Name: molecular spring constant analysis by biomembrane force probe spectroscopy
Uploaded: 2021-11-20T13:00:00
Description: Watch this Scientific Journal Video about Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy at JoVE.com

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.). Lining Arnold Ju is an Australian Research Council DECRA fellow (DE190100609).

1. Obtain Analyzable DFS Events
<ol>
	<li>Start the experiment in the software (e.g., LabVIEW VI) for the BFP control and parameter setting (Figure S1A).</li>
	<li>Observe the repetitive probe bead-target bead/cell touches in the software for BFP Monitor (Figure S1B).</li>
	<li>Test and achieve the adhesion frequency &#8804; 20% within the first 50 touches by tuning the impingement force and contact time, by which it ensures that &#8805; 89% of DFS adhesion event are mediated by single...

<ol>
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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. <strong class=...

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 the target. The Probe micropipette aspirates a RBC and a bead is glued at its apex via a biotin-streptavidin interaction. The bead is coated with the ligand of interest (Figure 1A). The Target micropipette aspirates either a cell or a bead bearing the receptor of interest, corresponding to the Bead-Cell (Figure 1B) and Bead-Bead (Figure 1C) modes, respectively5.
BFP construction, assembly and the DFS experimental protocols were described in detail previously1,6. Briefly, a BFP touch cycle consists of 5 stages: Approach, Impinge, Contact, Retract and Dissociate (Figure 1D). The horizontal RBC apex position is denoted as &#916;xRBC. At the beginning, the unstressed (zero-force) RBC deformation &#916;xRBC is 0 (Table 1). The Target is then driven by a piezotranslator to impinge on and retract from the Probe bead (Figure 1D). The RBC probe is first compressed by the Target with negative RBC deformation &#916;xRBC&#160;&#60; 0. In a Bond event, the Retract stage transitions from a compressive to a tensile phase with positive RBC deformation &#916;xRBC&#160;&#62; 0 (Figure 2C and D). According to Hooke&#39;s law, the BFP bearing force is able to be measured as F = kRBC&#160;&#215;&#160;&#916;xRBC, where kRBC (Table 1) is the RBC spring constant of the BFP. Upon bond rupture and the completion of one touch cycle, the probe bead returns to zero-force position with &#916;xRBC&#160;= 0 (Figure 1D).
To determine the kRBC, we measure and record the radii of the probe micropipette inner orifice (Rp), the RBC (R0) and the circular contact area (Rc) between the RBC and the probe bead (Figure 1A). Then kRBC is calculated according to the Evan&#39;s model (Eq. 1)7,8 using a LabVIEW program that acts as a virtual instrument (VI) to operate the BFP (Figure S1A)8,9.
<img alt="Equation 1" src="/files/ftp_upload/62490/62490eq01.jpg" /> (Eq. 1)
With a BFP established and DFS raw data obtained, hereby we present how to analyze the spring constant of ligand-receptor pair or cells. The DFS raw data on the interaction of the glycosylated protein Thy-1 and K562 cell bearing integrin &#945;5&#946;1 (Thy-1-&#945;5&#946;1; Figures 3A and 3B)10 and that of the fibrinogen and bead coated integrin &#945;IIb&#946;3 (FGN-&#945;IIb&#946;3; Figure 3C)11,12 have been used to demonstrate the Bead-Cell and Bead-Bead analysis modes, respectively.
BFP Experimental Preparation 
For details of BFP experimental preparation and instrumentation, please refer to the previously published protocols3. In brief, human RBC has been biotinylated using the Biotin-PEG3500-NHS in the carbon/bicarbonate buffer. Proteins of interest have been covalently coupled to the borosilicate glass beads using MAL-PEG3500-NHS in the phosphate buffer. To attach to the biotinylated RBC, the probe bead is also coated with streptavidin (SA) using the MAL-SA. Please see the Table of Materials and Table 2.
To assemble the BFP (Figure 1, left), the third micropipette termed &#39;Helper&#39; will be used to deliver the probe bead and glue it to the RBC&#39;s apex1,3. The covalent interaction between the SA coated probe bead and biotinylated RBC is much stronger than the ligand-receptor bond of interest. Thus, the Dissociate stage can be interpreted as the ligand-receptor bond rupture rather than the detachment of Probe bead from the RBC.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-Mercaptopropyltrimethoxysilane (MPTMS)</td>
			<td>Uct, Specialties, llc</td>
			<td>4420-74-0</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>BFP data acquisition VI</td>
			<td>LabVIEW</td>
			<td></td>
			<td>BFP control and parameter setting</td>
		</tr>
		<tr>
			<td>BFP data analysis VI</td>
			<td>LabVIEW</td>
			<td></td>
			<td>BFP raw data analysis</td>
		</tr>
		<tr>
			<td>Biotin-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5026-1</td>
			<td>RBC biotinylation</td>
		</tr>
		<tr>
			<td>Borosilicate Glass beads</td>
			<td>Distrilab Particle Technology, Netherlands</td>
			<td>9002</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A0336</td>
			<td>Ligand functionalization</td>
		</tr>
		<tr>
			<td>Camera VI</td>
			<td>LabVIEW</td>
			<td></td>
			<td>BFP monitoring</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>MAL-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5002-1</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Carbonate/bicarbonate buffer preparation; Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Tyrode&#8217;s buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic Monohydrate (NaH2PO4&#8226;H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Streptavidin-Maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>S9415</td>
			<td>Glass bead functionalization</td>
		</tr>
	</tbody>
</table>

molecular spring constant analysis by biomembrane force probe spectroscopy

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.

In this work, we have demonstrated the protocol of the BFP spring constant analysis. For the Bead-Cell analysis mode, we analyzed the kmol&#160;of the molecular bond between the glycosylated protein Thy-1 coated on the Probe bead and the integrin &#945;5&#946;1 expressed on the Target K562 cell (Thy-1-integrin &#945;5&#946;1; Figure 3A)10. The kcell is also derived from the Bead-Cell m...

Watch this Scientific Journal Video about Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy at JoVE.com

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

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.

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...

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