Development of DNA-based Fluorescent Molecular Tension Probes to Investigate Integrin Mediated Mechanical Forces Open Access

Zhang, Yun (2016)

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Mechanical stimuli profoundly alter cell functions and cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging. The aim of this dissertation is to describe the design and application of DNA hairpin force probes to study beta-3 integrin receptor involved mechanotransduction mechanisms in living cells. Chapter 1 introduces the cell adhesion molecules participating in mechanotransduction processes, mainly focusing on the integrin subfamily of adhesion receptors. This chapter also analyzes and compares the available tools and methods for directly measuring the forces transduced by integrins. Chapter 2 gives a detailed description of the development of a new class of molecular tension probes that employs a DNA hairpin as a ‘switch' element. This probe unfolds at a specific threshold force and reports tension in a digital rather than analogue manner. Application of the DNA-based probes in early focal adhesions reveals that alpha-v-beta-3 integrins prefer stiffer ligands at the leading tip of the focal adhesions. Chapter 3 explores the role of alpha-IIb-beta-3 integrin mechanics in platelet activation, which is critical in the process of clot formation. We found that lateral ligand mobility influenced platelet activation. Using the DNA-based probe, the first molecular tension map of alpha-IIb-beta-3 integrin was generated, revealing platelet mechanics with high spatial and temporal resolution. This work led to a proposed model for the role of mechanics in platelet activation and aggregation. Chapter 4 details the development of a DNA-origami tension sensor, aiming at increase the force threshold of current single hairpin molecular probes via multiple parallel sensors at the end of a DNA six-helix bundle. Force calibration and simulations revealed unexpected mechanical response of this origami structure, providing novel insights into DNA nanostructure mechanics. Biological application of this design was demonstrated through the measurement of platelet forces. Chapter 5 summarizes the thesis and discusses future directions for DNA based molecular tension probes. These tools are anticipated to impact the field of mechanotransduction by enabling force measurements with high spatial and temporal resolution.

Table of Contents

Chapter 1 Mechanotransduction by cell adhesion molecules and the methods to study cellular forces 1

1.1 Mechanotransduction by cell adhesion molecules 2

1.1.1 Examples of CAMs for cell-cell and cell-ECM interaction 2

1.1.2 Integrin CAMs 7

1.2 Methods for measuring cellular forces 12

1.2.1 Single cell force microcopy (SCFM) 13

1.2.2 Traction force microscopy based on the substrate deformation 14

1.2.3 FRET based molecular tension probes 16

1.3 Aims of the dissertation 25

1.4 References 26

Chapter 2 Design and synthesis of DNA-based molecular tension probes to study integrin forces during early cell adhesion. 42

2.1 Introduction 43

2.2 Results 46

2.2.1 Design and synthesis of DNA-based tension probes 46

2.2.2 Visualizing integrin tension 48

2.2.3 Selective mechanical response toward linear and cyclic RGD. 52

2.2.4 F1/2-encoded probes to analyze force distribution within FAs 53

2.3 Discussion 56

2.4 Materials and methods 57

2.4.1 Materials 57

2.4.2 DNA-based tension probes 58

2.4.3 F1/2 calculation for DNA hairpin 60

2.4.4 Force probes calibration by biomembrane force probe (BFP) 61

2.4.5 Quenching efficiency (QE) measurement for different reporter pairs 62

2.4.6 Calibration curve and determination of F factor and sensor density 63

2.4.7 Measurement of QE (1- IDA/ID) on glass substrate 63

2.4.8 Conversion of IDA/ID to calculate the fraction of unfolded hairpins 64

2.4.9 Imaging parameters 64

2.4.10 Surface preparation 65

2.4.11 Supported lipid membrane preparation 65

2.4.12 HPLC 66

2.4.13 MALDI-TOF mass spectroscopy 66

2.4.14 Fluorescence microscopy 67

2.4.15 Hairpin hybridization 67

2.4.16 Cell culture 68

2.5 References 68

Appendix 73

Chapter 3 Integrins harness mechanics to trigger platelet activation and aggregation 100

3.1 Introduction 101

3.2 Results 103

3.2.1 Lateral ligand fluidity alters platelet adhesion and spreading 103

3.2.2 pN forces are employed for fibrinogen binding and clot retraction 106

3.2.3 The spatial distribution of platelet tension is regulated by myosin II phosphorylation pathways 110

3.2.4 Correlation of phosphatidylserine exposure with platelet tension 112

3.3 Discussion 113

3.4 Materials and methods 117

3.4.1 Materials 117

3.4.2 Platelet isolation 118

3.4.3 Peptide synthesis 119

3.4.4 Tension signal radial intensity profiling and fitting 120

3.5 References 121

Appendix 126

Chapter 4 DNA origami-based tension probes 133

4.1 Introduction 134

4.2 Results 135

4.2.1 Fabrication of DNA origami based force sensors. 135

4.2.2 Calibration of DNA origami tension sensors with BFP 138

4.2.3 Computational modeling of tension thresholds 139

4.2.4 Application of origami tension probes in living cell system. 143

4.3 Discussion 145

4.4 Materials and methods 146

4.4.1 Materials 146

4.4.2 Preparation, purification and characterization of six-helix origami. 148

4.4.3 Preparation of origami sensor for BFP calibration. 149

4.4.4 Origami sensor calibration by BFP. 149

4.4.5 Platelet isolation 151

4.4.6 Surface Preparation. 151

4.4.7 AFM imaging. 152

4.5 References 153

Chapter 5 Summary and future directions 157

5.1 Summary 158

5.2 Future directions 160

5.2.1 Application of current DNA sensors 160

5.2.2 Advancing current DNA sensors 160

5.2.3 From 2D to 3D 161

5.3 Other contributions and curriculum vitae 162

5.4 References 164

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