Development of Fluorescence-based Molecular Tension Probes to Investigate Cellular Mechanical Forces Open Access
Jurchenko, Carol (2015)
Abstract
Mechanical forces are important in cellular development, normal morphogenesis, and wound healing. The mechanisms by which cells utilize tension to regulate biochemical events, however, are not well understood. In part, this is due to the limited availability of tools to study molecular mechanotransduction in live cells. The aim of this thesis is to describe the development and application of fluorescence-based sensors for mapping forces exerted by cell-surface proteins in living cells. Chapter 1 describes the historical context and biological motivation for developing molecular tension fluorescence microscopy (MTFM) probes. This chapter includes an analysis of the available methods for measuring cellular forces and case studies of model mechanotransduction pathways. Chapter 2 details the development of MTFM probes and their application in studying the forces associated with the initial stages of endocytosis of the ligand-activated epidermal growth factor receptor (EGFR). This work revealed that clathrin-mediated endocytosis of the EGFR is associated with pN scale forces and represents the first demonstration of a molecular probe to study forces applied by cell-surface receptors. Chapter 3 explores integrin receptor forces, which are important in cell adhesion. An MTFM probe consisting of a cyclic RGD peptide conjugated to a polyethylene glycol polymer was surface immobilized through streptavidin-biotin linkage. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ~106-108 times larger than integrin-RGD affinity, this work led to the discovery that integrin receptors in focal adhesions mechanically dissociate streptavidin-biotin tethered ligands. These results suggest that integrin-ligand complexes undergo a marked enhancement in stability when assembled within focal adhesions. In chapter 4, MTFM is used to examine the role of force in the activation of the Notch receptor, which plays a critical role in cell development. While the activation mechanism for Notch remains unclear, one widely accepted hypothesis involves force-mediated unfolding of the receptor that leads to cleavage by a metalloprotease, resulting in receptor activation. To validate this model, we engineered Notch MTFM probes and mapped Notch-ligand forces in live cells. Chapter 5 concludes the thesis by summarizing the work and discussing future directions for MTFM probes.
Table of Contents
Chapter 1. Mechanotransduction and Methods to Measure Cellular Tension........... 1
1.1 Role of molecular mechanotransduction in cell biology and biochemistry..... 2
1.1.1 A brief history of measuring molecular tension in live cells........... 2
1.1.2 Focal adhesions as a model mechanotransduction system........... 4
1.2 Emerging methods for measuring molecular tension..... 10
1.2.1 Genetically encoded tension sensors........... 14
1.2.2 Immobilized tension sensors........... 19
1.3 Outlook..... 24
1.4 References..... 29
Chapter 2. Visualizing mechanical tension across membrane receptors with a fluorescent sensor........... 46
2.1 Development of a fluorescence-based molecular tension sensor..... 47
2.1.1 Motivation for developing a molecular mechanosensor........... 47
2.1.2 Design of the sensor........... 48
2.2 Application of the sensor..... 48
2.3 Characterization of the sensor..... 50
2.4 Correlation of EGFR endocytosis with sensor signal..... 54
2.5 Materials and methods..... 56
2.5.1 Synthesis and characterization of streptavidin-quencher conjugates........... 56
2.5.2 Synthesis and characterization of EGF-PEG conjugates........... 57
2.5.3 Cell culture........... 58
2.5.4 Functionalization of glass substrate biosensors........... 59
2.5.5 Functionalization of supported lipid bilayers........... 60
2.5.6 Characterization of the zero-force sensor confirmation........... 61
2.5.7 Fluorescence microscopy........... 62
2.5.8 Image analysis........... 62
2.5.9 Quantitative force maps........... 63
2.5.10 Determination of EGFR phosphorylation and activation........... 65
2.5.11 Actin inhibition........... 65
2.5.12 CLC-eGFP transfection........... 65
2.6 References..... 66
Appendix 2..... 69
Chapter 3. Integrin-generated forces lead to streptavidin-biotin unbinding in cellular adhesions........... 80
3.1 Introduction..... 81
3.1.1 Mechanotransduction in the integrin pathway........... 81
3.1.2 Methods for studying adhesion forces........... 81
3.2 Design and synthesis of MTFM sensor..... 84
3.3 Surface characterization and density calibration..... 85
3.4 Integrin forces lead to MTFM sensor dissociation..... 89
3.5 Imaging integrin force dynamics..... 94
3.6 Generality of integrin-driven biotin dissociation and relationship to focal adhesions..... 96
3.7 Conclusion..... 97
3.8 Materials and methods..... 99
3.8.1 Reagents........... 99
3.8.2 Synthesis of cRGDfK(C)-QSY21-PEG23-biotin and cRGDfK(C)-A647-PEG23-biotin........... 99
3.8.3 Streptavidin labeling........... 100
3.8.4 Biotin functionalization of glass substrates........... 100
3.8.5 Surface density quantification........... 101
3.8.6 Determination of Förster distance between Alexa 647 and QSY21........... 104
3.8.6.1 Calibration A to obtain the Förster distance of Alexa 647 and QSY21..... 106
3.8.6.2 Calibration B to obtain the Förster distance of Alexa 647 and QSY21..... 108
3.8.7 cRGDfK(C)-peptide conjugate immobilization with streptavidin-biotin........... 110
3.8.8 Synthesis of cRGDfK α-thioester, 1........... 110
3.8.9 Synthesis of fluorescein-PEG24-biotin conjugate, 2........... 110
3.8.10 Conjugation of 1 and 2 to generate cRGDfK-Alexa 647-PEG24-fluorescein-biotin, 4........... 111
3.8.11 Covalent conjugation of cRGDfK(C)-A647-PEG24 to substrate........... 111
3.8.12 Cell culture........... 111
3.8.13 Live cell fluorescence microscopy imaging........... 112
3.8.14 Immunostaining protocols........... 113
3.9 References..... 113
Appendix 3..... 120
Chapter 4. Exploring the Mechanotransduction Model of Notch Receptor Activation........... 132
4.1 Brief overview of the Notch-Delta system..... 133
4.1.1 Description of the Notch receptor and its ligands........... 133
4.1.2 Events leading to Notch activation and translocation to the nucleus........... 135
4.2 Discussion of proposed mechanisms of Notch activation..... 137
4.3 Development of an MTFM probe to study Notch activation..... 138
4.3.1 DNA-based MTFM Notch probes........... 139
4.3.2 Binding of Dll1-expressing cells to a recombinant Notch 1 extracellular domain........... 141
4.3.3 Differentiating the effects of clustering and density in Dll1-expressing cells binding to NECD surfaces........... 144
4.4 Testing the ability of Dll1-expressing cells to exert a force on the Notch receptor..... 146
4.4.1 The streptavidin anchored DNA-based MTFM Notch sensor........... 146
4.4.2 The AuNP anchored DNA-hairpin MTFM sensor........... 149
4.5 Conclusions..... 155
4.6 Materials and methods..... 155
4.6.1 Cell Culture........... 155
4.6.2 Insect cell (Sf9) transfection and expression of NECD-mCherry........... 156
4.6.3 Mammalian cell (HEK293FT) transfection........... 156
4.6.4 Purification and biotinylation of expressed proteins........... 156
4.6.5 Synthesis of synthetic lipid bilayer surfaces........... 157
4.6.6 Synthesis of streptavidin anchored MTFM surfaces........... 158
4.6.7 Synthesis of AuNP-DNA MTFM surfaces........... 159
4.6.8 Microscopy methods........... 160
4.7 References..... 160
Chapter 5. Summary and future directions........... 165
5.1 Summary..... 166
5.2 Future directions..... 168
5.2.1 Advancing the MTFM probe design........... 168
5.2.2 Clustering and force........... 169
5.3 References..... 170
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