List of figures ............................................................................................................13

Abbreviations14

1 Introduction17

1.1 The nuclear envelope ...........................................................................................18
1.2 The nuclear pore complex ....................................................................................20
1.3 Nucleocytoplasmic transport.................................................................................24
1.4 Functions of the NE and NPCs other than transport.............................................27
1.5 The nuclear lamina ...............................................................................................28
1.6 Dynamics of the nuclear envelope during the cell cycle........................................29
1.6.1 Nuclear envelope breakdown ....................................................................29
1.6.2 Functions of NE and NPC components during mitosis ..............................31
1.6.3 Nuclear envelope and nuclear pore complex formation.............................31
1.7 MEL-28 and NET5 ................................................................................................37
1.7.1 MEL-28 ......................................................................................................37
1.7.2 NET5..........................................................................................................39
1.8 Aim of the thesis ...................................................................................................40

2 Results....................................................................................................................41

2.1 MEL-28 .................................................................................................................42
2.1.1 Generation of specific reagents against human MEL-28 ...........................42
2.1.2 Human MEL-28 is a nuclear envelope and kinetochore protein.................44
2.1.3 MEL-28 localizes to nuclear pores and the INM.........................................44
2.1.4 MEL-28 is required for NPC maintenance in human cells..........................49
2.1.5 Annulate lamellae can form independently of MEL-28...............................51
2.1.6 MEL-28 is required for postmitotic NPC assembly.....................................54
2.1.7 MEL-28 interacts with the Nup107-160 complex........................................57
2.1.8 Identification of proteins bound to MEL-28.................................................59
2.1.9 MEL-28 acts upstream of the Nup107-160 complex in NPC assembly......62
2.1.10 Mapping the NE-localization domain in MEL-28 ......................................64
2.1.11 Interplay with the Ran GTPase system....................................................67
2.1.12 Expression and purification of full length MEL-28 ....................................70
2.1.13 Recombinant MEL-28 rescues the MEL-28 depletion phenotype
in the first steps of NPC assembly...........................................................72
2.1.14 Investigating the function of the MEL-28 AT hook75
2.1.15 Inhibition of nuclear assembly with an excess of AT hook
recapitulates the MEL-28 depletion phenotype........................................78
2.1.16 The C-terminus of MEL-28 contains at least one
chromatin binding motif besides the AT hook..........................................82
2.1.17 MEL-28 binding to chromatin is regulated during the cell cycle ...............86 7
2.2 NET5.....................................................................................................................89
2.2.1 NET5 is a conserved transmembrane protein with a
well defined domain organization ..............................................................89
2.2.2 Human NET5 localizes to foci in the nuclear envelope..............................90
2.2.3 RNAi depletion of NET5 causes aberrant nuclear and cytoskeletal
morphology and folding of the nuclear envelope .......................................94
2.2.4 Mapping the sequence requirements for NET5 localization.......................97
2.2.5 Additional approaches and experiments ..................................................100

3 Discussion............................................................................................................102

3.1 MEL-28 ...............................................................................................................103
3.1.1 MEL-28 is a conserved NPC/INM protein in metazoa..............................103
3.1.2 MEL-28 is essential for NPC formation ....................................................104
3.1.3 MEL-28 interacts with the Nup107-160 complex......................................104
3.1.4 MEL-28 functions in postmitotic NPC formation
by recruiting nucleoporins to chromatin ...................................................105
3.1.5 Recombinant MEL-28 rescues Nup107-160 complex binding
to chromatin.............................................................................................107
3.1.6 The MEL-28 AT hook binds to chromatin and is important
for NPC formation....................................................................................107
3.1.7 Regulation of MEL-28 function by the GTPase Ran ................................110
3.1.8 The function of MEL-28 is regulated during the cell cycle........................110
3.1.9 Model for MEL-28 in postmitotic NPC formation ......................................111
3.2 NET5...................................................................................................................114
3.2.1 NET5 is a conserved transmembrane protein of the INM ........................114
3.2.2 Specific domains mediate correct NET5 localization ...............................114
3.2.3 Loss of NET5 perturbs nuclear integrity...................................................115
3.2.4 Future perspectives regarding NET5 .......................................................117

4 Materials and methods ........................................................................................118

4.1 Materials .............................................................................................................119
4.1.1 Chemicals and reagents ..........................................................................119
4.1.2 Commonly used buffers, solutions and media .........................................122
4.1.3 Materials ..................................................................................................123
4.1.4 Instruments ..............................................................................................124