Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Summary

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Abstract

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Introduction

Cell-cell adhesion is essential for the development and integrity of multicellular organisms and is mediated by a diverse array of cell surface molecules. Many of these adhesion molecules have been identified and characterized, though many remain to be discovered. Several methods are used to investigate the properties of cell adhesion molecules (CAMs), including cell sorting assays, cell aggregation assays^1-8 and biophysical methods, such as atomic force microscopy and surface force spectroscopy^9-12.

The complexity of even simplified in vitro systems using cell lines makes it difficult to determine the adhesive properties of a putative CAM. Typically, a molecule is considered a CAM if it induces cell aggregation when transfected into a non-adhesive cell line. However, it is clear that this is not direct evidence of adhesive activity. For instance, facilitating the cell surface delivery or stability of a CAM would also result in increased cell aggregation^1,13. Moreover, a true CAM may fail to mediate cell aggregation if the cell line lacks other co-factors required for cell surface delivery or stabilization.

To avoid these complicating factors, more direct assays can be employed that are based on the idea that adhesive interactions should be an intrinsic biochemical property of the extracellular domain. While beads were initially used to characterize Ng-CAM², these assays have been extended in order to investigate cadherin-mediated adhesion^12,14,15. Using fusion of the C-cadherin ectodomain-Fc fusions, the Gumbiner lab showed that multiple cadherin repeats contribute to homophilic interactions¹⁴. Using comparable bead aggregation assays, E-cadherin and N-cadherin adhesion have also been characterized^12,15, as have a number of protocadherins^1,15-18 and Dscam isoforms from Drosophila¹⁹. Here we describe a relatively simple and rapid assay for characterizing the adhesive activity of secreted, epitope-tagged ectodomains of putative homophilic CAMs (Figure 1). We have used this assay primarily to characterize members of the cadherin superfamily.

Protocol

1. Cell Preparation (Day -2 to 0)

1. Split HEK293 cells 1:5 using 0.05% Trypsin-EDTA solution and incubate in Growth Media at 37 °C with 5% CO₂ until 60 - 80% confluent (2 - 3 days). For each condition, culture cells in 2 x 100 mm dishes.

2. Cell Transfection (Day 1)

1. Transfect HEK293 cells with plasmid encoding Fc-fusion using a transfection reagent such as Lipofectamine. Alternative methods that result in comparable transfection efficiencies may also be used.
2. Return transfected cells to incubator for 24 hr.

3. Cell Propagation (Day 2)

1. Preheat Growth Media without Fetal Bovine Serum (-FBS) to 37 °C.
2. Rinse dishes 2x with 10 ml Growth Media -FBS, and return the dishes to the 37 °C incubator for 1 hr.
3. Rinse the cultures dishes one more time with 10 ml Growth Media -FBS, for a total of 3 washes.
4. Incubate the transfected HEK293 cells at 37 °C for another 48 hr without FBS before collecting media.

4. Bead Aggregation (Day 4)

1. To collect media from culture dishes, transfer media from each pair of dishes to 50 ml conical tubes and spin at 500 x g for 5 min to pellet cellular debris.
2. Filter media from 50 ml conical tube into a centrifugal filter using a 30 ml syringe and 0.45 µm syringe filter.
3. Spin centrifugal filters at 4,000 x g and 4 °C until volume of concentrated culture media is approximately 500 µl (approximately 15 min). Repeat until all culture media has been added and concentrated.
4. Add 1.5 µl of Protein G magnetic beads to 1 ml of ice cold Binding Buffer in a 1.5 ml microcentrifuge tube for each sample, place on a magnet and remove buffer. Immediately add the concentrated culture media to the Protein G magnetic beads.
5. Rotate tubes at 4 °C for 2 hr.
6. Place tubes on a magnet and remove media. Quickly wash beads twice with ice cold 1 ml Binding Buffer, and then resuspend beads in 300 µl Binding Buffer.
7. Split resuspended beads into 2 tubes, 150 µl into each tube, and then add 1.5 µl of 200 mM CaCl₂ or 200 mM EDTA for the “calcium” and “no calcium” conditions, respectively.
8. Transfer 100 µl from each condition to a depression well slide and collect micrographs using a transmitted light microscope (Figure 2). Collect images from 5 fields of view for each experiment at each desired time point.

5. Data Analysis

1. Using ImageJ, or comparable image analysis software, open one of the five image datasets using the Import/Image Sequence… from the File pulldown menu. These will be opened as an image stack.
2. In the Image/Properties… dialog box, change the image units to pixels and set Pixel Width and Pixel Height to 1.0.
3. Convert the images to binary using the Adjust/Threshold… command in the Image pulldown menu. Set the threshold to include pixels that contribute to beads or bead aggregates, but that exclude background and small particles. Apply to all images in the stack.
4. In the Set Measurements dialog box, check the Area and Stack Position boxes.
5. Run Analyze Particles… in the Analyze pulldown menu. This will generate a list of identified particles, including their size (area) and the image in which they were identified.
6. Repeat this process for which experiment and experimental condition.

Results

An example experiment is presented in Figure 2, which shows calcium-dependent bead aggregation by the ectodomain of N-cadherin fused to Fc (NcadEC-Fc). In the absence of calcium, beads exhibit little or no tendency to aggregate and there is no increase in aggregate size with time (Figure 1A,C). In the presence of calcium, beads coated with NcadEC-Fc show robust aggregation, with aggregate size increasing over time (Figure 1B,C). This experiment was repeated three times, ...

Discussion

Cell adhesion is an essential feature of multicellular life and is mediated by a broad array of cell surface proteins. Of these, the detailed adhesive properties are understood for only a relatively small proportion. Here, we have described a simple, rapid protocol for investigating the homophilic adhesive capacity of secreted ectodomains fused to a convenient epitope tag. This approach has a number of important advantages. First, stable cell lines are not required^14,20, as sufficient quantities of protein can...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pFc-N1 plasmid	Addgene	To be submitted
HEK293 cells	American Type Culture Collection	CRL-1573
DMEM	Mediatech - Corning	10-013-CV
Fetal Bovine Serum (FBS)	Sigma	F2442
100x Pen-Strep (10,000 U/ml)	Life Technologies	15140-122
0.05% Trypsin-EDTA	Life Technologies	25300054
Lipofectamine 2000	Life Technologies	11668030
Dynabeads – Protein G	Life Technologies	10003D
Bovine Serum Albumin	Sigma	A3294
Depression slides	Electron Microscopy Sciences	71878-06
Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane	Millipore	UFC901024
0.45 mm syringe filter	Sarstedt	83.1826
Dynamag-2 Magnet	Life Technologies	12321D
Fiji (ImageJ) Image Analysis Software	http://fiji.sc/Fiji
Table of Buffers/Solutions
Growth Media	DMEM + 10% FBS + Pen-Strep	Filter sterilize and store at 4 °C.
Growth Media –FBS	DMEM + Pen-Strep	Filter sterilize and store at 4 °C.
Binding Buffer	50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA	Vortex until dissolved, keep on ice.