Development of Nanoparticle-based Tools to Investigate Cell Mechanotransduction at the Living/Nonliving Interface Público
Liu, Yang (2016)
Published
Abstract
Mechanical forces play an essential role in regulating cell functions and cell fate, ranging from proliferation, differentiation to migration and apoptosis. Despite great significance, the lacking of molecular tools to interrogate these forces has hindered the biological community to fully understand the underlying mechanisms of mechanotransduction. In this dissertation, multiple gold nanoparticle (AuNP) tension probes and actuators are introduced for the study of mechanotransduction at the surface of living cells. Chapter 1 briefly reviews the role of mechanical forces in cell biology and several common techniques that have recently emerged for measuring cell forces. This chapter also emphasizes the design and biological applications of synthetic tension probes for molecular tension fluorescence microscopy (MTFM). Chapter 2 describes the systematic development of a gold nanoparticle tension probe for visualizing forces exerted by integrin receptors. For the first time, integrin forces were quantified with molecular tension probes during initial cell spreading and focal adhesion maturation. Chapter 3 reports the technological advancement f a nanopatterned tension probe arrays and its application in understanding how nanoscale clustering of RGD ligands alters the mechano-regulation of their integrin receptors. We found that the mechanism of sensing ligand spacing is force-mediated. Chapter 4 details the recent development of DNA-based nanoparticle tension probes for studying the mechanism of how chemo-mechanical coupling influences immune function. With unprecedented sensitivity, we provided the first pN tension maps of individual TCR-pMHC complexes during Tcell activation. Importantly, naive CD8 T cells recruit TCRs, co-receptor CD8, and adhesion molecules such as LFA-1 to achieve a force of 12-19 pN, which is indispensable for enhanced antigen fidelity. Chapter 5 introduces the development of a novel optomechanical acatutor that can specifically deliver pN forces to cell surface receptors. We showed that a variety of cell behaviors, such as cell adhesion, migration and T-cell activation, are mechanosensitive and can be remotely controlled by our opt omechanicalswitch. Chapter 6 summaries of the thesis and provides a future outlook on developing the next generation of molecular tension probes and actuators. We believe that these optical methods and tools will likely transform the study of mechanotransduction in biology.
Table of Contents
Chapter 1: Development of molecular tools to study mechanotransduction at the cell surface. 1
1.1 Introduction. 2
1.1.1 Examples of cell mechanotransduction. 2
1.1.2 The molecular foundation of mechanotransduction. 4
1.2 Current methods to measure cellular forces. 6
1.2.1 Polymer network deformation (TFM and mPADs). 6
1.2.2 Single molecule force spectroscopy (SMFS). 8
1.3 Molecular Tension Probes. 9
1.3.1 Genetically-encoded and immobilized tension probes. 9
1.3.2 Guidelines for the design of immobilized tension probes. 10
1.3.3 Selecting the key components of immobilized tension probes. 12
1.3.3.1 Mechanical Springs. 12
1.3.3.2 Spectroscopic rulers. 14
1.3.3.3 Immobilization strategies. 16
1.3.4 Biological applications of immobilized tension probes. 17
1.4 Aim and scope of the dissertation. 19
1.5 References. 21
Chapter 2: Development of gold nanoparticle-based tension probes for mechano-imaging of integrin-mediated forces. 31
2.1 Introduction. 32
2.2 Results and discussion. 34
2.2.1 Synthesis and characterization of the MTFM probes. 34
2.2.2 Immobilization and characterization of the AuNP-MTFM sensors on glass substrate. 38
2.2.3 Live-cell imaging of integrin-mediated forces. 39
2.2.4 Visualization of integrin forces initial and mature focal adhesions. 41
2.3 Conclusion. 43
2.4 Materials and methods. 44
2.4.1 Materials. 44
2.4.2 Synthesis of gold nanoparticles. 45
2.4.3 Negative staining TEM. 45
2.4.4 Ensemble fluorescence measurement. 46
2.4.5 HPLC. 46
2.4.6 MALDI-Mass Spectroscopy. 46
2.4.7 Optical Microscopy. 46
2.4.8 AFM imaging. 47
2.4.9 Cell culture. 47
2.4.10 Paxillin-immunostaining. 48
2.4.11 Synthesis and quantification of dsDNA-AuNP for NSET calibration. 48
2.5 References. 51
2.6 Appendix. 56
Chapter 3: Utilizing nanopatterned MTFM to investigate the impact of integrin clustering on force transmission. 70
3.1 Introduction. 71
3.2 Results and discussion. 75
3.2.1 Fabrication and characterization of the nanopatterned AuNP probes. 75
3.2.2 Co-localizing different FA markers with integrin tension. 80
3.2.3 The impact of ligand spacing on integrin tension. 81
3.3 Conclusion. 86
3.4 Materials and methods. 88
3.4.1 Materials. 88
3.4.2 Fabrication of BCMN patterned gold nanoparticle (AuNP) arrays. 89
3.4.3 Scanning Electron Microscopy. 90
3.4.4 Ensemble fluorescence measurements. 90
3.4.5 HPLC. 90
3.4.6 MALDI-Mass Spectrometry. 90
3.4.7 Optical Microscopy. 91
3.4.8 AFM imaging. 91
3.4.9 Cell culture and transfection. 92
3.5 References. 93
3.6 Appendix. 100
Chapter 4: Investigating T-cell mechanics with DNA-based AuNP tension probes. 117
4.1 Introduction. 118
4.2 Results. 120
4.2.1 Fabrication and characterization of the DNA-based nanoparticle tension sensors. 120
4.2.2 Piconewton forces are transmitted by individual TCR complexes prior to T cell activation. 122
4.2.3 TCR-ligand tension requires co-receptor engagement and is modulated by adhesion molecules. 123
4.2.4 T cells harness mechanical forces as a checkpoint of antigen quality. 126
4.3 Discussion and Conclusion. 130
4.4 Materials and methods. 133
4.4.1 Chemical reagents. 133
4.4.2 Antibodies. 134
4.4.3 MHC. 134
4.4.4 OT-1 cell harvesting and purification. 135
4.4.5 DNA sequences. 136
4.4.6 F1/2 calculation for 35% GC content hairpin (12 pN). 136
4.4.7 Surface Preparation. 137
4.4.8 Bulk fluorescence measurement. 139
4.4.9 HPLC. 140
4.4.10 MALDI-mass spectrometry. 140
4.4.11 Calcium imaging. 140
4.4.12 Fluorescence immunostaining. 141
4.4.13 Drug Inhibition. 141
4.4.14 AFM imaging. 142
4.4.15 DNA labeling. 142
4.4.16 Optical microscopy. 142
4.5 References. 144
4.6 Appendix. 150
4.7 Supplementary Note 1. 166
4.8 Supplementary Note 2. 166
Chapter 5: Development of Nanoscale Optomechanical Actuators for Controlling Mechanotransduction in Living Cells. 168
5.1 Introduction. 169
5.2 Results and discussion. 170
5.2.1 Fabrication and characterization of OMA nanoparticles. 170
5.2.2 Optical modulation of integrin-mediated cell adhesion, protrusion and migration. 174
5.2.3 Actuation of T-cell receptors with OMA nanoparticles. 178
5.3 Conclusion. 180
5.4 Materials and methods. 181
5.4.1 Gold Nanorod preparation. 181
5.4.2 Polymerization and encapsulation of AuNRs with pNIPMAm. 182
5.4.3 Characterization of OMAs. 183
5.4.4 Determination of Dynamics of OMA nanoparticles. 184
5.4.5 Determination of cRGDfK peptide density on OMA nanoparticles. 185
5.4.6 Quantitative fluorescence calibration curve using supported lipid bilayers. 185
5.4.7 Determination of the F factor. 186
5.4.8 Determination of FAM density on OMA nanoparticles surface. 186
5.4.9 Estimation of OMA nanoparticle collapse-driven forces using molecular tension sensor. 187
5.4.10 Simulations. 188
5.4.11 Surface Preparation. 189
5.4.12 Synthesis of RGDfK-N3. 191
5.4.13 Cell culture and transfection. 191
5.4.14 Microscopy and optomechanics experiments. 192
5.4.15 Calcium imaging. 193
5.4.16 Determination of F-actin displacement using TIRF-based nanometry. 194
5.5 References. 196
5.6 Appendix. 201
Chapter 6: Summary and Perspective. 224
6.1 Summary. 225
6.2 Perspective. 228
6.3 Other contributions. 231
6.4 References. 234
About this Dissertation
| School | |
|---|---|
| Department | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Palavra-chave | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Development of Nanoparticle-based Tools to Investigate Cell Mechanotransduction at the Living/Nonliving Interface () | 2018-08-28 10:41:05 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|