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

<div><p>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.</p></div

IgG surrogate antigens tethered by H12-D-domain enhance the accumulation of BCR and pSyk into B cell immunological synapse than the ones tethered by streptavidin.

<p>(A) Statistical quantification for the mean fluorescence intensity (mFI) of biotin and Alexa 568-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes by either H12-D-domain (red color) or streptavidin (blue color). Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (B) Statistical quantification for the accumulation of human IgM-BCRs into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of IgM-BCRs by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Statistical quantification for the accumulation of pSyk signaling molecules into the immunological synapse as measured by the mFI of BCR within the immunological synapse from Ramos human B cells that were placed on PLB membranes presenting the same amount of IgG surrogate antigen as shown in A that were tethered by either H12-D-domain or streptavidin. Each dot represents a single measurement for the mFI of pSyk by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.</p

H12-D-domain construct efficiently tethers the IgG surrogate antigens on PLB membranes and these tethered IgG surrogate antigens induce the formation of BCR and surrogate antigen microclusters.

<p>(A) Shown are representative TIRFM images of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes through H12-D-domain. The Alexa 647-conjugated goat IgG anti human IgM surrogate molecules were pre-incubated (100 or 0 nM) with the PLB membranes containing H12-D-domain. Bar is 1.5 µm. (B) Statistical quantification for the mean fluorescence intensity (mFI) of Alexa 647-conjugated goat IgG anti human IgM surrogate antigens tethered on the surface of PLB membranes. Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Shown are representative two-color TIRFM images of BCR (green) or the surrogate antigen (red) microclusters within the contact interface of human Ramos B cell with the PLB membranes tethering the Alexa 647-conjugated goat IgG anti human IgM surrogate antigens. Also shown are the merged images. Bar is 1.5 µm. (D) Statistical quantification for the mFI of BCR microclusters (top panel) or surrogate antigen microclusters (lower panel) within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.</p

The IgG surrogate antigens tethered on PLB membranes induce the recruitment of pSyk into the B cell immunological synapse and upregulate the surface activation marker CD69 on mice primary B cells.

<p>(A) Shown are representative two-color TIRFM images of BCR (green) or pSyk (red) microclusters within the immunological synapse of human Ramos B cells placed on PLB membranes tethering goat IgG anti-human IgM surrogate antigen (top panel) or purified control IgG from goat sera (lower panel) for 10 min. Also shown are the merged images. Bar is 1.5 µm. (B) Statistical quantification for the mFI of pSyk microclusters within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Flow cytometry analysis for B cell surface activation marker CD69 from mouse primary B cell after a 24-hour incubation on PLB membranes tethering IgG surrogate antigens or the control membranes.</p

Exploring binding sites of anti-MPER.

<p>(A) Schematic representation of peptides used for scanning the binding sites of anti-MPER. (B) Results of anti-MPER binding to peptides in ELISA. MAbs 2F5 and 4E10 were used as control. The concentration of each antibody was 0.2 µg/ml.</p

H12-D-domain construct efficiently tethers the IgG surrogate antigens on PLB membranes in a clear-cut dose dependent manner.

<p>(A) Shown are representative TIRFM images of FITC-conjugated mouse IgG anti-chicken IgM surrogate antigens tethered on the surface of PLB membranes through H12-D-domain construct. The FITC-conjugated mouse IgG anti-chicken IgM surrogate molecules were pre-incubated at different concentration (100, 50, 25 or 0 nM) with the PLB membranes containing H12-D-domain. Bar is 1.5 µm. (B) Statistical quantification for the mFI of FITC-conjugated mouse IgG anti-chicken IgM surrogate antigens tethered on the surface of PLB membranes. Each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons. (C) Shown are representative TIRFM images of IgG surrogate antigen microclusters within the contact interface of chicken DT40 B cell with the PLB membranes tethering FITC-conjugated mouse IgG anti-chicken IgM surrogate antigens. Bar is 1.5 µm. (D) Statistical quantification for the mFI of surrogate antigen microclusters within the B cell immunological synapse. Each dot shows one measurement from a single cell. Bars represent means ± SD. Two-tailed t tests were performed for statistical comparisons.</p

Schematic presentation of the amino acid sequences of H12-D-domain and its linker function of tethering IgG surrogate antigens on Ni²⁺-containing PLB membranes.

<p>(A) All the domains within the protein A molecule from <i>Staphylococcus aureus </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063735#pone.0063735-Forsgren1" target="_blank">[36]</a>. One protein A molecule has five 56 to 61 aa residue Ig binding domains (domain A, B, C, D and E respectively). The amino acid sequences of D domain of Protein A are given. Also given are the amino acid sequences of the modified version of H12-D-domain used in the new method. H12 stands for HHHHHHHHHHHH polyhistidine tag. To eliminate the Fab VH-3 region binding capability of D domain, we chose to mutant the DD (aa36–37) to AA (aa36–37) in the H12-D-domain construct. (B) A cartoon to present the function of H12-D-domain construct to link the intact whole IgG surrogate antigen molecule on the surface of Ni²⁺-containing PLB membranes on supported glass coverslips.</p

H12-D-domain mediated PLB membranes only tether whole IgG, but not Fab, F(ab’)₂ or IgM molecules.

<p>(A) Shown are representative TIRFM images of Alexa 568-conjugated goat whole IgG, F(ab’)₂ or Fab fragments of goat IgG molecules, or the biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. Bar is 1.5 µm. (B) Statistical quantification for the mFI of Alexa 568-conjugated goat whole IgG, F(ab’)₂ or Fab fragments of goat IgG molecules tethered on the surface of PLB membranes with pre-attached H12-D-domain. (C) Statistical quantification for the mFI of biotin-conjugated mouse IgM molecules tethered on the surface of PLB membranes with or without pre-attached H12-D-domain. The PLB membranes tethering the biotin-conjugated mouse IgM molecules were further incubated with the Alexa 568-conjugated streptavidin for TIRFM imaging. In both B and C, each dot represents a single measurement for the mFI of the tethered IgG surrogate antigens by Image J software. Bars represent means±SD. Two-tailed t tests were performed for statistical comparisons.</p

Neutralizing activity of anti-MPER antibodies against Env-mediated syncytium-formation and infection by HIV-1 pseudoviruses, laboratory-adapted and primary strains.

<p>Neutralizing activity of anti-MPER antibodies against Env-mediated syncytium-formation and infection by HIV-1 pseudoviruses, laboratory-adapted and primary strains.</p

Immunogen design and protein purification.

<p>(A) Schematic representation of gp41 and NCMs. The gp41 molecule consists of fusion peptide (FP), N-terminal heptad repeat (NHR), loop (immunodominant) region, C-terminal heptad repeat (CHR), membrane-proximal external region (MPER), and cytoplasm tail (CT). (B) The amino acid sequences of NCMs. The sequence of N36 and C34 are in brown and blue, respectively. The epitopes for 2F5 (ELDKWA) and 4E10 (NWFDIT) are highlighted in red and green, respectively. The mutations of T569A and I675V are in boldface. (C) NCMs may form 6HB with exposed MPER tails. The epitopes for 2F5 and 4E10 are in red and green, respectively. (D) SDS-PAGE analysis of purified NCMs. The estimated molecular weight of NCMs is 11.8 kD. (E) Chemical cross-linking assay of NCM. NCM(IV), NCM(TA) and NCM(TAIV) showed similar results (data not shown).</p