Engineered coiled-coil protein domains as probes for molecular mechanobiology Restricted; Files Only

Abstract

Cells experience and transmit piconewton-scale forces that regulate essential processes such as adhesion, migration, and signal transduction. Understanding these forces and their molecular consequences remains a central challenge in mechanobiology. In this dissertation, we introduce the development of molecular tension sensors coiled-coil motifs. These probes allow one to detect transient intracellular forces and connect cellular mechanical forces to biochemical functionalities. Chapter 1 introduces the current state of mechanotransduction research and existing techniques for measuring cellular forces, highlighting the need for programmable and sensitive tools to detect and utilize molecular tension. Chapter 2 describes the development of coiled-coil tension sensors (CCTS), a set of digital responsive, tunable probes that quantify mechanical forces in living cells with defined threshold responses. Chapter 3 presents the coiled-coil mechanical switch (CCMS), a force-gated reconstitution system that converts intracellular tension into luminescent or fluorescent outputs with up to 24-fold signal enhancement. Building on CCMS framework, Chapter 4 integrates CCMS with APEX2 proximity labeling to identify the mechanome associated with load-bearing vinculin populations. Together, these chapters advance the field from force detection to force-activated biochemistry, establishing coiled-coil based platforms for mapping and engineering cellular responses to mechanical cues with molecular precision. Finally, Chapter 5 summarizes the work in this dissertation and discusses its future outlook.

Table of Contents

Table of Content

CHAPTER 1.       INTRODUCTION OF MECHANOTRANSDUCTION, MOLECULAR FORCE DETECTION TOOLS AND MECHANICAL SWITCHES.

1.1 Introduction of Mechanotransduction with a Focus on Focal Adhesions

1.1.1 The Role of Focal Adhesion Protein Complex

1.1.2 Key FA Proteins: Talin, Vinculin, and FAK

1.2 Tools for Measuring Cellular Forces

1.2.1 Cellular Scale Force Measurement

1.2.2 Molecular Tension Sensors

1.2.2.1 Extracellular Tension Sensors

1.2.2.2 Intracellular Tension Sensors

1.2.2.3 Programmable Biomaterial Design for Tension Sensors

1.3 Synthetic Molecular Switches

1.3.1 Biochemical Switches

1.3.2 Mechanical Switches

1.4 Aim and Scope of This Dissertation

1.5 References

CHAPTER 2.       DEVELOPMENT OF DIGITAL AND TUNABLE GENETICALLY ENCODED TENSION SENSORS BASED ON ENGINEERED COILED-COILS.

2.1 Abstract

2.2 Introduction

2.3 Results and Discussion

2.3.1 Design and Geometry Considerations for Creating a Vinculin CCTS

2.3.2 CCTSs can be Engineered in a Predictable Manner to Detect Vinculin Tension

2.3.3 Force Calibration of CCs Reveals Thresholds of CCTSs

2.3.4 Analysis of CCTS Data Reveals Dynamic Sub-populations of Vinculin Experiencing F>9.9 pN

2.4 Conclusion

2.5 Materials and Methods

2.5.1 Generation of tension sensor and force insensitive constructs.

2.5.2 Generation of His-AviTag-CC expression constructs.

2.5.3 His-AviTag-CCs expression and purification.

2.5.4 Cell culture and transfection.

2.5.5 Fluorescence microscopy of cultured cells.

2.5.6 Sensitized Förster resonance energy transfer index analysis.

2.5.7 Molecular Dynamics (MD) Protocol:

2.5.8 Primer modification and purification.

2.5.9 Assembling biotin-protein–DNA conjugates.

2.5.10 Single-molecule force spectroscopy calibration.

2.5.11 FRET efficiency calculations using spectrofluorometry.

2.6 Acknowledgement

2.7 Appendix

2.7.1 Supplementary data

Movie 2.1: Spin Movie of Clover-S0-mRuby2 3D rendering structure predicted by AlphaFold2.

Movie 2.2: Representative SMD simulation showing CC-S0 unfolding dynamics.

2.7.2 Role of CC orientation in CCTS

2.7.3 Molecular dynamics simulation of coiled-coil stability.

2.7.4 Analysis of Single-molecule Optical Tweezer Data

2.8 References

CHAPTER 3.       COILED-COIL MECHANICAL SWITCH FOR PICONEWTON INTRACELLULAR FORCE DETECTION.

3.1 Abstract

3.2 Introduction

3.3 Results and Discussion

3.3.1 Overall CCMS design

3.3.2 NanoLuc CCMS responds to traction forces

3.3.3 Ratiometric CCMS engineered from split mNeonGreen for mapping vinculin forces.

3.4 Conclusion

3.5 Materials and Methods

3.5.1 Recombinant DNA construct.

3.5.2 Cell culture and transfection

3.5.3 Generation of stable cell lines using lentivirus

3.5.4 Luminescent assays

3.5.5 Flow cytometry

3.5.6 DBCO-Modified Surface Preparation

3.5.7 Synthesis of Hydrogel Precursors (Tetra-PEG DBCO)

3.5.8 Rheology: Time Sweep Experiment

3.5.9 Fluorescence microscopy

3.5.10 Ratiometric CCMS Imaging data analysis

3.5.11 Immunofluorescence staining

3.6 Acknowledgement

3.7 Appendix

3.7.1 Supplementary data

3.8 References

CHAPTER 4.       COILED-COIL AS MECHANICAL SWITCH THAT ENABLES IDENTIFICATION OF MECHANOME ASSOCIATED WITH HIGH FORCE VINCULIN POPULATION

4.1 Abstract

4.2 Introduction

4.3 Results and Discussion

4.3.1 Design of APEX CCMS

4.3.2 Developing APEX2 CCMS workflow

4.3.3 Identify adhesome using CCMS through proteomics

4.3.4 AFAP1 is associated to high force FAs to assist in the formation of stress fibers

4.4 Conclusion

4.5 Materials and Methods

4.5.1 Recombinant DNA construct.

4.5.2 Cell culture and transfection

4.5.3 Fluorescence microscopy

4.5.4 Biotin-phenol labeling

4.5.5 Immunofluorescence staining

4.5.6 Proteomics sample preparation.

4.5.7 LC-MS/MS

4.5.8 Proteomics data analysis.

4.6 Acknowledgement

4.7 Apendix

4.8 References

CHAPTER 5.       SUMMARY AND PERSPECTIVE

5.1 Summary

5.2 Perspective

5.2.1 Further development of CCTS and CCMS

5.2.2 Future applications

5.3 Closing Remarks

5.4 References

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biophysics, Biomechanics Chemistry, Analytical Chemistry, Molecular
Keyword	Mechanotransduction FRET Genetically encoded tension sensor
Committee Chair / Thesis Advisor	Khalid Salaita, Emory University
Committee Members	Vincent Conticello, Emory University Laura Finzi, Emory University

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