Inner nuclear membrane protein targeting
studied by quantitative live cell imaging and
RNAi screening
Referees:
Prof. Dr. Iain Mattaj
Prof. Dr. Oliver Gruss
3
http://d-nb.info/1075553911
Table of Contents
Summary
6
Zusammenfassung
7
Table of contents
1
Introduction
1.1
2
The Nuclear Envelope (NE): an overview
1.1.1
The scaffold of the metazoan nucleus: the nuclear lamina
1.1.2
The nuclear pore complex (NPC): structure and functions
13
15
1.2
Nuclear Membrane Proteins
1.2.1
Outer Nuclear Membrane Proteins (ONMPs)
1.2.2
Inner Nuclear Membrane Proteins (INMPs)
1.2.3
The role of INMPs in disease
18
18
20
23
1.3
Nuclear Envelope Dynamics in Mitosis and Interphase
1.3.1
NE Remodeling During Mitosis: Role of Nuclear Membrane Proteins
1.3.2
NE Remodeling during Interphase
25
25
1.4
Targeting of INM Proteins In Interphase
1.4.1
Insertion of INM Proteins in the membrane
1.4.2
The Diffusion-Retention Model
1.4.3
NPC permeability barrier for membrane proteins
1.4.4
Active/facilitated INM protein targeting
1.4.5
Role of NPC Components in INM Protein Targeting
1.4.6
Importance of subcellular trafficking in INM protein targeting
1.4.7
NPC-independent trafficking pathways?
29
30
30
32
33
35
36
37
1.5
Aim of the research
39
12
28
Results
2.1
2.2
2.3
2.4
Generation of a novel reporter to study INM protein trafficking
42
42
2.1.1
ER trapping of LBR by increasing its N-terminal cytoplasmic domain
2.1.2
Inducible cis-cleavage of the retention domain allows
44
INM localization of LBR
46
2.1.3
^Controlled induction of Target-INM targeting by inhibitor removal
50
2.1.4
Target-INM strategy can be applied to other INMPs
52
A quantitative assay to study LBR targeting after siRNA gene knock down
2.2.1
General screening pipeline and implementation
52
56
2.2.2
Identification of genes implicated in LBR targeting process
61
A predictive mathematical model for INM proteins targeting
61
2.3.1
Model description
62
2.3.2
Parameters estimation for Control siRNA
2.3.3
Determination of best model for a predictable phenotype
65
67
2.3.4
The model clusters genes hit into 3 distinct classes
72
Validation of model prediction
72
2.4.1
NPCs densities partially account for reduction of targeting
2.4.2
The LMNA KD phenotype is explained by increased retention in the NE 74
76
2.4.3
NUP93 KD cells also lose size selectivity for soluble dextrans
9
3
Material and Methods
3.1
3.2
3.3
3.4
3.5
3.6
4
Materials
Molecular Biology
Sample Preparation
Imaging
Image analysis
Mathematical modelling
84
85
91
95
96
101
Discussion
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
Target-INM: a new reporter for inner nuclear membrane
protein targeting
A quantitative assay to study kinetics of INM protein targeting
INM protein targeting is a slow process
The first siRNA screen for INM protein trafficking
INM protein targeting is dependent on the number of NPCs
INM protein targeting is limited by available nuclear retention sites
The Nup93-based complexes function as size controllers of the
NPC for membrane proteins
Size selectivity for soluble proteins and membrane both involve Nup93
but are likely controlled by distinct mechanisms
The ER and NE proteins Nesprin-1 and SIGMAR1 affect LBR targeting
The diffusion retention model is sufficient to explain LBR targeting
112
114
115
116
117
118
118
119
120
120
Abbreviations
122
Bibliography
123
Acknowledgments
135
10