EXPANDING THE MOLECULAR TOOLKIT TO MEASURE CELL FORCE: FROM SCAFFOLD SYNTHESIS TO MEASURING MECHANOTRANSDUTION Open Access
Bender, Rachel (Spring 2023)
Abstract
Mechanical communication is a concept central to all life. Cells transmit and transduce piconewton level
forces with the extracellular matrix and it is these forces that guide cell function. Through a process known
as mechanotransduction, cells use the receptors that coat their cell membrane to convert mechanical cues
from their surroundings into biochemical responses that control the force generating machinery inside the
cell. Foundational work focused on studying the forces generated by entire cells by measuring the amount
of deformation they caused on different substrates. Indeed, the techniques used in these studies are still
widely used today; however, the focus of this dissertation is on another, more refined, class of techniques
known as molecular force sensors which provide information on the forces mediated through discrete
interactions. One class of these sensors are those that are immobilized onto a scaffold and measure the
forces generated by external cell receptors. In Chapter 2, we describe the characterization of the transcyclooctene/
tetrazine reaction for producing scaffolds to immobilize biomolecules such as molecular force
sensor. We demonstrate that these scaffolds are degradation resistant and can be homogenously
functionalized with molecular sensors for measuring the integrin generated forces of fibroblasts. In Chapter
3, we use these scaffolds to immobilize a new class of force probes constructed of peptide nucleic acids
(PNA), a synthetic nucleic acid that is resistant to enzyme degradation and binds with a high affinity to
other oligonucleotides. We demonstrate that PNA-based force sensors improve the resolution of tension
imaging in aggressive cancer cell lines, and likely report on the upper levels of integrin mediated cell force.
Finally, in Chapter 4, we describe the synthesis of a new reversible shearing DNA probe to study the effect
that molecular force extension curves have on mechanotransduction. We demonstrate that integrins are
sensitive to the geometries of their ligands and are capable of detecting abrupt changes in resistive force
that occur throughout the extracellular matrix. In summary, this work contributes new tools for studying
cell mechanical forces over extended time and force ranges and expands our understanding of the role
ligand geometry plays in receptor mediated mechanotransduction.
Table of Contents
DISTRIBUTION AGREEMENT
APPROVAL SHEET
ABSTRACT COVER PAGE
ABSTRACT
COVER PAGE
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF SCHEMES
CHAPTER 1: Introduction
1.1. Cell structure and mechanotransduction
1.2. Measuring cell forces using synthetic tension sensors
1.2.1 Fluorescence in synthetic tension sensors
1.2.2 Force response behavior of synthetic tension sensors
1.2.3 Analog surface immobilized tension sensors
1.2.4 Digital surface immobilized tension sensors
1.2.4.1 DNA hairpin sensors
1.2.4.2 DNA rupture sensors
1.3 Limitations of existing nucleic acid force sensors
1.3.1 Biostability
1.3.2 Measuring “real-time” cell integrin forces > 19 pN
1.4 Dissertation scope and outline
CHAPTER 2: Surface tethering of biomolecules using the reaction between transcyclooctene and tetrazine
2.1. Abstract
2.2. Introduction
2.3. Results
2.3.1 TCO/Tz iEDDA surfaces are more homogenous than Biotin/STVD surfaces
2.3.2 TCO/Tz iEDDA surfaces are more resistant to degradation than Biotin/STVD surfaces
2.3.3 TCO/Tz iEDDA surfaces are superior scaffolds for studying mechanotransduction
2.4. Discussion
CHAPTER 3 PNA tension probes expand the measurable force range of nucleic acid integrin force sensing technology
3.1 Abstract
3.2 Introduction
3.2.1 Protein-based force sensors
3.2.2 Peptide nucleic acids (PNA)
3.2.3 Characterization of PNA
3.3 Results
3.3.1 Synthesis and design of PNA force sensors
3.3.2 Characterization of PNA force sensors
3.3.3 PNA sensors report on the forces generated by fibroblasts
3.3.4 PNA sensors report on the upper levels of integrin force generated by muscle cells
3.3.5 PNA force sensors report on cancer cell mechanics with improved resolution over DNA sensors
3.4 Discussion
CHAPTER 4: Cell adhesion receptors detect the force-extension curve of their ligands
4.1 Abstract
4.2 Introduction
4.2.1 Design of molecular force sensors
4.2.2 Modeling polymer behavior: force extension graphs
4.2.3 Polymer force-extension behaviors
4.2.4 Worm-like chain model
4.2.5 Molecular springs with secondary structure
4.2.6 How force influence the kinetics and thermodynamics of unfolding
4.2.7 Mechanotransduction depends on outside-in and inside-out signaling
4.2.8 Integrins detect the force-extension curve of their ligands
4.3 Results
4.3.1 Synthesis and design of reversible probes
4.3.2 RU and RS probes have identical DG’s of unfolding but differ in their molecular extension curves
4.3.3 Platelet integrin forces unfold RU but not RS probes
4.3.4 Fibroblast integrins unfold RS and RU probes in a time-dependent fashion
4.3.5 Fibroblasts show enhanced mechanical signal when cultured on RUcRGD probes compared to RScRGD probes
4.3.6 Focal adhesions have lower turnover rates in fibroblasts cultured on RUcRGD
4.4 Discussion
CHAPTER 5: Conclusions and future outlook
5.1 Summary of Advances
5.1.1 Discussion of biological limitations
5.2 Outstanding questions and directions in developing nucleic acid
nanotechnology for mechanobiology applications
5.3 Areas of exploration in mechanobiology
APPENDEX A SUPPORTING INFORMATION
A.1 Characterizing the trans-cyclooctene and tetrazine iEDDA reaction for use in surface modifications for mechanobiology applications
A.1.1 Materials
A.1.2 Equipment
A.1.3 Methods
A.1.3.1 Synthesis of tension probes
A.1.3.2 Surface preparation
A.1.3.3 DNA hybridization
A.1.3.4 Imaging chamber assembly
A.1.3.5 Cell culture
A.1.3.6 Image acquisition and analysis
A.1.4 Figures, tables, and schemes
A.2 PNA tension probes expand the measurable force range of nucleic acid force sensing technology
A.2.1 Materials
A.2.2 Equipment
A.2.3 Methods
A.2.3.1 PNA oligomer synthesis
A.2.3.2 Tension probe synthesis
A.2.3.3 Surface preparation
A.2.3.4 DNA hybridization
A.2.3.5 Imaging chamber assembly
A.2.3.6 Cell culture
A.2.3.7 Image acquisition and analysis
A.2.4 Note 1 – van’t Hoff analysis of PNA duplexes
A.2.5 Figures, table, and schemes
A.3 Cell adhesion receptors detect the force-extension curve of their ligands
A.3.1 Materials
A.3.2 Equipment
A.3.3 Methods
A.3.3.1 Synthesis of substructures for tension probes
A.3.3.2 Synthesis of DNA tension probes
A.3.3.3 Synthesis of fluorescently labeled human fibronectin
A.3.3.4 Electron Spray Ionization (ESI) mass spectroscopy
A.3.3.5 Characterization of reversible probes
A.3.3.6 Surface preparation
A.3.3.7 DNA hybridization
A.3.3.8 Imaging chamber assembly
A.3.3.9 Fibroblast staining
A.3.3.10 Platelet handling
A.3.3.11 Cell culture
A.3.3.12 Cell transfection
A.3.3.13 Image acquisition and analysis
A.3.4 Note 2 – van’t Hoff analysis of RS and RU probes
A.3.5 Note 3 – oxDNA simulation
A.3.6 Figures, tables, and schemes
APPENDEX B: GLOSSARY AND COMMON ABBREVIATIONS
APPENDEX C: PERMISSIONS
REFERENCES
About this Dissertation
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