VU Research Portal
Deconstruction of septin assembly
Szuba, A.
2019
document version
Publisher's PDF, also known as Version of record
Link to publication in VU Research Portal
citation for published version (APA)
Szuba, A. (2019). Deconstruction of septin assembly.
General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain
• You may freely distribute the URL identifying the publication in the public portal ?
Take down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
E-mail address:
vuresearchportal.ub@vu.nl
Contents
1 Introduction 1
1.1 Cell shape control 1
1.1.1 Plasma membrane 1
1.1.2 Cytoskeleton 2
1.2 Septins 6
1.2.1 The septin family of proteins 6
1.2.2 Hierarchical assembly of septins into higher-order structures 7
1.2.3 Septin functions 10
1.3 Septins at the cell cortex 11
1.3.1 Septins and cell rigidity 11
1.3.2 Septin interactions with the plasma membrane 11
1.3.3 Septin-actin interaction 12
1.4 Goal and approach of this thesis 13
1.5 Thesis outline 13
2 Quantifying septin self-assembly by electron microscopy-based mass
mapping 15
2.1 Introduction 16
2.2 Materials and methods 18
2.2.1 Septin complex purification 18
2.2.2 Protein characterization 20
2.2.3 TIRF imaging of septins 22
2.2.4 Mass mapping by STEM 24
2.2.5 Atomic force microscopy (AFM) 25
2.3 Results 26
2.3.1 TIRF microscopy investigation of septin polymerization 26
2.3.2 Mass mapping of septin filaments and bundles 29
2.4 Discussion 35
2.5 Supplemental material 37
Contents
3 Membrane-templated assembly of septins 41
3.1 Introduction 42
3.2 Materials and Methods 44
3.2.1 Septin preparation 44
3.2.2 Fluorescence microscopy of septins on supported lipid bilayers 44 3.2.3 Transmission electron microscopy of septins on lipid
mono-layers 46
3.2.4 Cryo-electron microscopy of septins on lipid vesicles 46
3.2.5 Atomic force microscopy of septins on supported lipid bilayers 47
3.3 Results 49
3.3.1 Fluorescence imaging of septins on supported lipid bilayers 49
3.3.2 Electron microscopy imaging of septins on lipid monolayers 54
3.3.3 Electron microscopy imaging of septins on lipid vesicles 57
3.3.4 Atomic force microscopy imaging of septin-membrane
inter-actions 59
3.4 Discussion 68
3.4.1 Septins form rigid membrane-anchored networks 68
3.4.2 Towards a model of septin assembly on membranes 69
3.4.3 Biological implications 73
3.5 Supplemental material 73
4 Binding and self-assembly of septins on model biomembranes 77
4.1 Introduction 77
4.2 Methods 80
4.2.1 Vesicle preparation 80
4.2.2 Septin preparation 81
4.2.3 QCM-D 81
4.2.4 Combined QCM-D/SE measurements 82
4.2.5 AFM force indentation 84
4.3 Results 85
4.3.1 Septins bind charged lipid membranes through electrostatic
interactions 85
4.3.2 Mass transport limits the rate of septin adsorption 88
4.3.3 Dependence of septin adsorption on septin concentration 89
4.3.4 Impact of septin polymerization on membrane binding 91
4.3.5 Septin organization revealed through analysis of
hydrody-namically trapped water content 94
4.3.6 Mechanical properties of the septin films 95
4.3.7 Thickness of the membrane-bound septin films 98
4.4 Discussion 102
4.4.1 Comparison of QCM-D and SE data with AFM measurements102
4.4.2 Towards a model for membrane-templated septin assembly 103
4.5 Supplemental material 107
Contents
4.5.1 Figures 107
4.5.2 Tables 115
5 Summary and Outlook 119
5.1 Summary 119
5.2 Outlook 121
Acknowledgements 127
Bibliography 131
