Abstract
Mechanical forces between cells and their extracellular
matrix (ECM) are mediated by hundreds of different
receptors. These biophysical interactions play fundamental roles in
processes ranging from cellular development to tumor progression.
However, mapping the spatial and temporal dynamics of tension among
various receptorligand pairs remains a significant challenge.
Chapter 1 of this dissertation gives a brief overview of the
history of studying mechanical signal transduction and summarizes
the state of the art techniques to answer long-standing questions.
In Chapter 2, the development of a synthetic strategy to generate
modular tension probes combining the native chemical ligation (NCL)
reaction with solid phase peptide synthesis (SPPS) is described. In
principle, this approach accommodates virtually any peptide or
expressed protein amenable to NCL. A small library of tension
probes displaying different ligands was generated for mapping
integrin and cadherin tension. It was also a first demonstration of
long-term (~3 days) molecular tension imaging. In chapter 3,
we describe the development of a thermo-responsive enzymatic
nanoreactor that can regulate reaction rates by tuning the
phase transition of a polymer hydrogel. Enzymes were
embedded into hydrogel nanoparticles that are
thermoresponsive. When the temperature is raised above
the lower critical solution temperature (LCST) of the
polymer, the water is expelled from the hydrogel, which dampens
the enzymatic turnover rate. This approach provides a
tool to modulate enzymatic reactions using external stimuli and
could significantly improve our ability to control chemical
reactions on-demand.
Table of Contents
Table of Contents
Chapter 1: Introduction of mechanotransduction and cell
adhesion 16
1.1. Mechanotransduction in cell biology 17
1.2. Focal adhesion and mechanotransduction 19
1.3. RGD motif and synergy binding site on fibronectin
24
1.4. Other receptor systems 26
1.5. Methods for studying live cell mechanical properties
26
1.6. Current molecular-scale tension sensors 28
2.1.1 Genetically encoded tension sensors 29
2.1.2 Living-nonliving interface tension sensors 29
1.7. Outlook 32
Chapter 2: A General approach to generate ultra-robust and
highly modular molecular tension probes 38
2.1 Motivation and designing the tension sensor 39
2.1.3 The mechanism of quantifying the tension signal 41
2.1.4 Design and synthesis of the tension sensor 49
2.1.5 Methods and material 54
2.1.6 Determination of quenching efficiency 57
2.2 Integrin tension lead to biotin-streptavidin dissociation
in cell adhesion 61
2.2.1 Experiment findings 61
2.2.2 Discussion 65
2.3 Design and synthesis of the covalent, ultra-stable tension
sensor 69
2.3.1 Covalent linkage via click chemistry 69
2.3.2 Fluorophore selection 71
2.3.3 Design and synthesis of MTFM sensor 73
2.4 The development of zwitterionic silane to prevent
non-specific interaction of cells and glass surfaces 81
2.4.1 Biofouling in bioscience studies 81
2.4.2 Methods to prevent biofouling 82
2.4.3 Zwitterionic SBS passivation method 83
2.4.4 Synthesis of SBS 84
2.4.5 Preparation of the glass surface 84
2.4.6 Materials and Methods. 87
2.4.7 Surface crowding caused by passivation molecules
87
2.4.8 Comparison of SBS and traditional PEG passivation
88
2.5 Tension map of fibroblast cells 91
2.5.1 Determine the surface density of MTFM tension sensor
91
2.5.2 Determine the quenching efficiency of the MFTM sensor
94
2.5.3 Integrin tension fluorescent map 96
2.5.4 Verifying tension reversibility by actin inhibitors
97
2.5.5 Tension causing by integrin mediated focal adhesion
99
2.5.6 Quantification of tension 100
2.6 Long term tension signal analysis 107
2.7 Tension generated by binding fibronectin synergy ligand
PHSRN 109
2.7.1 Fibronectin III 9th and 10th domain 109
2.7.2 The effect of synergy site in cell mechanics 109
2.8 Cell-cell adhesion through Cadherin 111
2.8.1 Tension map of cadherin 111
2.8.2 Antibody mapping of cadherin 111
2.9 Materials and Methods 114
2.9.1 Reagents 114
2.9.2 Peptide synthesis 115
2.9.3 HPLC 115
2.9.4 MALDI-TOF Mass spectroscopy 115
2.9.5 Cu-TBTA preparation 115
2.9.6 Fluorescence microscopy 115
2.9.7 Cell culture. 116
2.9.8 Preparation of small unilamellar vesicles phospholipids
116
2.9.9 Assembly of supported lipid membranes 117
2.10 References 118
Chapter 3: Thermo-responsive enzymatic nanoreactor to regulate
reaction rate 130
3.1. Motivation and designing the thermo-regulated enzymatic
nanoreactor 131
3.1.1 Introduction 131
3.1.2 Design and mechanism of the thermo-regulated nanoenzyme
reactor 137
3.1.3 Thermoresponsive polymers 139
3.1.3.1 LCST 140
3.1.3.2 pNIPAM and pNIPMAM 141
3.1.3.3 Glucose oxidase and horse radish peroxidase 142
3.2. Synthesis of enzymatic nanoreactors 144
3.2.1 Synthesis of polyNIPAM and polyNIPMAM nanoparticles with
alkyne functional groups 144
3.2.2 Modification of nanoparticles with NHS ester functional
groups 146
3.2.3 Characterization of nanoparticles 146
3.2.3.1 TEM 146
3.2.4 Conjugation of enzymes on the nanoparticles 147
3.2.4.1 Fluorescent modification of enzyme for quantification
purpose 147
3.2.4.2 Covalent conjugation of enzymes 149
3.2.4.3 Fluorescent image of nanoactuators 149
3.2.4.4 Mapping the distribution of enzymes in the particle
using 5nm gold 150
3.2.5 Determination of the number of enzymes on the hydrogel
particle by fluorescence 152
3.2.5.1 DLS analysis of particle volume transition with
temperature 155
3.3. Kinetics of enzymatic reaction actuation 156
3.3.1 Results and discussion 156
3.3.2 Real-time recording of enzymatic reaction inhibition
160
3.4. Conclusion and outlook 161
3.5. References 162
Chapter 4: Summary and future directions 174
Appendix 191
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