An Approach to Generate Ultra-Robust and Highly Modular Molecular Tension Probes and Thermo-Responsive Enzymatic Nanoreactors to Regulate Reaction Rate Open Access

Chang, Yuan (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/6q182k869?locale=en
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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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