A New and Robust Method of Tethering IgG Surrogate Antigens on Lipid Bilayer Membranes to Facilitate the TIRFM Based Live Cell and Single Molecule Imaging Experiments

Yinsheng Xu,Zhengpeng Wan,Wanli Liu,Xin Chen,Liling Xu,Shaosen Zhang,Joseph J Barchi,Xingwang Zhao,Yilin Fan

doi:10.1371/journal.pone.0063735

Abstract

Our understanding of cell-cell interactions has been significantly improved in the past years with the help of Total Internal Reflection Fluorescence Microscope (TIRFM) in combination with an antigen presenting system supported by planar lipid bilayer (PLB) membranes, which are used to mimic the extensive receptor and ligand interactions within cell-cell contact interface. In TIRFM experiments, it is a challenge to uniformly present ligand molecules in monomeric format on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of Ni2+-containing PLB membranes. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain) to tether IgG surrogate antigens on Ni2+-containing PLB membranes. We systematically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens tethered by H12-D-domain show better lateral mobility and are more uniformly distributed on PLB membranes than the ones tethered by streptavidin. Neither IgM molecules, nor Fab or F(ab’)2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust method to tether IgG surrogate antigens or other molecules fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.

Highlights

Cell-cell contact based information exchanges play key roles in maintaining the function of various types of organismal systems, for instance, the neurological system and the immunological system
We introduce an alternative method to tether IgG molecules or other recombinant proteins fused with an IgG Fc portion on the surface of planar lipid bilayer (PLB) membranes
Through the atomic level interaction analyses, the recent studies of Olaf and colleagues [38] showed that for each of these five Ig binding domains, the Glutamine (Q) 9 and 10 in H1 helical bundle are critical for the binding to Fc portion of IgG molecules, while the Aspartate (D) 36 and 37 in the linker region between H2 and H3 helical bundle are critical for the binding to the Fab VH3 region of both IgM and IgG molecules (Fig. 1A)

Summary

Introduction

Cell-cell contact based information exchanges play key roles in maintaining the function of various types of organismal systems, for instance, the neurological system and the immunological system. Within cell-cell contact interfaces, extensive receptor and ligand interactions are established. Neither clear is how these interactions initiate and modulate cross membrane signal transduction. These types of questions have been attracting the research interests of many laboratories. To visualize these receptor and ligand interactions within such cell-cell contact interface, a variety of advanced fluorescence microscope techniques have been applied to the related studies [1–4]. Within each hierarchy of these SMAC structures, distinct immune receptor and ligand interactions show up and play different roles in maintaining an appropriate extent of immune cell activations [9–11]

Objectives

Methods

Results

Conclusion