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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

Abstract

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

Introduction

As a live-cell DFS technique, BFP engineers a human red blood cell (RBC; Figure 1) into an ultrasensitive and tunable force transducer with a compatible spring constant range at 0.1-3 pN/nm1,2,3. To probe ligand-receptor interaction, BFP enables DFS measurements at ~1 pN (10-12 N), ~3 nm (10-9 m), and ~0.5 ms (10-3 s) in force, spatial, and temporal resolution4,5. Its experimental configuration consists of two opposing micropipettes, namely the Probe and t....

Protocol

1. Obtain Analyzable DFS Events

  1. Start the experiment in the software (e.g., LabVIEW VI) for the BFP control and parameter setting (Figure S1A).
  2. Observe the repetitive probe bead-target bead/cell touches in the software for BFP Monitor (Figure S1B).
  3. Test and achieve the adhesion frequency ≤ 20% within the first 50 touches by tuning the impingement force and contact time, by which it ensures that ≥ 89% of DFS adhesion event are mediated by single.......

Representative Results

In this work, we have demonstrated the protocol of the BFP spring constant analysis. For the Bead-Cell analysis mode, we analyzed the kmol of the molecular bond between the glycosylated protein Thy-1 coated on the Probe bead and the integrin α5β1 expressed on the Target K562 cell (Thy-1-integrin α5β1; Figure 3A)10. The kcell is also derived from the Bead-Cell m.......

Discussion

In summary, we have provided a detailed data analysis protocol for preprocessing the DFS raw data and deriving molecular spring constants in the BFP Bead-Bead and Bead-Cell analysis modes. Biomechanical models and equations required for determining molecular and cellular spring constants are presented. Albeit different integrins are studied, the kmol measured by the Bead-Bead mode and the Bead-Cell mode possesses significant range differences (Figure 3A vs.

Acknowledgements

We thank Guillaume Troadec for helpful discussion, Zihao Wang for hardware consultation, and Sydney Manufacturing Hub, Gregg Suaning and Simon Ringer for support of our lab startup. This work was supported by Australian Research Council Discovery Project (DP200101970 - L.A.J.), NSW Cardiovascular Capacity Building Program (Early-Mid Career Researcher Grant - L.A.J.), Sydney Research Accelerator prize (SOAR - L.A.J.), Ramaciotti Foundations Health Investment Grant (2020HIG76 - L.A.J.), National Health and Medical Research Council Ideas Grant (APP2003904 - L.A.J.), and The University of Sydney Faculty of Engineering Startup Fund and Major Equipment Scheme (L.A.J.). Lini....

Materials

NameCompanyCatalog NumberComments
3-Mercaptopropyltrimethoxysilane (MPTMS)Uct, Specialties, llc4420-74-0Glass bead functionalization
Anhy. Sodium Phosphate Dibasic (Na2HPO4)Sigma-AldrichS7907Phosphate buffer preparation
BFP data acquisition VILabVIEWBFP control and parameter setting
BFP data analysis VILabVIEWBFP raw data analysis
Biotin-PEG3500-NHSJenKemA5026-1RBC biotinylation
Borosilicate Glass beadsDistrilab Particle Technology, Netherlands9002Glass bead functionalization
Bovine serum albuminSigma-AldrichA0336Ligand functionalization
Camera VILabVIEWBFP monitoring
D-glucoseSigma-AldrichG7021Tyrode’s buffer preparation
HepesSigma-AldrichH3375Tyrode’s buffer preparation
MAL-PEG3500-NHSJenKemA5002-1Glass bead functionalization
Potassium Chloride (KCl)Sigma-AldrichP9541Tyrode’s buffer preparation
Sodium Bicarbonate (NaHCO3)Sigma-AldrichS5761Carbonate/bicarbonate buffer preparation; Tyrode’s buffer preparation
Sodium Carbonate (Na2CO3)Sigma-AldrichS2127Carbonate/bicarbonate buffer preparation
Sodium Chloride (NaCl)Sigma-AldrichS7653Tyrode’s buffer preparation
Sodium Phosphate Monobasic Monohydrate (NaH2PO4•H2O)Sigma-AldrichS9638Phosphate buffer preparation
Streptavidin-MaleimideSigma-AldrichS9415Glass bead functionalization

References

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Molecular Spring ConstantBiomembrane Force ProbeForce SpectroscopySingle Molecule BindingHooke s LawRed Blood CellLigand ReceptorContact AreaMicropipetteForce time DataApproachRetractCompressive PhaseTensile Phase

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