ClyA anchors correctly folded GFP to the outer surface of E. coli and to the surface of s-MVs

To determine the topology of the ClyA-GFP and GFP-ClyA chimeras, we probed the surface accessibility of GFP on whole cells and on vesicles. Previous studies showed that a fraction of the secreted ClyA remains located on the bacterial cell surface.¹⁴ Likewise, we observed that both ClyA-GFP and GFP-ClyA were localized to the cell surface as evidenced by the accessibility of the GFP moiety to cross-reacting antibodies. Specifically, positive immunofluorescent- and immunogold-labeling using anti-GFP antibodies was detected for JC8031 cells expressing ClyA fused with GFP but not in the cases of unfused ClyA or unfused GFP (Fig. 3, shown for unfused GFP and ClyA-GFP).

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Figure 3

Microscopic analysis of ClyA expression. JC8031 cells grown at 37°C in LB were induced to express (a) GFP and (b) ClyA-GFP. For immunofluorescence microscopy, cells were treated with mouse monoclonal anti-GFP and subsequently with rhodamine-conjugated anti-mouse IgG. Panels show phase contrast microscopy and fluorescence microscopy using green and red emission filters as indicated. For immunoelectron microscopy, cells were treated with mouse monoclonal anti-GFP and subsequently with gold-conjugated anti-mouse IgG. Arrows indicate the 25 nm gold particles. The bars are equal to 500 nm.

To determine if the GFP associated with vesicles was similarly accessible to cross-reacting antibodies, immunofluorescent labeling of s-MVs was performed but only a weak immunofluorescent signal above background could be seen in this analysis (data not shown). This prompted us to explore a more sensitive and quantitative surface plasmon resonance (SPR)-based strategy for detecting vesicle-associated GFP. For this, biotinylated anti-E. coli antibodies (test channel) and bovine serum albumin (BSA, reference channel) were coupled to an SPR sensor chip through streptavidin binding. To verify that the SPR surface could capture our s-MVs, we introduced solutions containing varying amounts of intact s-MVs from fraction 7 above, where the initial concentration of s-MVs in this fraction was 13.5 ± 1.34 µg/µl. Following introduction of ClyA-GFP-containing s-MVs to the SPR, we immediately noticed that the test channel coated with anti-E. coli antibodies but not the BSA-coated reference channel was highly fluorescent (Fig. 4a), even after several PBS wash steps, indicating specific capture of our s-MVs on the SPR surface. SPR binding revealed concentration-dependent shifts in the SPR wavelength over a range of s-MV concentrations (0.02–0.70 µg/µl) (Fig. 4b and c). Importantly, there was no measurable change in SPR wavelength upon treatment of surface-captured s-MVs with PBS (data not shown), indicating that the fluorescent vesicles were stably and tightly bound to the immobilized anti-E. coli antibody. Finally, to determine whether our SPR strategy was suitable for detecting OMV-associated antigens, we introduced anti-GFP monoclonal antibodies into the test channel that contained surface-captured s-MVs displaying active GFP. Specific binding between anti-GFP antibodies and vesicle-associated GFP was confirmed by a marked increase in the SPR wavelength in the test channel (Fig. 4d, black line). Controls were performed where these s-MVs were treated with non-specific anti-His6× monoclonal antibodies or where s-MVs displaying unfused ClyA were captured on the SPR surface and treated with anti-GFP antibodies; both cases resulted in no detectable increase in the SPR wavelength (shown for anti-His6× treatment of Cly-GFP s-MVs in Fig. 4d, gray line).

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Figure 4

Detection of vesicles and vesicle-associated antigens via immuno-SPR. (a) Fluorescence microscopy analysis of the binding of fluorescent s-MVs (at a concentration of 0.35 µg/µl) to anti-E. coli antibody in the test channel and to the BSA-treated surface in the reference channel following 20 min of s-MV binding and 20 min PBS rinse. The measurements indicated represent the size of the PDMS master. (b) Overlaid SPR sensorgrams showing concentration-dependent binding of OMVs to immobilized anti-E. coli antibodies. For each binding experiment, 200 µl of OMV-containing samples (diluted to the concentrations indicated) was introduced to the test or reference channel for 20 min, followed by a 20 min PBS rinse. The SPR signal was recorded as wavelength shift (nm) versus time and plotted as a “sensorgram”. All binding experiments were performed at 25°C ± 1°C with a flowrate of 10 µl/min. Each vesicle sample was assayed in triplicate and the standard error was determined to be less than 5%. (c) Vesicle standard curve generated using the SPR immunosensor. The steady-state SPR signal change was calculated by subtracting the average SPR signal during the PBS wash step following OMV binding from the average SPR signal collected during the initial PBS wash step prior to OMV addition. The equation y = 0.92ln(x) + 3.63 with an R² value of 0.95 describes the fit of a straight line through the logarithm of the data and was determined using SigmaPlot. The results are the average of the values calculated for the SPR signal change from three independent binding measurements with error bars showing ± standard error. It is noteworthy that the lower detection limit for this system was determined to be 0.01 µg/µl (10% of the compensated SPR wavelength shift in the standard curve) and that for vesicle concentrations ≥0.18 µg/µl, SPR wavelength shifts >2.5 nm were recorded, which is ~10× greater than the baseline signal. (d) Representative sensorgrams for antibody binding to s-MV surface-displayed GFP in test and reference channels. Channels were prepared identically so that fluorescent s-MVs were captured in both channels. The change in SPR signal over time was measured following addition of 1 µg/µl anti-GFP (black line) or 1 µg/µl anti-his6× (gray line) monoclonal antibody to surface-captured s-MVs. Antibody binding proceeded for 20 min followed by a 10 min PBS rinse. Each antibody was assayed in triplicate and the standard error was determined to be less than 5%.

We next performed Proteinase K (PK) susceptibility assays on ClyA-GFP and GFP-ClyA vesicles to determine whether the immuno-accessible GFP was protected by the vesicle structure. When s-MVs derived from JC8031 cells expressing ClyA-GFP or GFP-ClyA were incubated with PK in the absence of membrane-disrupting detergent, vesicle-associated fluorescence was completely abolished (Fig. 5a, shown for ClyA-GFP) suggesting that the majority of the functional GFP was surface exposed and not protected by the vesicle structure. Consistent with this, Western blot analysis confirmed that nearly all of the s-MV-associated ClyA-GFP was degraded to a lower molecular weight anti-GFP or anti-ClyA cross-reacting species upon treatment with PK (Fig. 5b, lanes 1–3 and 7–9). Interestingly, we observed a significant amount of PK-resistant material following identical treatment of GFP-ClyA s-MVs (Fig. 5b, lanes 4–6 and 10–12) that persisted even after incubation with 2–5× higher PK concentrations for 2× longer durations (data not shown). Since these PK-treated s-MVs were non-fluorescent but contained a considerable portion of PK-resistant GFP-ClyA, we conclude that only a fraction of the fusion localizes with functional GFP tethered outside the OMV while the remainder adopts an inactive conformation that is protected by the vesicle structure. Possible reasons for this include a localization defect caused by the relatively high level of expression for GFP-ClyA compared to ClyA-GFP and/or the apparent instability of the fusion as evidenced by the multiple anti-ClyA cross-reacting bands seen in the absence of PK (Fig. 5b, lane 10). For both chimeras, complete proteolytic digestion of GFP by PK occurred when membranes were disrupted by the addition of 1% SDS (Fig. 5b). Control experiments using purified, soluble ClyA-GFP showed that the protein was PK-sensitive both in the presence or absence of the detergent (data not shown).

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Figure 5

Biochemical and genetic analysis of ClyA localization. Proteinase K susceptibility of OMV-tethered GFP as determined by: (a) fluorescence microscopy of vesicles generated from JC8031 cells expressing ClyA-GFP treated with Proteinase K and SDS as indicated; and (b) Western blot of vesicles generated from JC8031 cells expressing ClyA-GFP or GFP-ClyA. Blots were probed with mouse anti-GFP (left panels) or anti-ClyA serum (right panels). Molecular weight (MW) ladder is marked at left. An equivalent number of vesicles was used in all cases. (c) Western blot analysis of periplasmic and OMV fractions from JC8031 (dsbA+) and JC8031 dsbA::Kan cells (dsbA-) expressing either ClyA-GFP or ClyA-His6 as indicated. (d) Immunofluorescence (left and center panels) of wt JC8031 (dsbA+, top) and JC8031 dsbA::Kan cells (dsbA-, bottom) expressing pClyA-GFP or pClyA-His6 as indicated and fluorescence (right panel) of vesicles derived from the same cells as indicated. For immunofluorescence, cells were treated with either mouse monoclonal anti-GFP or anti-polyhistidine antibodies and subsequently with rhodamine-conjugated anti-mouse IgG.

Periplasmic disulfide bond-forming machinery is required for localization of ClyA and ClyA fusions in OMVs

In previous work, ClyA in the periplasm was shown to adopt a monomeric conformation owing to an intramolecular disulfide bond formed between the cysteine residues at positions 87 and 285 in the polypeptide.³⁶ The presence of the disulfide bond was sufficient to prevent oligomerization of ClyA and inactivate its native haemolytic activity. In agreement with this finding, Wai et al. reported that DsbA, the enzyme responsible for catalyzing the formation of disulfide bonds in periplasmic proteins, was absent from OMVs containing ClyA and that the absence of DsbA was necessary for ClyA to oligomerize into its haemolytic conformation.¹⁴ Contrary to the findings of Wai et al., we found that DsbA was co-localized in vesicles containing the ClyA fusions (see Fig. 1 above). Thus, we hypothesized that oxidized, monomeric ClyA might be favorable for efficient cell surface and vesicle localization of our fusions. To test our hypothesis, we compared expression of ClyA-GFP in strain JC8031 and an isogenic dsbA::Kan mutant derived from JC8031. Western blot analysis revealed that while both strains accumulated similar amounts of ClyA-GFP in the periplasm, only in cells with DsbA present was localization of ClyA-GFP observed (Fig. 5c). This was corroborated by a complete lack of fluorescence seen for OMVs derived from JC8031 dsbA::Kan cells expressing ClyA-GFP (Fig. 5d). Immunofluorescent staining of these same cells revealed that localization of ClyA-GFP to the bacterial cell surface was also dependent upon DsbA (Fig. 5d). To our surprise, DsbA-dependent vesicle localization was also observed for unfused ClyA suggesting that the periplasmic redox state is a critical factor in regulating the efficiency of protein localization into vesicles under the conditions tested here. Interestingly, we observed very little, to no, apparent cytotoxicity for naked OMVs from plasmid-free JC8031 cells, consistent with earlier studies,¹⁴ and also for s-MVs containing ClyA-His6, ClyA-GFP or GFP-ClyA (data not shown) suggesting that ClyA in our vesicles was not in its haemolytically-active, oligomeric conformation.

Engineered ClyA-GFP s-MVs are useful for visualizing vesicle interactions with eukaryotic cells

Previous studies have shown that vesicles derived from pathogenic E. coli or non-pathogenic E. coli strains can associate with eukaryotic cells.^{20; 33} Thus, we next investigated whether s-MVs functionalized with ClyA-GFP were suitable for tracking vesicle association with eukaryotic cells. Previous attempts at this focused on loading GFP into the lumen of vesicles following its transport into the periplasm by the twin-arginine translocation (Tat) pathway.³³ However, GFP-containing OMVs were only weakly fluorescent and could not be tracked in host cells by microscopy, likely due to the low yield of GFP transport to the periplasm via the Tat system. To determine if s-MVs engineered with ClyA-GFP were bright enough for tracking studies, we performed vesicle-host cell co-incubation assays. Punctate green staining was observed following a 30 min incubation of HeLa cells with ~150 µg vesicles purified ClyA-GFP s-MVs and the intensity of this staining increased as the incubation time between HeLa cells and ClyA-GFP s-MVs increased (Fig. 6a). These results imply that vesicles persisted on the cell surface or had fused directly to the target cell membrane. To corroborate this, HeLa cells that had been incubated with purified ClyA-GFP-containing s-MVs were stained with a fluorescent version of the cell surface marker wheat germ agglutinin (WGA) and then washed with PBS. Confocal microscopy revealed that ClyA-GFP OMVs co-localized with WGA on the exterior of the cell (data not shown). Next, we explored the fate of ClyA-GFP vesicles by examining whether the appearance of punctate fluorescence was temperature dependent, a hallmark of cellular internalization.^{20; 37} HeLa cells incubated with vesicles containing ClyA-GFP at 4°C exhibited very low levels of cell-associated fluorescence (compare Fig. 6b). However, when HeLa cells were incubated with ClyA-GFP s-MVs at 4°C for 3 h and then shifted to 37°C for 3 hr, strong cell fluorescence was observed (Fig. 6b), leaving open the possibility that some s-MVs may be internalized0 at 37°C. A key factor in endocytosis is ganglioside M1 (G_M1), which is a eukaryotic cell surface receptor for enterotoxins such as LT and cholera toxin (CT) and is required for endocytosis of LT-containing OMVs derived from non-pathogenic E. coli.²⁰ Therefore, we tested whether the observed fluorescence of HeLa cells incubated with ClyA-GFP s-MVs was G_M1-dependent. Indeed, fluorescence associated with HeLa cells was significantly decreased following incubation with purified ClyA-GFP s-MVs that had been pretreated with G_M1 (Fig. 6c). Moreover, incubation with G_M1-treated vesicles resulted in a small number of large fluorescent clusters and much less punctate green fluorescence than was observed for HeLa cells incubated with untreated ClyA-GFP OMVs (Fig. 6c). Thus, it appears that G_M1 cell surface receptors may play an important role in mediating interactions between HeLa cells and engineered vesicles. Finally, to assay the cytotoxic effect of vesicles on target cells, we analyzed how cultured HeLa cells were affected by equivalent amounts of different vesicle preparations. In general, vesicles containing ClyA-His6 or ClyA-GFP exhibited virtually no detectable cytotoxicity (Fig. 6d), consistent with a monomoric, DsbA+ conformation¹⁴ for both ClyA and ClyA-GFP in our vesicles.

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Figure 6

Interaction of ClyA-GFP vesicles with HeLa cells. (a) OMVs containing ClyA-GFP were incubated with HeLa cells at 37°C for 30 min or 3 h as indicated. Fixed cells were stained with 0.5 mg/mL ethidium bromide (EtBr, upper panels) and visualized by fluorescence microscopy. (b) Temperature dependence of OMV-HeLa cell interactions was examined by incubation of HeLa cells with GFP-ClyA OMVs at the temperatures indicated. An equivalent number of OMVs (~150 µg) was used in all cases. (c) Untreated (− G_M1) or pretreated (+ G_M1) OMVs from JC8031 cells expressing ClyA-GFP were incubated with HeLa cells at 37°C for 3 h. Fixed cells were stained with 0.5 mg/mL ethidium bromide (EtBr, upper panels) and visualized by fluorescence microscopy. An equivalent number of OMVs (~150 µg) was used in all cases. (d) Cytotoxicity of vesicles as measured using the MTS assay with HeLa cell cultures. % viability is reported as the viability of vesicle-treated HeLa cells normalized to the viability following treatment with PBS. HeLa cells were treated with vesicle solutions derived from plasmid-free JC8031 cells and JC8031 cells expressing ClyA-His6, ClyA-GFP (dark gray). An equivalent number of OMVs (~150 µg) was used in all cases. Each sample was assayed in triplicate with error bars showing ± standard error.

Cell culture

Human epithelial cervical carcinoma (HeLa) cells were obtained from the American Type Culture Collection (ATCC # CCL-2) and grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 10% NuSerum, and 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 95% air, 5% CO₂. For fluorescence microscopy experiments, cells were grown on 12-mm circular glass coverslips for two days prior to experimentation.

Subcellular fractionation

Cytoplasmic and periplasmic fractions from cells expressing fusion proteins were generated by the cold osmotic shock procedure⁵³ and the pellet remaining after removal of the soluble fraction was collected as the insoluble fraction.

Isolation of bacterial vesicles

Vesicles were isolated from late log-phase bacterial cultures grown aerobically at 37°C in LB broth (unless otherwise indicated) essentially as described previously.¹⁴ Briefly, bacterial cells were removed by centrifugation at 5,000×g for 15 min at 4°C and the cell-free supernatants were filtered through a 0.2 µm-pore-size vacuum filter. Vesicles were collected from the filtered supernatant by ultracentrifugation at 141,000×g for 2 h at 4 °C in a 28 Ti rotor (Beckman Instruments, Inc.) and the pellet containing OMVs was carefully removed and suspended in PBS (pH 7.0). Vesicle preparations were plated on LB agar to confirm complete removal of bacterial cells. Vesicles preparations were kept at −20°C.

Outer membrane vesicles fractionation

Separation of pelleted outer membrane vesicle samples was performed as described.⁸ Briefly, vesicles were isolated as described above but suspended in 50 mM HEPES (pH 6.8), adjusted to 45% Optiprep (Sigma) in 0.15 ml and transferred to the bottom of 12-ml ultracentrifugation tubes. Different Optiprep/HEPES layers were sequentially added as follows: 0.9 ml 35%, 0.9 ml 30%, 0.66 ml 25%, 0.66 ml 20%, 0.33 ml 15% and 0.33 ml 10%. Gradients were centrifuged (180,000×g, 180 min, 4°C). A total of 10 fractions of equal volumes were sequentially removed and analysed by SDS-PAGE.

Vesicle characterization

The amount of vesicles in purified cell-free supernatant was determined by measuring the total protein concentration or the dry mass of vesicles according to published protocols.⁵⁴ Particle size distribution and zeta potential of vesicle samples containing approximately 30 µg/mL total protein in 1 mL of PBS were measured in a Nanosizer Nano ZS instrument (Malvern Instruments) using standard protocols. Malvern Dispersion Technology Software was used for data acquisition and analysis, applying the general purpose algorithm for calculating size distributions and the Smoluchowski approximation for determining zeta potential.

Protein assays

Whole cells, OMVs and subcellular fractions were assayed for Bla, LacZ and OPH activity using nitrocefin (Sigma), ONPG (Sigma) and paraoxon (Sigma), respectively, according to standard spectrophotometric assays.^{55; 56; 57} Cells expressing anti-digoxin scFv were labeled with Dig-BODIPY and analyzed by flow cytometry as described.⁵⁸ Total protein concentration was assayed using the BCA Protein Assay kit (Pierce). Protease accessibility assays were performed as described but with Proteinase K.³³ Briefly, vesicles were treated at 37°C for 30 min in 20 mM Tris HCl (pH 8.0) with PK (0.1 mg/ml) in either the absence or presence of 1% SDS. In parallel control experiments, ClyA-GFP and GFP-ClyA that had been purified by IMAC were similarly treated with PK. Following the incubation, all samples were placed on ice and 1 mM PMSF was added to quench all proteolysis, and the samples were analysed by SDS-PAGE. Western blotting was performed as described by Chen et al.⁴⁸ using the following primary antibodies: anti-ClyA (kindly provided by Sun Nyunt Wai, Umeå University, Sweden), anti-GFP (Sigma), anti-GroEL (Sigma), anti-polyhistidine (Sigma), anti-OmpA and anti-DsbA (kindly provided by Jon Beckwith, Harvard Medical School). Membranes were developed on film using Immuno-Star™ HRP Substrate Kit (Bio-Rad).

Surface plasmon resonance (SPR)

The SPR configuration consisted of a sensor chip, an optical measuring unit, a flow cell and a syringe pump and was similar to that developed previously.^{59; 60} The SPR chip was SF10 glass with a thin layer (50 nm) of gold that was attached to a prism made of SF10 using index matching oil. Two microfluidic channels (reference and test) made of polydimethylsiloxane (PDMS) were placed on the SPR sensor chip and screw-clamped to seal the channels. Single-wavelength light was obtained by passing white light from a Xe-lamp (Oriel) through a monochromater (Oriel). Light with a bandwidth less than 1 nm passed through the polarizer, where only p-polarized light was transmitted. The reflected light intensity (RI) was measured with a CCD camera (Sony) that showed high sensitivity around 600 nm. When the incident angle of the beam was fixed to 60 degrees, treatment of the sensor chip with PBS resulted in an SPR wavelength of about 600 nm. The SPR wavelength was obtained at each pixel by fitting the RI versus wavelength data to a second-order polynomial equation and the resulting SPR wavelengths, covering a pre-defined region of interest, were averaged. Before each measurement, the RI of s-polarized light was recorded at each pixel for reference. The reliable detection limit of the SPR sensor was measured to be less than 0.2 nm.

Vesicle and vesicle antigen detection using SPR

An SPR chip for detection of vesicles and vesicle-associated antigens was performed as follows. First, an alkanethiol monolayer was self-assembled on the 50-nm gold layer of the sensor chip surface with a mixed solution (1:2 molar ratio) of 10 mM 11-mercaptoundecanoic acid (11-MUA) and 6-mercapto-1-hexanol (6-MCH) as described previously.^{61; 62} The hydroxyl-terminated self-assembled monolayer (SAM), which cannot be activated by N-hydroxysuccinimide (NHS) and N-ethyl-N’-(3-diethylaminopropyl) carbodiimide (EDC), was used as a spacer to construct the sensor surface. Second, following activation of the terminal carboxylic groups of the mixed SAM with a 1:1 mixture of 0.1 M NHS and 0.4 M EDC for 10 min, streptavidin (SA; 200 µg/ml; MP Biomedicals) in 10 mM sodium acetate buffer (pH 5.5) was injected and allowed to covalently couple for 15–20 min followed by PBS rinsing and blocking with 1.0 M ethanolamine hydrochloride (pH 8.5) for 10 min. This resulted in a large increase in the SPR wavelength (data not shown). Because of the close relationship that exists between electrostatic binding and covalent binding of SA with carboxylic terminated SAMs, this increase in SPR signal is a decent estimate of the extent of covalent binding of SA to the SAMs.⁶¹ Third, following a thorough PBS wash, biotinylated rabbit anti-E. coli antibody (140 µg/ml in PBS; Cortex Biochem) was injected over the SA surface for 20 min and unbound biotinylated anti-E. coli antibody was removed by washing with PBS for 10 min. This resulted in an exponential increase in the SPR wavelength that was characteristic of electrostatic binding between SA and biotin-conjugated proteins (data not shown). As control, bovine serum albumin (BSA; 140 µg/ml in sodium acetate buffer) was added in place of anti-E. coli antibody to the SA-coated reference channel of the SPR sensor chip and, as expected, no detectable SPR wavelength shift was observed following introduction of BSA or, subsequently, anti-E. coli antibody (data not shown).

Fluorescence microscopy

For immunofluorescence studies, E. coli cells that had been induced to express GFP or ClyA-GFP were washed three times in PBS, incubated at 4°C overnight with mouse anti-GFP (or anti-polyhistidine) diluted 1:500, pelleted, washed three times with PBS, incubated for 1 h with rhodamine-labeled goat anti-mouse IgG (Molecular Probes) diluted 1:100, pelleted and washed three more times with PBS. Finally, cells were examined by a Zeiss Axioskop 40 fluorescent microscope with Spotflex color digital camera and filter sets for GFP (485 nm for excitation and 505 nm for emission) and Rhodamine (540 nm for excitation and 600 nm for emission). For fluorescent studies of OMV interactions with eukaryotic cells, HeLa cells grown on glass coverslips were washed in OptiMEM (Life Technologies) without serum and then treated as described under Results. Following treatment, cells were fixed with 3.7% formalin in PBS, washed three times in PBS, permeabilized in PBS/0.1% Triton X-100, stained with 0.5 mg/mL ethidium bromide in PBS, and finally washed three times in PBS. Coverslips were mounted onto glass slides using Vectashield Hardset mounting medium (Vector Laboratories) prior to wide-field epifluorescence analysis. For WGA studies, non-permeabilized cells were incubated with 1 µg/ml Texas red WGA (Molecular Probes) for 1 h at 4°C. For G_M1 experiments, ~150 µg of vesicles were preincubated with 10 µg G_M1 (Sigma) for 30 min at 25°C.

Electron microscopy

Ultrastructural analysis of vesicles was performed by negative staining technique as described previously.⁶³ For immunogold labeling, a 10-µL suspension of induced E. coli cells was collected, washed and applied to 400-mesh Formvar- and carbon-coated copper grids (Electron Microscopy Sciences) and incubated for 1 h with anti-GFP diluted 1:500. Cells were washed with PBS, incubated for 1 h with 25 nm colloidal gold-conjugated goat anti-mouse IgG (Electron Microscopy Sciences) diluted 1:100, washed again, negatively stained with 0.25% phosphotungstic acid (PTA, Electron Microscopy Sciences) with 0.01% BSA in water and viewed using a FEI/Philips Morgagni transmission electron microscope.

We thank Kyung Hun Yoonand Michael Shuler (Cornell University) and Sang Hun Lee, Tai Hyun Park and Sung June Kim (Seoul National University) for their assistance with the SPR studies. We also thank Harris Bernstein, Wilfred Chen, George Georgiou, Koreaki Ito, Roland Lloubes, Virginia Miller, Sun Nyunt Wai and Joel Weiner for their generous gifts of strains, plasmids and antibodies. This material is based upon work supported by the Ladies Auxiliary to the Veterans of Foreign Wars Cancer Research Fellowship (to A.M.D.), a New York State Health Research Science Board (HRSB) Breast Cancer Research Fellowship (to A.M.D.), the National Institutes of Health under Grant R21 NIBIB EB005669 (to D.A.P. and M.P.D), and a NYSTAR James D. Watson Young Investigator Award (to M.P.D.).