Effect of Hn33 on DrBoNT transcytosis across HBE cell monolayers.

<p>A constant amount of DrBoNT-800 (13.3 nM) alone, or varying concentrations of Hn33 (66.7–666.7 nM) were mixed before adding to the apical medium. SEB was added instead of Hn33 as a negative control. After incubating at 37°C, 5% CO₂, aliquots of basal medium were removed and scanned on the Odyssey imaging system. Results were the average of three inserts ± standard deviation. Significant differences (ρ<0.05) between BoNT alone and BoNT with Hn33 are noted (*).</p

Labeled DrBoNT internalization in the presence of labeled Hn33.

<p>Polarized HBE cell monolayers were incubated with DrBoNT-488 and Hn33-PacBlue for 1.5 h. Cells were fixed and then imaged. (a) Hn33-PacBlue (red); (b) DrBoNT-488 (green), and (c) Hn33-PacBlue and DrBoNT-488 merged. Arrows point to the punctations showing colocalization. (Manders coefficient = 0.6) Red bar = 2 μM.</p

Movement of DrBoNT and Hn33 across polarized HBE cell monolayers via transcytosis.

<p>Varying concentrations (A) DrBoNT-800 (6.6–266.7 nM) or (B) Hn33-680 (33–666.7 nM) were added to the apical medium on HBE cell monolayers. After 2, 4, and 8 h incubations (37°C, 5% CO₂), aliquots of the basal medium were scanned on an Odyssey imaging system. Labeled protein standards were used to convert density measurements to nM concentrations. Results were the average of three inserts ± standard deviation. Statistical analysis was performed using a Student’s t-test.</p

Confocal microscopic images showing effects of DrBoNT and Hn33 on HBE cell monolayer integrity.

<p>HBE cells were cultured on transwell inserts until polarized monolayers developed. DrBoNT-800 (100 nM) and Hn33-680 (600 nM) were added to the apical medium and incubated for 24 h. Cell monolayers were stained with an antibody that recognizes the tight junction protein ZO-1 (green). Nuclear DNA was stained with Hoechst 33342 (blue) and actin with Texas Red phalloidin (red). Cells were imaged using confocal microscopy. (A) Cell monolayers without DrBoNT and Hn33 (control); (B) cell monolayers treated with DrBoNT and Hn33; C and D: Gray-scale images of A and B respectively, showing tight junctions. White bar = 10 μM.</p

Comparison of cell binding of WGA-594, DrBoNT-488, DrBoNT with Hn33 and Hn33 alone.

<p>The mean fluorescence intensities of cells for each treatment were measured and the background subtracted. The results were the average of five cells ± standard deviation. Statistical analysis using an unpaired, two-tailed Student’s t-test showed that binding of DrBoNT alone was significantly different from that of WGA, DrBoNT in the presence of Hn33, and Hn33 alone (ρ<0.05). Statistical analysis using a one way ANOVA did not show significant differences between the binding of WGA, DrBoNT in the presence of Hn33, and Hn33 alone (ρ>0.05).</p

BoNT/A complex internalized faster than BoNT/A in HT-29 intestinal epithelial cells.

<p>Cells grown on coverslips were incubated with 300 nM BoNT/A-488 (A) or BoNT/A complex-488 (B) for 30 min, washed with HBSS and labeled plasma membrane with WGA-594 (red), washed, fixed and mounted for confocal microscopy as described in methods. Images obtained using a 63X oil immersion objective. Z-stack images showed BoNT/A only on the cell surface at 30 min, while at the same time, BoNT/A complex showed internalization into the cell compartment. Optical slices were viewed from basal to apical side of the cells.</p

Effect of Hn33 on DrBoNT internalization.

<p>Polarized HBE cell monolayers were incubated with DrBoNT-488 (150 nM) in the absence or the presence of unlabeled Hn33 (700 nM) for 1.5 h and imaged using confocal microscope. Z-stacks of the cells were obtained using a 63X oil immersion objective with a 2X magnification. (a) shows the mid-section of control cells without the addition of any proteins; (b) shows the surface section of cells treated with DrBoNT-488 in the absence of Hn33; and (c) shows the mid-section of the cells treated with DrBoNT-488 in the presence of Hn33. DrBoNT-488 (green) is internalized in the presence of Hn33 via small vesicles as indicated by the white arrows (c), while it is localized to the surface of the cells in the absence of Hn33 (b). Cell membranes were labeled with WGA-594 (red).</p

Comparison of DrBoNT internalization in the absence or presence of Hn33.

<p>The mean fluorescence intensities of cells for each treatment were measured and the background subtracted. The results were the average of five cells ± standard deviation. Significant difference (ρ<0.05) was observed between DrBoNT with Hn33 and DrBoNT-488 alone.</p

Binding analysis of DrBoNT and Hn33 using surface plasmon resonance.

<p>The DrBoNT and Hn33 binding rate constant was determined using a Biacore T100 SPR. The SPR-derived steady-state binding of DrBoNT to Hn33 protein immobilized by amine coupling to a carboxymethylated dextran-coated CM3 sensor chip.</p

Detection of Hn33-680 in basal medium after Hn33-680 and unlabeled Hn33 were added to the apical medium.

<p>Detection of Hn33-680 in basal medium after Hn33-680 and unlabeled Hn33 were added to the apical medium.</p