DNA Nanotechnology as an Emerging Tool to Investigate Cell Mechanics: From Studying T Cell Mechanotransduction to Development of Catalytic Amplification Assay for Readout of Cellular Forces Público
Ma, Pui-Yan (Summer 2019)
Published
Abstract
T cells are key players in adaptive immune system, and their activation is crucial for maintaining functional defense mechanisms that destroy infected or cancer cells. Productive T cell activation only occurs when T cells form physical contact with opposing cells that present cognate antigens on their surfaces. This initial antigen recognition by T cell receptor triggers a series of phosphorylation events that ultimately activates T cell signaling and functions. Recent work has demonstrated externally applied forces can activate T cells. However, whether T cells generate internal forces transmitted to the surface receptors and whether these forces have a role in regulating T cell signaling and functions remain unexplored.
This thesis presents two separate lines of studies pertaining to development of biophysical tools that harness mechanical properties of DNA to study cell mechanics. In the first part of this thesis, I developed and applied DNA-based force probes that report (or physically cap) forces generated by the surface receptors of T cells with high spatial, temporal and force resolution. Work presented in Chapter 2 revealed that T cell receptor transmits pN forces to its antigen at artificial cell-cell junctions, using hybrid supported lipid bilayer as a model cell surface. Importantly, these forces persist when T cell receptor-antigen complexes spatially reorganize at the junction. In Chapter 3, I showed that T cell’s LFA-1 adhesion receptor is a mechanosensor, where lateral mobility of cognate ICAM-1 ligand controls T cell-substrate interaction. I demonstrated LFA-1 can transmit forces >19 pN upon cell adhesion and migration, with a distinct force pattern compared to T cell receptor forces. Lastly, I discovered that LFA-1 mechanically communicates with T cell receptor to achieve fine-tuned T cell spreading, early signaling and functional responses. In the second part of this thesis (Chapter 4), I developed the first catalytic amplification assay for readout of integrin forces generated by fibroblasts. Additionally, I examined its potential in screening drugs that impair cell mechanics in a high throughput format. Overall, this thesis showcases the potential uses of engineered DNA structures as novel biophysical tools to revolutionize cellular force measurement.
Table of Contents
Chapter 1. Introduction.................. 1
1.1.Molecular mechanism of T cell activation..................................... 2
1.2.Signal regulation by LFA-1 adhesion receptor on the T cell membrane.................................... 7
1.3.Role of mechanical forces in T cell activation.............................. 9
1.4.A succinct overview justifying the need to study mechanotransduction of cells.... 12
1.5.Traditional methods to measure cellular forces............................ 14
1.6.Molecular methods to measure cellular forces............................ 16
1.6.1. Reversible analog probes 17
1.6.2. Reversible digital probes. 22
1.6.2. Irreversible digital probes 29
1.7.Thesis overview......................... 35
Chapter 2. Ratiometric Tension Probes for Mapping Receptor Forces and Clustering at Intermembrane Junctions............ 38
2.1.Introduction............................... 39
2.2.Results and discussion............... 43
2.2.1. Design of ratiometric tension probes............................ 43
2.2.2. Validating ratiometric tension probes using biotinylated silica beads................................ 44
2.2.3. Ratiometric tension probes map T cell receptor forces and clustering................................... 48
2.2.4. T cell receptors transmit forces at the cSMAC ................ 52
2.2.5. TCR force transmission is enhanced by limiting the ligand mobility .................................... 53
2.2.6. TCR forces and clustering are driven by myosin IIA ......... 53
2.3.Conclusion................................. 56
2.4.Materials and methods.............. 56
2.4.1. Materials.......................... 56
2.4.2. Transgenic mice, T cell activation and purification......... 57
2.4.3. General experimental...... 58
2.4.4. Oligonucleotide synthesis 59
2.4.5. Fluorescence labelling of streptavidin................................ 59
2.4.6. Synthesis of biotin-functionalized particles............. 59
2.4.7. Functionalization of AuNP with DNA tension probes or DNA duplexes..................................... 60
2.4.8. Preparation of small unilamellar vesicle.................... 61
2.4.9. Supported lipid bilayer (SLB) formation and functionalization........................ 61
2.4.10. Drug treatment.............. 63
2.4.11. Image acquisition and analysis...................................... 63
2.5. Appendix.................................. 64
Chapter 3. LFA-1 integrins are piconewton “mechanical rheostats” that tune T cell signaling and function........................................... 76
3.1.Introduction............................... 77
3.2.Results....................................... 79
3.2.1. LFA-1 dependent T cell spreading and TCR signaling favor low ligand mobility.......... 79
3.2.2. DNA-based force sensors reveal that different LFA-1 subsets transmit a spectrum of pulling forces within distinct compartments of T cells spreading on ICAM-1.... 83
3.2.3. Multiplexed DNA based tension probes report spatiotemporal dynamics of TCR and LFA-1 forces...................... 89
3.2.4. Mechanochemical stabilization of LFA-1-ICAM-1 bonds potentiates TCR triggered T cell activation............................ 90
3.2.5. Mechanical communication between TCR and LFA-1 augments T cell signaling and function...................................... 95
3.3.Discussion and conclusion........ 97
3.4.Materials and methods............ 101
3.4.1. Reagents........................ 101
3.4.2. Antibodies..................... 102
3.4.3. General experimental.... 102
3.4.4. Oligonucleotide synthesis 103
3.4.5. OT-1 cell harvesting and purification.............................. 103
3.4.6. Plasmids........................ 104
3.4.7. Lentiviral production and generation of stable expressing soluble, biotinylated-recombinant mouse ICAM-1 cell lines........ 104
3.4.8. Purification of biotinylated ICAM-1s from suspension culture 105
3.4.9. Fluorescence microscopy 106
3.4.10. Preparation of DNA hairpin-based molecular tension probes on glass surfaces.......... 106
3.4.11. Preparation of ICAM-1 TGT substrates........................ 108
3.4.12. Preparation of multiplexed TGT substrates for co-presentation of ICAM-1 and pMHC... 108
3.4.13. Preparation of small unilamellar vesicle.................. 109
3.4.14. SLB formation............. 109
3.4.15. Immunofluorescence staining.................................... 110
3.4.16. IL-2 ELISA................. 110
3.5.Appendix................................. 112
Chapter 4. Mechanically Induced Catalytic Amplification Reaction for Readout of Receptor‐Mediated Cellular Forces............................ 125
4.1.Introduction............................. 126
4.2.Results and discussion............. 129
4.2.1. Characterization of the efficiency and specificity of RCA on surfaces functionalized with DNA primers........................... 129
4.2.2. “Turn-off” TGT probes report force-driven primer exposure by integrins.............. 130
4.2.3. MCR amplifies exposed primers as a result of integrin tension..................................... 132
4.2.4. MCR detects drug that directly impairs integrin tension 135
4.2.5. MCR screens antibodies that impair integrin mechanics 136
4.3.Conclusion............................... 138
4.4.Materials and methods............ 139
4.4.1. Materials........................ 139
4.4.2. Cell culture.................... 140
4.4.3. General experimental.... 140
4.4.4. Functionalization of oligonucleotides...................... 140
4.4.5. Solution-based RCA...... 143
4.4.6. Fabrication of gold thin films for DNA anchoring........ 144
4.4.7. Preparation of oligonucleotide modified gold film.......................................... 144
4.4.8. Quantification of the density of surface-immobilized DNA........................................ 145
4.4.9. Preparation of glass surfaces labelled with cRGDfK 145
4.4.10. Preparation of gold nanoparticles decorated with dsDNA..................................... 146
4.4.11. Mechanically-induced catalytic amplification reaction (MCR)..................................... 147
4.4.12. Dose-dependent inhibition of integrin mediated tension quantified by MCR.................. 148
4.4.13. F-actin staining............ 148
4.4.14. Antibody blocking assay 149
4.4.15. Image acquisition and analysis.................................... 149
4.5.Appendix................................. 150
Chapter 5. Conclusion and future outlooks........................................ 163
Bibliography................................ 168
Vita…............................................ 198
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