JCS007260 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Confocal images of RBL cells stimulated by lipid patterns with DNP-cap-DPPE and DiI for 15 minutes at room temperature show almost all cells contacting the pattern have Alexa Fluor-488 IgE (left panel) concentrated on the ligand-containing features (right panel). Bar, 10 μm.

• Supplemental Figure S2 -

  Fig. S2. Confocal images of RBL cells stimulated by soluble antigen show lack of colocalization between Alexa Fluor-588 IgE (green) and Alexa Fluor-568 Annexin V (red). Cells were stimulated by DNP-BSA for 5 minutes at 37°C, then washed in cold PBS twice and incubated with Alexa 568-annexin V (1:40 dilution) at room temperature for 15 minutes in BSS.

• Supplemental Figure S3 -

  Fig. S3. Confocal images of RBL cells show colocalization between CD63-EGFP and Lysotracker Red (LS). Bar, 10 μm.

• Supplemental Figure S4 -

  Fig. S4. Lack of apparent dependence of fusion location on the timing of the fusion events. Location is quantified as the distance between the fusion site and the nearest patterned feature.

• Supplemental Figure S5 -

  Fig. S5. CTB-GM1 (green) and transferrin receptors (red) colocalize intracellularly (yellow) in the perinuclear endosomal compartment of RBL mast cells. For immunocytochemical labeling of transferrin receptors, cells in suspension were labeled with Alexa Fluor-488 CTB for 1.5 hours at 37°C, fixed with 4% paraformaldehyde in PBS for 15 minutes, then quenched in PBS with 10 mg/ml BSA, permeabilized with 0.1% Triton X-100 and incubated with 5 μg/ml anti-CD71 (OX26, PharMingen Inc, San Diego CA), followed by Alexa Fluor-568 labeled goat anti-mouse IgG1 (Molecular Probes, Eugene OR). Bar, 10 μm.

• Supplemental Figure S6 -

  Fig. S6. EGFP-Rab11 (green) and Alexa Fluor-555−CTB-GM1 (red) colocalize intracellularly (yellow) in the perinuclear endosomal compartment of RBL mast cells. Bar, 10 μm.

• Supplemental Figure S7 -

  Fig. S7. CD63-EGFP (green) in secretory lysosomes and Alexa Fluor-555−CTB-GM1 (red) localize to distinct regions within RBL mast cells. Bar, 10 μm.

• Supplemental Figure S8 -

  Fig. S8. Antibody bound to CTB shows concentration over lipid patterns, suggesting the CTB-GM1 labeled endosomes that are delivered to the plasma membrane undergo fusion at those targeted locations. For surface labeling of CTB by antibody, FITC-CTB (5 ?g/ml; Sigma) was added to cell aliquots for 1 hour at 37°C just prior to the experiment, then cells were washed twice and resuspended in BSS, incubated with the patterned bilayers for 10 minutes at 37°C, followed by 15 ?g/ml Alexa Fluor-488−anti-FITC IgG (Molecular Probes). Cells were then imaged by confocal fluorescence microscopy. Bar, 10 μm.

• Movie 1 -

  Movie 1. RBL cell with secretory lysosomes labeled by CD63-EGFP (green) and lysotracker (red) undergoes stimulated exocytosis as observed by confocal fluorescence microscopy. Movie captures events within 10 minutes after stimulation. Image sequences were obtained at 3 seconds per frame and displayed at eight frames per second. Length of the movie is 100 frames, i.e. 5 minutes.

• Movie 2 -

  Movie 2. RBL cell with secretory lysosomes labeled by CD63-EGFP undergoes stimulated exocytosis as observed by TIRFM. Boundaries of patterned antigen are shown in green. Inserted red squares indicate where exocytosis occurs just before these events. Movie captures events within 30 minutes after stimulation. Image sequences were obtained by TIRFM at a frame rate of 0.4 seconds per frame and displayed at 10 frames per second. Length of the movie is 100 frames, i.e. 40 seconds.

• Movie 3 -

  Movie 3. Three secretory vesicles labeled by CD63-EGFP fuse with the plasma membrane at the same location within a period of 40 seconds. Movie captures events within 30 minutes after stimulation. Boundaries of patterned antigen are shown in green. Image sequences were obtained by TIRFM at a frame rate of 0.2 seconds per frame and displayed at five frames per second. Length of the movie is 220 frames, i.e. 44 seconds.

• Movie 4 -

  Movie 4. Colchicine inhibits dynamic transport of secretory lysosomes labeled with CD63-EGFP (compare with Movie 2). Image sequences were obtained by TIRFM at a frame rate of 0.4 seconds per frame and displayed at 10 frames per second. Length of the movie is 150 frames, i.e. 1 minute.