A toolbox based on DNA nanotechnology to investigate cell receptor mechanics Open Access
Ma, Rong (Spring 2021)
Abstract
Mechanical forces exist widely at the interplay between a cell and extracellular matrix, or between cells, and are critical in mediating diverse biological functions, ranging from proliferation and differentiation, to cell activation and cancer development. To study these mechanical forces, innovative tools are needed. Thus, as one of the most intensively studied biopolymers, nucleic acids are becoming an important force-responsive material for the study of mechanobiology. Various platforms functionalized with DNA constructs have been designed to sense and manipulate receptor forces due to their well understood mechanical characteristics and highly modular composition. This dissertation introduces the current state of mechanobiology and mechano-immunology understanding, as well as the fundamental principles of DNA mechanics. Following an overview of the classic design and applications of DNA-based tools, this dissertation describes four unique DNA-based methods to investigate cell receptor mechanics from different perspectives and discusses the limitations of current techniques. Finally, future enrichment of the DNA mechanotechnology toolbox is envisioned.
Table of Contents
Chapter 1. Mechanotransduction at the cell surface and methods to study receptor forces 1
1.1. Introduction 2
1.2. Mechanotransduction at the cell surface 3
1.2.1. Mechanotransduction through integrins 3
1.2.2. Mechanotransduction through T cell receptor 4
1.3. Methods to study receptor forces 10
1.3.1. DNA mechanics 12
1.3.2. DNA-based molecular force sensing 15
1.4. Aim and scope of the dissertation 20
1.5. References 23
Chapter 2. DNA probes that store mechanical information reveal transient piconewton forces applied by T cells 29
2.1. Introduction 30
2.2. Results and Discussion 33
2.2.1. Preparation and Characterization of DNA hairpin tension probe substrate 33
2.2.2. Hybridization between the DNA hairpin tension probes and the locking strand in a cell-free system 33
2.2.3. Unlocking the tension probes with toehold-mediated strand displacement in a cell-free system 34
2.2.4. Confirming mechanically selective hybridization and toehold-mediated unlocking of the probes with T cells 34
2.2.5. Confirming the stability of locked probes 35
2.2.6. Multiple rounds of mechanical information storage of TCR forces 35
2.2.7. Mechanical information storage of TCR forces in migratory T cells 37
2.2.8. TCR force signal enhancement by the mechanical information storage 38
2.2.9. Detection and analysis of the TCR forces with altered peptide ligands 39
2.2.10. Detection and analysis of the PD1 forces 43
2.3. Conclusion 44
2.4. Supplementary note 46
2.5. Materials and methods 48
2.5.1. Oligonucleotides 48
2.5.2. Reagents 50
2.5.3. Cells 51
2.5.4. Equipment 52
2.5.5. Labeling oligonucleotides with dyes 52
2.5.6. Biotinylated dimeric ICAM-1 expression 53
2.5.7. Surface preparation 53
2.5.8. Microscopy 55
2.6. Appendix 60
2.7. References 76
Chapter 3. Investigating the TCR force regulatory network with DNA tension probes and ensemble measurement of TCR-pMHC force lifetime based on DNA hybridization kinetics 81
3.1. Introduction 82
3.2. Establishing a model that describes the system 87
3.3. Results and Discussion 89
3.3.1. TCR force lifetime measurement with antiCD3ε, pMHC N4 and pMHC Q4 89
3.3.2. Effect of co-receptor engagement on TCR force and force lifetimes 93
3.3.3. The effect of cytoskeleton network on TCR force and force lifetime 99
3.4. Conclusion 106
3.5. Materials and methods 108
3.5.1. Oligonucleotides 108
3.5.2. Reagents 109
3.5.3. Equipment 110
3.5.4. Oligonucleotide preparation 111
3.5.5. HPLC 111
3.5.6. Transgenic mice and T cell preparation 111
3.5.7. Substrate preparation 112
3.5.8. Microscopy 114
3.5.9. Flow cytometry 114
3.5.10. Data analysis 115
3.5.11. Model 116
3.6. Appendix 120
3.7. References 143
Chapter 4. Towards the identification of mechanically active T cells, TCRs, and antigens with mechanically selective proximity tagging 148
4.1. Introduction 149
4.2. Results and Discussion 152
4.2.1. Hybridization on 2D substrate is not affected by the presence of proximity labeling enzyme 152
4.2.2. Mechanically selective proximity biotinylation 154
4.2.3. High throughput detection of the biotinylated mechanically active T cells 156
4.3. Conclusion 159
4.4. Materials and methods 160
4.4.1. Oligonucleotides 160
4.4.2. Reagents 161
4.4.3. Equipment 162
4.4.4. Mice and cells 163
4.4.5. Oligonucleotide preparation 163
4.4.6. Fluorescence labeling of streptavidin 164
4.4.7. Substrate preparation 165
4.4.8. Microscopy 167
4.4.9. Proximity biotinylation and flow cytometry 168
4.5. Appendix 169
4.6. References 176
Chapter 5. Molecular tension probe based on force-induced DNA peeling mechanism maps integrin forces with microscopy and enables force-based cell high throughput identification 179
5.1. Introduction 180
5.2. Results and discussion 184
5.2.1. Design and preparation of the DNA tension probes based on peeling mechanism 184
5.2.2. Visualizing the integrin forces in fibroblasts 184
5.2.3. Quantitative analysis of integrin tension on peeling probe in comparison with 56 pN TGT 187
5.2.4. Peeling probe revealed that the formation of perinuclear actin requires sustained force transmission, and the associated focal adhesions exert force > 41 pN 188
5.2.5. High throughput detection of cells with higher integrin mechanical activity. 190
5.3. Conclusion 193
5.4. Materials and methods 195
5.4.1. Oligonucleotides 195
5.4.2. Reagents 196
5.4.3. Equipment 197
5.4.4. Oligonucleotide preparation 198
5.4.5 DNA tension probe substrate preparation 199
5.4.6. Cell culture 200
5.4.7. Fluorescence microscopy 200
5.4.8. Flow cytometry 200
5.4.9. General experiments 200
5.4.10. Data analysis 201
5.5. Appendix 203
5.6. References 214
Chapter 6. Summary and future outlook 218
6.1. Summary 219
6.2. Future outlook for cell receptor mechanics studies with current techniques 221
6.2.1. Direct observation of catch-bond in TCR 221
6.2.2. Neoantigen and neoantigen-specific T cell identification 222
6.2.3 Elucidating the TCR “mechanome” to better understand the T cell triggering mechanism 222
6.2.4. Elucidating the mechanical regulation network and predicting mechanical plasticity with transcriptomics 223
6.2.5. Applying mechanically selective hybridization-based methods to integrins 223
6.2.6. Other directions 224
6.3. Future outlook for DNA mechano-technology 224
6.4. Concluding remarks 225
6.5. References 227
About this Dissertation
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Primary PDF
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A toolbox based on DNA nanotechnology to investigate cell receptor mechanics () | 2021-05-10 08:56:20 -0400 |
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Supplemental Files
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Movie A3.1 (locking of TCR-antiCD3e force) | 2021-05-10 08:56:26 -0400 |
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Movie A3.2 (locking of TCR-pMHC N4 force) | 2021-05-10 08:56:38 -0400 |
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Movie A3.3 (locking of TCR-pMHC Q4 force) | 2021-05-10 08:56:47 -0400 |
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Movie A3.4 (locking of TCR-N4 force with CD28-B7-1) | 2021-05-10 08:56:55 -0400 |
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Movie A3.5 (locking of TCR-pMHC N4 force with LFA-1-ICAM-1) | 2021-05-10 08:57:02 -0400 |
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Movie A3.6 (locking of TCR-pMHC N4_CK666) | 2021-05-10 08:57:10 -0400 |
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Movie A3.7 (locking of TCR-pMHC N4_Blebb force) | 2021-05-10 08:57:16 -0400 |
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Movie A3.8 (locking of TCR-pMHC N4_Jas force) | 2021-05-10 08:57:24 -0400 |
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Movie A3.9 (locking of TCR-pMHC N4_DMSO force) | 2021-05-10 08:57:31 -0400 |
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Movie A2.1 (erasing of the locked tension signal) | 2021-05-10 08:57:40 -0400 |
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Movie A2.2 (photobleaching control) | 2021-05-10 08:57:48 -0400 |
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Movie A2.3 (scrambled control1 for unlocking) | 2021-05-10 08:57:57 -0400 |
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Movie A2.4 (scrambled control2 for unlocking) | 2021-05-10 08:58:08 -0400 |
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Movie A2.5 (migratory OT-1 cell) | 2021-05-10 08:58:17 -0400 |
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