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 adhesiv....

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. Prehe....

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.