Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Summary

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

Abstract

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.

Introduction

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 ad....

Protocol

1. Oligonucleotide labeling and hybridization

1. Reduction of Protein G disulfide bonds
   1. Dissolve 10 mg of Protein G (ProtG) in 1 mL of ultrapure water.
      NOTE: The Protein G here is modified with a single cysteine residue at the C-terminus and an N-terminus poly-histidine tag.
   2. Buffer exchange ≥20 µL of ProtG (10 mg/mL) into 1x PBS (pH 7.2) with a P6 column.
   3. Measure the protein concentration following buffer exchange.
      NOTE: Typical concentration of 7-8 mg/mL.
   4. Prepare 120 mM Tris(2-carboxyethyl)phosphine (TCEP) by dissolving 3 mg of TCEP into 90 µL of 1x PBS (pH 7.2) fol....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
4-channel drill guide	Custom made		3D printed with ABS filament
4-holes slide	Custom made		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.
Acetone	VWR	BDH1101-4LP
Acrylic spacer	Custom made		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.
Aluminium chip holder	Custom made		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.
Aminosilane	AlfaAesar	L14043	CAS 1760-24-3
Antibiotic/antimycotic solution	Cytiva HyClone	SV3007901	Pen/Strep/Fungiezone
Beads, ProtG coated polystyrene	Spherotech	PGP-60-5
Bovine serum albumin	VWR	332
Buffer, DNA PAINT			0.05% Tween-20, 5 mM Tris, 75 mM MgCl2, 1 mM EDTA
Buffer, T50M5			10mM Tris, 50 mM NaCl, 5 mM MgCl2
Buffer, Rolling			HBSS with 2mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 0.1% BSA
Buffer, Wash			10 mM Tris, 50 mM NaCl, 5 mM MgCl2 and 2 mM CaCl2, 0.05% Tween 20
Calcium chloride	VWR	BDH9224
Cell culture flasks	VWR	10062-868
Concentrated sulfuric acid	VWR	BDH3072-2.5LG	95-98%
Coverslip holding tweezers	Techni-Tool	758TW150
Diamond-coated drill bits	Abrasive technology	C5250510	0.75 mm diamond drill
DNA, amine-ssDNA (top strand)	IDT DNA	Custom oligo	CCGGGCGACGCAGGAGGG /3AmMO/
DNA, biotin-ssDNA (bottom strand)	IDT DNA	Custom oligo	/5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG
DNA, imager strand for DNA-PAINT	IDT DNA	Custom oligo	GAGGGAAA TT/3Cy3Sp/
DNA, imager strand for permanent labelling	IDT DNA	Custom oligo	CCGGGCGACGCAGG /3Cy3Sp/
Double-sided tape	Scotch	237	3/4 inch width, permanent double-sided tape
EDTA	Thermofisher	15575020	0.5 M EDTA, pH 8.0
Epoxy	Gorilla	42001	5 minute curing time
Fetal Bovine Serum (FBS)	Avantor	97068-085
GelGreen	Biotium	41005
Glacial acetic acid	VWR	BDH3094-2.5LG
Glass, Coverslips	Fisher Scientific	12-548-5P
Glass, Microscope slide	VWR	48300-026	75 mm x 25 mm x 1 mm
Glass, Staining jar	VWR	74830-150	Wheaton Staining Jar (900620)
Hanks' Balanced Salt solution (HBSS)	Lonza	04-315Q
Hemocytometer	Sigma-Aldrich	Z359629-1EA
HL-60 cells	ATCC	CCL-240
Humidity chamber slide support	Custom made		3D printed with ABS filament
Hydrogen peroxide	VWR	BDH7690-1	30%
Imidazole	Sigma-Aldrich	I2399
Inlets/outlets	Custom made		Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm  polyethylene tubing (Tygon, I.D. 1/32” O.D. 3/32”) into each hollow setscrew
Iscove Modified Dulbecco Media (IMDM)	Lonza	12-722F
Magnesium chloride	VWR	BDH9244
Magnetic Ni-NTA beads	Invitrogen	10103D
Mailer tubes	EMS	EMS71406-10
Methanol	VWR	BDH1135-4LP
Micro Bio-Spin P-6 Gel Columns	Biorad	7326200	In SSC Buffer
PEG	Laysan Bio	MPEG-SVA-5000
PEG-biotin	Laysan Bio	Biotin-PEG-SVA-5000
Potassium hydroxide	VWR	470302-132
Protein, Protein G	Abcam	ab155724	N-terminal His-Tag and C-terminal cysteine
Protein, P-selectin-Fc	R&D System	137-PS	Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF
Protein, Streptavidin	Cedarlane	CL1005-01-5MG
Pump Syringe	Harvard Apparatus	704801
Sodium bicarbonate	Ward’s Science	470302-444
Sodium chloride	VWR	97061-274
Sulfo-SMCC	Thermofisher	22322
Syringe	Hamilton	81520	Syringes with PTFE luer lock, 5 mL
Syringe needles	BD	305115	Precision Glide 26 G, 5/8 Inch Length
TCEP	Sigma-Aldrich	C4706-2G
Tris	VWR	BDH4502-500GP
Tubing, Adaptor	Tygon	ABW00001	Formulation 3350, I.D. 1/32”; O.D. 3/32”
Tubing, Polyethylene	BD Intramedic	427406	Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm
Tween-20	Sigma-Aldrich	93773