Protein-based Tension Probes: From Mapping Integrin Adhesion Forces to the Mechanopharmacology of Smooth Muscle Cells Open Access

Galior, Kornelia Delfina (2017)

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Cells constantly sense and translate mechanical forces into biochemical signals as part of their normal physiology, thus regulating processes that range from cell adhesion to differentiation and migration. Misregulation in the ability to sense and respond to mechanical cues contributes to a number of diseases including cancer cell invasion and atherosclerosis. While much is known about the identity and spatial localization of proteins within the cell, there is limited knowledge about the mechanical state of proteins. This dissertation presents the development and application of protein-based sensors to map and better understand the forces that regulate mechanotransduction pathways at the single cell level.

Chapter 1 describes the biological motivation for studying mechanical forces in living cells. The chapter also compares different methods that were developed to understand the coupling between mechanical forces and biochemical signaling.

Chapter 2 describes the development of protein-based tension sensors for mapping forces exerted by the cell receptors. These sensors were engineered to measure the high magnitude traction forces applied by integrin receptors. The probes were designed with a I27 titin domain as a spring and this was flanked by a fluorophore and a quencher and immobilized onto a gold nanoparticle substrate. We observed unfolding of titin. In addition, by introducing a molecular disulfide "clamp" within the core of the titin-based sensor and monitoring the kinetics of disulfide reduction, we obtained the first quantitative measurement of integrin forces within mature adhesions.

Chapter 3 describes an application of the protein-based tension sensors for investigating the molecular mechanics associated with asthma. We discovered that asthmatic airway smooth muscle cells have a higher contractile response than normal smooth muscle cells and that this response is further enhanced in the presence of inflammatory inducers. Using the signal generated by the titin tension probe, we demonstrate the first example of conducting a mechanopharmacology measurement that uses pN scale forces as an output. The effective dose for albuterol, a bronchodilator, was quantified for human samples from patients with a history of asthma. The EC50 varied as a function of nicotine and NGF treatment in a manner that was patient-dependent. Thus, our approach represents an important step toward personalized mechanomedicine.

Chapter 4 explores the role of integrin-mediated tension in two cellular adhesion structures: focal adhesions and invadopodia using titin-based molecular tension probes. We quantified the integrin tension from cell lung cancer tumor spheroids. Interestingly, cells tagged as being leaders in collective invasion were found to have higher levels of integrin tension. By mapping the integrin forces in invadosomes, we found that the integrin tension is localized around the invadosome rings.

Chapter 5 concludes the thesis with a summary and an outlook for the future design and application of molecular based tension sensors.

Table of Contents

Table of Contents

Chapter 1: Current methods for measuring cell-generated contractile forces1

1.1 Introduction 2

1.1.1 The importance of mechanics in biology 2

1.1.2 Integrin-mediated mechanical sensing 3

1.2 Current techniques to probe cell mechanics 6

1.2.1 Polymer network deformation (TFM and mPADs) 6

1.2.2 Single molecular force spectroscopy (SMFS) 7

1.2.3 Molecular tension fluorescence microscopy (MTFM) 9

1.3 Aim and scope of the dissertation 13

1.5 References 15

Chapter 2: Using protein engineering to map high-magnitude integrin forces21

2.1 Introduction 22

2.2 Results and discussion 25

2.2.1 Engineering titin as a tension probe to map integrin forces 25

2.2.2 Integrin forces irreversibly unfold sfGFP 28

2.2.3 Integrin forces unfold I27 through RGD in fibronectin 30

2.2.4 Covalently locked I27 resists complete mechanical unfolding by integrin forces 33

2.2.5 Kinetic measurements of S-S reduction to determine integrin forces 36

2.2.6 The rate of I27 unfolding is integrin subtype-dependent 39

2.3 Conclusion 40

2.4 Materials and methods 44

2.4.1 Reagents 44

2.4.2 Cell culture 44

2.4.3 Transfection 45

2.4.4 Antibody blocking 45

2.4.5 Protein engineering 45

2.4.6 Protein expression with UAA incorporation 46

2.4.7 Dye labeling 46

2.4.8 AuNP surface preparation 46

2.4.9 AFM imaging 47

2.4.10 Optical microscopy 47

2.4.11 Data analysis 47

2.4.12 Heat map generation 48

2.4.13 Ensemble fluorescence measurements for calculating quenching efficiency 48

2.5 References 50

2.6 Appendix 56

2.7 Supplementary Note 1 65

Chapter 3: Molecular tension probes as a tool to study the mechanopharmocology of airway smooth muscle cells67

3.1 Introduction 68

3.2 Results and discussion 71

3.2.1 Myosin dependent contractile forces in human airway smooth muscle cells are transmitted across integrin receptors 71

3.2.2 Human ASM cells isolated from asthmatic patients have an enhanced integrin tension phenotype 74

3.2.3 Nicotine promotes enhancement of integrin forces and stimulates the release of matrix metalloproteinases 77

3.2.4 Role of nicotinic acetylcholine receptor (nAChR) in the transmission of contractile forces 79

3.2.5 Albuterol dose-dependently inhibits contractile forces 82

3.3 Conclusion 85

3.4 Materials and methods 86

3.4.1 Materials 86

3.4.2 Cell culture and transfection 87

3.4.3 Antibody blocking 87

3.4.4 Protein engineering and dye labeling 87

3.4.5 Fabrication of protein-AuNP surfaces 88

3.4.6 Fluorescence immunostaining protocol 89

3.4.7 Drug treatment 89

3.4.8 Kinetic experiment for force measurement 89

3.4.9 Live cell fluorescence microscopy imaging 90

3.5 References 91

3.6 Appendix 100

Chapter 4: Biological applications of MTFM sensors in cancer biology109

4.1 Introduction 110

4.2 Results and discussion 111

4.2.1 Titin-based MTFM sensor reveals that leader and follower cell lines isolated from a 3D tumor spheroids have different mechanical phenotypes 111

4.2.2 Leader cell invasion is mediated through focal adhesion signaling pathway 114

4.2.3 Myosin-mediated integrin tension regulates invadosome ring dynamics 116

4.2.4 Invadosome movement in the cytoplasm is highly dynamic and is regulated by the integrins and focal adhesion signaling pathway 120

4.3 Conclusion 121

4.4 Methods and materials 123

4.4.1 Reagents 123

4.4.2 Cell culture 123

4.4.3 Antibody blocking 123

4.4.4 Protein expression and dye labeling 124

4.4.5 Fabrication of protein-AuNP surfaces 124

4.4.6 Live cell fluorescence microscopy imaging 125

4.5 References 126

4.6 Appendix 129

Chapter 5: Conclusions and perspectives131

5.1 Summary 132

5.2 Future directions 134

5.3 Other contributions 138

5.4 References 139

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