Name: Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay (Video) | JoVE Journal
Uploaded: 2021-09-27T15:00:00.000+00:00
Duration: 8 min 24 s
Description: Learn the adhesion footprint assay to image molecular adhesion in cell rolling using TIRF microscopy. This protocol enables quantification of physiological molecular forces and spatial mapping of individual adhesion events.

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5 minutes

This work was supported by the Canada Foundation of Innovation (CFI 35492), Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2017-04407), New Frontiers in Research Fund (NFRFE-2018-00969), Michael Smith Foundation for Health Research (SCH-2020-0559), and the University of British Columbia Eminence Fund.

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The authors declare no conflict of interest.

The adhesion footprint assay enables visualization of the molecular adhesion events between PSGL-1 and P-selectin during cell rolling adhesion. This process is initiated by P-selectin-mediated capturing followed by rolling under fluidic shear stress. Potential issues during the experiment usually involve poor cell rolling or missing fluorescent tracks even when cells roll well. These problems are often resulting from quality controls at the critical steps in the protocol, as listed in the troubleshooting table (T...

The rolling adhesion cascade describes how circulating cells tether to and roll along the blood vessel wall1. Passive rolling is primarily mediated by selectins, a major class of cellular adhesion molecules (CAMs)1. Under the shear flow of blood, leukocytes expressing P-selectin glycoprotein ligand-1 (PSGL-1) form highly transient bonds with P-selectin, which may be expressed on the surface of inflamed endothelial cells. This process is critical for leukocytes to migrate to a site of inflammation2. In addition, PSGL-1 is also a mechanosensitive receptor capable of triggering the subsequent firm adhesion stage of the rolling adhesion cascade upon its engagement with P-selectin3.
Genetic mutations affecting CAM function can severely affect the immune system, such as in the rare disease of leukocyte adhesion deficiency (LAD), where malfunction of adhesion molecules mediating rolling leads to severely immunocompromised individuals4,5,6. In addition, circulating tumor cells have been shown to migrate following a similar rolling process, leading to metastasis7,8. However, because cell rolling is fast and dynamic, conventional experimental mechanobiology methods are unsuitable for studying molecular interactions during cell rolling. While single-cell and single-molecule manipulation methods like atomic force microscopy and optical tweezer were able to study molecular interactions such as P-selectin&#39;s force-dependent interaction with PSGL-1 at the single-molecule level9, they are unsuitable for investigating live adhesion events during cell rolling. Additionally, the interaction characterized in vitro cannot directly answer the question about molecular adhesion in vivo. For instance, what molecular tension range is biologically relevant when cells are functioning in their native environment? Computational methods such as adhesive dynamics simulation10 or simple steady-state model11 have captured certain molecular details and how they influence the rolling behavior but are highly dependent on the accuracy of the modeling parameters and assumptions. Other techniques such as traction force microscopy can detect forces during cell migration but do not provide sufficient spatial resolution or quantitative information on molecular tension. None of these techniques can provide direct experimental observations of the temporal dynamics, spatial distribution, and magnitude heterogeneity of molecular forces, which directly relate to cell function and behavior in their native environment.
Therefore, implementing a molecular force sensor capable of accurately measuring selectin-mediated interactions is crucial to improving our understanding of rolling adhesion. Here, we describe the protocol for the adhesion footprint assay12 where PSGL-1 coated beads are rolled on a surface presenting p-selectin functionalized tension gauge tethers (TGTs)13. These TGTs are irreversible DNA-based force sensors that result in a permanent history of rupture events in the form of fluorescence readout. This is achieved through the rupturing of the TGT (dsDNA) and then subsequent labeling of the ruptured TGT (ssDNA) with a fluorescently labeled complementary strand. One major advantage of this system is its compatibility with both diffraction-limited and super-resolution imaging. The fluorescently labeled complementary strand can either be permanently bound (&#62;12 bp) for diffraction-limited imaging or transiently bound (7-9 bp) for super-resolution imaging through DNA PAINT. This is an ideal system to study rolling adhesion as the TGTs are ruptured during active rolling, but the fluorescence readout is analyzed post-rolling. The two imaging methods also provide the user with more freedom to investigate rolling adhesion. Typically, diffraction-limited imaging is useful for extracting molecular rupture force through fluorescence intensity13, whereas super-resolution imaging allows for quantitative analysis of receptor density. With the ability to investigate these properties of rolling adhesion, this approach provides a promising platform for understanding the force-regulation mechanism on the molecular adhesion of rolling cells under shear flow.

<table><tbody><tr><td>4-channel drill guide</td><td>Custom made</td><td></td><td>3D printed with ABS filament</td></tr><tr><td>4-holes slide</td><td>Custom made</td><td></td><td>Drill clean microscope slide using a Dremel with diamond coated drill bits on a 4-channels drill guide which has a layout that matches with the centers of the 8-32 threaded holes on the aluminum clamp.</td></tr><tr><td>Acetone</td><td>VWR</td><td>BDH1101-4LP</td><td></td></tr><tr><td>Acrylic spacer</td><td>Custom made</td><td></td><td>Cut two blocks of acrylic sheets with the dimension of 40 mm x 30 mm x 2.5 mm. On each block, drill two 3 mm holes that are precisely aligned with the 4-40 holes on the aluminium holder.</td></tr><tr><td>Aluminium chip holder</td><td>Custom made</td><td></td><td>Machine anodized aluminium block into a C-shaped holder with the outer dimension of 640 mm x 500 mm x 65 mm and the opening dimension of 400 mm x 380 mm x 65 mm. Inlets and outlets are tapped with 8-32 thread.</td></tr><tr><td>Aminosilane</td><td>AlfaAesar</td><td>L14043</td><td>CAS 1760-24-3</td></tr><tr><td>Antibiotic/antimycotic solution</td><td>Cytiva HyClone</td><td>SV3007901</td><td>Pen/Strep/Fungiezone</td></tr><tr><td>Beads, ProtG coated polystyrene</td><td>Spherotech</td><td>PGP-60-5</td><td></td></tr><tr><td>Bovine serum albumin</td><td>VWR</td><td>332</td><td></td></tr><tr><td>Buffer, DNA PAINT</td><td></td><td></td><td>0.05% Tween-20, 5 mM Tris, 75 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM EDTA</td></tr><tr><td>Buffer, T50M5</td><td></td><td></td><td>10mM Tris, 50 mM NaCl, 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Buffer, Rolling</td><td></td><td></td><td>HBSS with 2mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 10 mM Hepes, 0.1% BSA</td></tr><tr><td>Buffer, Wash</td><td></td><td></td><td>10 mM Tris, 50 mM NaCl, 5 mM MgCl&lt;sub&gt;2&lt;/sub&gt; and 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.05% Tween 20</td></tr><tr><td>Calcium chloride</td><td>VWR</td><td>BDH9224</td><td></td></tr><tr><td>Cell culture flasks</td><td>VWR</td><td>10062-868</td><td></td></tr><tr><td>Concentrated sulfuric acid</td><td>VWR</td><td>BDH3072-2.5LG</td><td>95-98%</td></tr><tr><td>Coverslip holding tweezers</td><td>Techni-Tool</td><td>758TW150</td><td></td></tr><tr><td>Diamond-coated drill bits</td><td>Abrasive technology</td><td>C5250510</td><td>0.75 mm diamond drill</td></tr><tr><td>DNA, amine-ssDNA (top strand)</td><td>IDT DNA</td><td>Custom oligo</td><td>CCGGGCGACGCAGGAGGG /3AmMO/</td></tr><tr><td>DNA, biotin-ssDNA (bottom strand)</td><td>IDT DNA</td><td>Custom oligo</td><td>/5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG</td></tr><tr><td>DNA, imager strand for DNA-PAINT</td><td>IDT DNA</td><td>Custom oligo</td><td>GAGGGAAA TT/3Cy3Sp/</td></tr><tr><td>DNA, imager strand for permanent labelling</td><td>IDT DNA</td><td>Custom oligo</td><td>CCGGGCGACGCAGG /3Cy3Sp/</td></tr><tr><td>Double-sided tape</td><td>Scotch</td><td>237</td><td>3/4 inch width, permanent double-sided tape</td></tr><tr><td>EDTA</td><td>Thermofisher</td><td>15575020</td><td>0.5 M EDTA, pH 8.0</td></tr><tr><td>Epoxy</td><td>Gorilla</td><td>42001</td><td>5 minute curing time</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Avantor</td><td>97068-085</td><td></td></tr><tr><td>GelGreen</td><td>Biotium</td><td>41005</td><td></td></tr><tr><td>Glacial acetic acid</td><td>VWR</td><td>BDH3094-2.5LG</td><td></td></tr><tr><td>Glass, Coverslips</td><td>Fisher Scientific</td><td>12-548-5P</td><td></td></tr><tr><td>Glass, Microscope slide</td><td>VWR</td><td>48300-026</td><td>75 mm x 25 mm x 1 mm</td></tr><tr><td>Glass, Staining jar</td><td>VWR</td><td>74830-150</td><td>Wheaton Staining Jar (900620)</td></tr><tr><td>Hanks&#039; Balanced Salt solution (HBSS)</td><td>Lonza</td><td>04-315Q</td><td></td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629-1EA</td><td></td></tr><tr><td>HL-60 cells</td><td>ATCC</td><td>CCL-240</td><td></td></tr><tr><td>Humidity chamber slide support</td><td>Custom made</td><td></td><td>3D printed with ABS filament</td></tr><tr><td>Hydrogen peroxide</td><td>VWR</td><td>BDH7690-1</td><td>30%</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Inlets/outlets</td><td>Custom made</td><td></td><td>Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm&amp;nbsp; polyethylene tubing (Tygon, I.D. 1/32&amp;rdquo; O.D. 3/32&amp;rdquo;) into each hollow setscrew</td></tr><tr><td>Iscove Modified Dulbecco Media (IMDM)</td><td>Lonza</td><td>12-722F</td><td></td></tr><tr><td>Magnesium chloride</td><td>VWR</td><td>BDH9244</td><td></td></tr><tr><td>Magnetic Ni-NTA beads</td><td>Invitrogen</td><td>10103D</td><td></td></tr><tr><td>Mailer tubes</td><td>EMS</td><td>EMS71406-10</td><td></td></tr><tr><td>Methanol</td><td>VWR</td><td>BDH1135-4LP</td><td></td></tr><tr><td>Micro Bio-Spin P-6 Gel Columns</td><td>Biorad</td><td>7326200</td><td>In SSC Buffer</td></tr><tr><td>PEG</td><td>Laysan Bio</td><td>MPEG-SVA-5000</td><td></td></tr><tr><td>PEG-biotin</td><td>Laysan Bio</td><td>Biotin-PEG-SVA-5000</td><td></td></tr><tr><td>Potassium hydroxide</td><td>VWR</td><td>470302-132</td><td></td></tr><tr><td>Protein, Protein G</td><td>Abcam</td><td>ab155724</td><td>N-terminal His-Tag and C-terminal cysteine</td></tr><tr><td>Protein, P-selectin-Fc</td><td>R&amp;amp;D System</td><td>137-PS</td><td>Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF</td></tr><tr><td>Protein, Streptavidin</td><td>Cedarlane</td><td>CL1005-01-5MG</td><td></td></tr><tr><td>Pump Syringe</td><td>Harvard Apparatus</td><td>704801</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Ward&amp;rsquo;s Science</td><td>470302-444</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>97061-274</td><td></td></tr><tr><td>Sulfo-SMCC</td><td>Thermofisher</td><td>22322</td><td></td></tr><tr><td>Syringe</td><td>Hamilton</td><td>81520</td><td>Syringes with PTFE luer lock, 5 mL</td></tr><tr><td>Syringe needles</td><td>BD</td><td>305115</td><td>Precision Glide 26 G, 5/8 Inch Length</td></tr><tr><td>TCEP</td><td>Sigma-Aldrich</td><td>C4706-2G</td><td></td></tr><tr><td>Tris</td><td>VWR</td><td>BDH4502-500GP</td><td></td></tr><tr><td>Tubing, Adaptor</td><td>Tygon</td><td>ABW00001</td><td>Formulation 3350, I.D. 1/32&amp;rdquo;; O.D. 3/32&amp;rdquo;</td></tr><tr><td>Tubing, Polyethylene</td><td>BD Intramedic</td><td>427406</td><td>Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>93773</td><td></td></tr></tbody></table>

imaging molecular adhesion in cell rolling by adhesion footprint assay

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

The protocol above describes the experimental procedure of the adhesion footprint assay. The general experiment workflow is illustrated in Figure 1, from the flow chamber assembly (Figure 1A) to the surface functionalization (Figure 1B) and experiment and imaging steps (Figure 1C).
Figure 2 is a representative result for the ProtG-ssDNA bioconjug...

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

imaging-molecular-adhesion-in-cell-rolling-by-adhesion-footprint-assay

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Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

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Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

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Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

Learn the adhesion footprint assay to image molecular adhesion in cell rolling using TIRF microscopy. This protocol enables quantification of physiological molecular forces and spatial mapping of individual adhesion events.

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

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