Name: fluorescence biomembrane force probe: concurrent quantitation of receptor-ligand kinetics and binding-induced intracellular signaling on a single cell
Uploaded: 2015-08-04T13:00:00
Description: Watch this Scientific Journal Video about Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell at JoVE

Research related to this paper and the development of the fBFP technology in the Zhu lab were supported by NIH grants AI044902, AI077343, AI038282, HL093723, HL091020, GM096187, and TW008753. We thank Evan Evans for inventing this empowering experimental tool, and members of the Evans lab, Andrew Leung, Koji Kinoshita, Wesley Wong, and Ken Halvorsen, for helping us to build the BFP. We also thank other Zhu lab members, Fang Kong, Chenghao Ge and Kaitao Li, for their helps in the instrumentation development.

This protocol follows the guidelines of and has been approved by the human research ethics committee of Georgia Institute of Technology.
1. Human RBCs Isolation, Biotinylation and Osmolarity Adjustment
Note: Step 1.1 should be performed by a trained medical professional such as a nurse, with an Institutional Review Board approved protocol.
<ol>
	<li>Obtain 8-10 &#181;l (one drop) of blood from finger prick and add to 1 ml of the carbonate/bicarbonate buffer (<stro...

<ol>
	<li>Aplin, A. E., Howe, A., Alahari, S. K., Juliano, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+and+signal+modulation+by+cell+adhesion+receptors:+the+role+of+integrins,+cadherins,+immunoglobulin-cell+adhesion+molecules,+and+selectins.">Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins.</a> Pharmacological reviews. 50, 197-263 (1998).</li><li>Davis, M. M., Bjorkman, P. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+antigen+recognition+at+the+cell+membrane.">T cell antigen recognition at the cell membrane.</a> Molecular immunology. 52, 155-164 (2012).</li><li>Zarnitsyna, V., Zhu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+triggering:+insights+from+2D+kinetics+analysis+of+molecular+interactions.">T cell triggering: insights from 2D kinetics analysis of molecular interactions.</a> Physical biology. 9, 045005 (2012).</li><li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscope.">Atomic Force Microscope.</a> Physical Review Letters. 56, 930-933 (1986).</li><li>Marshall, B. 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J., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+catch+bonds+between+an+integrin+and+its+ligand.">Demonstration of catch bonds between an integrin and its ligand.</a> The Journal of cell biology. 185, 1275-1284 (2009).</li><li>Yago, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+bonds+govern+adhesion+through+L-selectin+at+threshold+shear.">Catch bonds govern adhesion through L-selectin at threshold shear.</a> The Journal of cell biology. 166, 913-923 (2004).</li><li>Yago, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+glycoprotein+Ibalpha+forms+catch+bonds+with+human+WT+vWF+but+not+with+type+2B+von+Willebrand+disease+vWF.">Platelet glycoprotein Ibalpha forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF.</a> The Journal of clinical investigation. 118, 3195-3207 (2008).</li><li>Chesla, S. E., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+two-dimensional+receptor-ligand+binding+kinetics+by+micropipette.">Measuring two-dimensional receptor-ligand binding kinetics by micropipette.</a> Biophysical journal. 75, 1553-1572 (1998).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetics+of+two-dimensional+TCR+and+pMHC+interactions+determine+T-cell+responsiveness.">The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness.</a> Nature. 464, 932-936 (2010).</li><li>Heinrich, V., Wong, W. P., Halvorsen, K., Evans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+biomolecular+interactions+by+fast+three-dimensional+tracking+of+laser-confined+carrier+particles.">Imaging biomolecular interactions by fast three-dimensional tracking of laser-confined carrier particles.</a> Langmuir : the ACS journal of surfaces and colloids. 24, 1194-1203 (2008).</li><li>Chen, W., Evans, E. A., McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+receptor-ligand+interactions+between+surfaces+by+thermal+fluctuations.">Monitoring receptor-ligand interactions between surfaces by thermal fluctuations.</a> Biophysical journal. 94, 694-701 (2008).</li><li>Evans, E., Ritchie, K., Merkel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+force+technique+to+probe+molecular+adhesion+and+structural+linkages+at+biological+interfaces.">Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.</a> Biophysical. 68, 2580-2587 (1995).</li><li>Evans, E., Leung, A., Heinrich, V., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+switching+and+coupling+between+two+dissociation+pathways+in+a+P-selectin+adhesion+bond.">Mechanical switching and coupling between two dissociation pathways in a P-selectin adhesion bond.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 11281-11286 (2004).</li><li>Ju, L., Dong, J. -. f., Cruz, M. A., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-terminal+Flanking+Region+of+the+A1+Domain+Regulates+the+Force-dependent+Binding+of+von+Willebrand+Factor+to+Platelet+Glycoprotein+Ib.">The N-terminal Flanking Region of the A1 Domain Regulates the Force-dependent Binding of von Willebrand Factor to Platelet Glycoprotein Ib.</a> Journal of Biological Chemistry. 288, (2013).</li><li>Chen, W., Lou, J., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forcing+switch+from+short-+to+intermediate-+and+long-lived+states+of+the+alphaA+domain+generates+LFA-1/ICAM-1+catch+bonds.">Forcing switch from short- to intermediate- and long-lived states of the alphaA domain generates LFA-1/ICAM-1 catch bonds.</a> The Journal of biological chemistry. 285, 35967-35978 (2010).</li><li>Chen, W., Lou, J., Evans, E. A., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+force-regulated+conformational+changes+and+ligand+dissociation+from+a+single+integrin+on+cells.">Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells.</a> The Journal of cell biology. 199, 497-512 (2012).</li><li>Judokusumo, E., Tabdanov, E., Kumari, S., Dustin, M. L., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensing+in+T+lymphocyte+activation.">Mechanosensing in T lymphocyte activation.</a> Biophysical journal. 102, L5-L7 (2012).</li><li>Bashour, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+and+CD3+have+complementary+roles+in+T-cell+traction+forces.">CD28 and CD3 have complementary roles in T-cell traction forces.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 2241-2246 (2014).</li><li>Nesbitt, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+glycoprotein+Ib/V/IX+and+integrin+alpha+IIbbeta+3-dependent+calcium+signals+cooperatively+regulate+platelet+adhesion+under+flow.">Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow.</a> The Journal of biological chemistry. 277, 2965-2972 (2002).</li><li>Mazzucato, M., Pradella, P., Cozzi, M. R., De Marco, L., Ruggeri, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+cytoplasmic+calcium+signals+in+a+2-stage+platelet+activation+process+induced+by+the+glycoprotein+Ibalpha+mechanoreceptor.">Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibalpha mechanoreceptor.</a> Blood. 100, 2793-2800 (2002).</li><li>Lefort, C. T., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+arrest+by+LFA-1+activation.">Neutrophil arrest by LFA-1 activation.</a> Frontiers in immunology. 3, 157 (2012).</li><li>Liu, B., Chen, W., Evavold, B. D., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+dynamic+catch+bonds+between+TCR+and+agonist+peptide-MHC+triggers+T+cell+signaling.">Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling.</a> Cell. 157, 357-368 (2014).</li><li>Lou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-enhanced+adhesion+regulated+by+a+selectin+interdomain+hinge.">Flow-enhanced adhesion regulated by a selectin interdomain hinge.</a> The Journal of cell biology. 174, 1107-1117 (2006).</li><li>Fiore, V. F., Ju, L., Chen, Y., Zhu, C., Barker, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+catch+of+a+Thy-1-alpha5beta1+syndecan-4+trimolecular+complex.">Dynamic catch of a Thy-1-alpha5beta1+syndecan-4 trimolecular complex.</a> Nature communications. 5, 4886 (2014).</li><li>Chen, W., Zarnitsyna, V. I., Sarangapani, K. K., Huang, J., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Receptor-Ligand+Binding+Kinetics+on+Cell+Surfaces:+From+Adhesion+Frequency+to+Thermal+Fluctuation+Methods.">Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods.</a> Cellular and molecular bioengineering. 1, 276-288 (2008).</li><li>Marshall, B. T., Sarangapani, K. K., Lou, J., McEver, R. 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A successful fBFP experiment entails a few critical considerations. First, for the force calculation to be reliable, the micropipette, the RBC, and the probe bead should be aligned as close to coaxial as possible. The projection of the RBC inside the pipette should be about one probe pipette diameter so that the friction between the RBC and the pipette is negligible. For a typical human RBC, the optimal pipette diameter is 2.0-2.4 &#181;m, which yields a best fit of Equation 117,30. Second, to ensure measureme...

Cell-to-cell and cell-to-extracellular matrix (ECM) adhesion is mediated by binding between cell surface receptors, ECM proteins, and/or lipids1. Binding allows cells to form functional structures1, as well as recognize, communicate, and react to the environment1-3. Unlike soluble proteins (e.g., cytokines and growth factors) that bind from a three-dimensional (3D) fluid phase onto the cell surface receptors, cell adhesion receptors form bonds with their ligands across a narrow junctional gap to bridge two opposing surfaces that constrain molecular diffusion in a two dimensional (2D) interface4-7. In contrast to 3D kinetics that are commonly measured by traditional binding assays (e.g., surface plasmon resonance or SPR), 2D kinetics have to be quantified with specialized techniques such as atomic force microscopy (AFM)8-10, flow chamber11,12, micropipette13,14, optical tweezers15 and biomembrane force probe (BFP)16-21.
More than merely providing physical linkage for cellular cohesion, adhesion molecules are a major component of the signaling machinery for the cell to communicate with its surroundings. There has been increasing interest in understanding how ligand engagement of adhesion molecules initiates intracellular signaling and how the initial signal is transduced inside the cell. Intuitively, properties of receptor-ligand binding can impact the signals it induces. However, it is difficult to dissect mechanistic relationships between the extracellular interaction and intracellular signaling events using traditional ensemble of biochemical assays because of their many limitations, e.g., a poor temporal resolution and the complete lack of spatial resolution. Existing methods that allow both biophysical (2D receptor-ligand binding kinetics) and biochemical (signaling) observations on live cells include substrates of tunable rigidity22, elastomer pillar arrays23 and flow chamber/microfluidic devices incorporated with fluorescence capability24-26. However, readouts of signaling and receptor-ligand binding have to be obtained separately (most often by different methods), making it difficult to dissect temporal and spatial relations of bond characteristics with signaling events.
Conventional BFP is an ultrasensitive force spectroscopy with high spatiotemporal resolution17. It uses a flexible red blood cell (RBC) as a force sensor, enabling measurement of single-molecule 2D kinetics, mechanical properties and conformational changes14,16,19-21,27-29. A fluorescent imaging based BFP (fBFP) correlates the receptor-ligand binding kinetics with the binding-triggered cell signaling at single-molecule scale. With this setup, in situ cell signaling activities in the context of surface mechanical stimulation was observed in T-cells27. The fBFP is versatile and can be used for studies of cell adhesion and signaling mediated by other molecules in other cells.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Table 1: Reagents/Equipment</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</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>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>Phosphate 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 Bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>MAL-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5002-1</td>
			<td>Bead functionalization</td>
		</tr>
		<tr>
			<td>Biotin-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5026-1</td>
			<td>RBC biotinylation</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N6261</td>
			<td>RBC osmolarity adjustment</td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide (NH4OH)</td>
			<td>Sigma-Aldrich</td>
			<td>A-6899</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>BDH</td>
			<td>67-56-1</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide (H2O2)</td>
			<td>J. T. Barker</td>
			<td>Jan-86</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Acetic Acid (Glacial)</td>
			<td>Sigma-Aldrich</td>
			<td>ARK2183</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>3-MERCAPTOPROPYLTRIMETHOXYSILANE(MPTMS)</td>
			<td>Uct Specialties, llc</td>
			<td>4420-74-0</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Borosilicate Glass beads</td>
			<td>Distrilab Particle Technology</td>
			<td>9002</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Streptavidin&#8722;Maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>S9415</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A0336</td>
			<td>Ligand functionalizing</td>
		</tr>
		<tr>
			<td>Fura2-AM</td>
			<td>Life Technologies</td>
			<td>F-1201</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Quantibrite PE Beads</td>
			<td>BD Biosciences</td>
			<td>340495</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>BD LSR II</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Capillary Tube 0.7-1.0mm x 30&#34;</td>
			<td>Kimble Chase</td>
			<td>46485-1</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>sutter instrument</td>
			<td>P-97</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Pipette microforce</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Fisher Scientific</td>
			<td>BP2629-1</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-G</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micro-injector</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>BD&#160;</td>
			<td>309602</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Narishige</td>
			<td>HI-7</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Home-designed mechanical parts and adaptors fabrications using CNC machining.&#160;</td>
			<td>Biophysics Instrument</td>
			<td>All parts are customized according to the CAD designs.</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Microscope (TiE inverted)</td>
			<td>Nikon</td>
			<td>MEA53100</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Objective CFI Plan Fluor 40x (NA 0.75, WD 0.72mm, Spg)</td>
			<td>Nikon</td>
			<td>MRH00401</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Camera, GE680, 640x480, GigE, 1/3&#34; CCD, mono</td>
			<td>Graftek Imaging</td>
			<td>02-2020C</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Prosilica GC1290 - ICX445, 1/3&#34;, C-Mount, 1280x960, Mono., CCD, 12 Bit ADC</td>
			<td>Graftek Imaging</td>
			<td>02-2185A</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Manual submicron probehead with high resolution remote control</td>
			<td>Karl Suss</td>
			<td>PH400</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Anti-vibration table (5&#8217; x 3&#8217;)</td>
			<td>TMC</td>
			<td>77049089</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>3D manual translational stage</td>
			<td>Newport</td>
			<td>462-XYZ-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SolidWorks 3D CAD software</td>
			<td>SOLIDWORKS Corp.</td>
			<td>Version 2012 SP5</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>LabVIEW software</td>
			<td>National Instruments</td>
			<td>Version 2009</td>
			<td>BFP system, BFP program</td>
		</tr>
		<tr>
			<td>3D piezo translational stage</td>
			<td>Physik Instrumente</td>
			<td>M-105.3P</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Linear piezo accuator</td>
			<td>Physik Instrumente</td>
			<td>P-753.1CD</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Micromanager software</td>
			<td></td>
			<td>Version 1.4</td>
			<td>fBFP system, fluorescence imaging program</td>
		</tr>
		<tr>
			<td>Dual Cam (DC-2)</td>
			<td>Photometrics</td>
			<td>77054724</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Dual Cam emission filter (T565LPXR)</td>
			<td>Photometrics</td>
			<td>77054725</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Fluorescence Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA-R2 C10600-10B</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Plastic paraffin film (Parafilm)</td>
			<td>Bemis Company, Inc</td>
			<td>PM996</td>
			<td>bottle sealing</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Table 2: Buffer solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate/bicarbonate buffer (pH 8.5)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>8.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>10.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer (pH 6.5-6.8)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaPhosphate monobasic&#160;&#160;&#160;NaH2PO4&#8226;H2O</td>
			<td>27.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. NaPhosphate dibasic&#160;&#160; Na2HPO4</td>
			<td>28.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2-5% buffer (pH 7.2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>20.77g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>2.38g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>0.13g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>0.71g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>9.70g/L</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence biomembrane force probe: concurrent quantitation of receptor-ligand kinetics and binding-induced intracellular signaling on a single cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

The BFP technique was pioneered by the Evans laboratory in 1995 17. This picoforce tool has been extensively used to measure interactions of proteins immobilized on surfaces, so as to analyze two-dimensional kinetics of adhesion molecules interacting with their ligands16,19,20,30, to measure molecular elasticity21,29, and to determine protein conformational changes21. For an fBFP, an additional set of epi-fluorescence-related devices with the corresponding software system (<str...

Watch this Scientific Journal Video about Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell at JoVE

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

fluorescence-biomembrane-force-probe-concurrent-quantitation-receptor

Research

JoVE Journal

Bioengineering

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...