Figure 3.

Regulation of CCL21 Secretion

(A) TIRF microscopy of CCL21-mCherry vesicles (white) of a single LEC after addition of 5 μM ionomycin. Yellow arrows indicate vesicles at time point 0″, secretion events are highlighted by white arrows. Scale bar, 2 μm. See Movie S2.

(B) Quantification of mCherry intensities of CCL21ΔC-mCherry expressing LEC culture supernatant subsequent to 6′ control or 5 μM ionomycin treatment. Bar graph shows mean ± SD of pooled samples from 3 independent experiments, n = 5 (control) and 6 (ionomycin).

(C) Quantification of mCherry intensities of culture supernatants of CCL21ΔC-mCherry expressing LECs 4 hr after washout of 1 hr long control or 10 μM BAPTA-AM treatment. BAPTA-AM is a cell-permeant Ca chelator, which is trapped in cells and binds intracellular calcium via carboxylic acid functional groups thus severely decreasing intracellular-free calcium. Bar graph shows mean ± SD of pooled samples from three independent experiments, n = 7 for each condition. Intensities of (B) and (C) are normalized to media background, which is set as 1.

(D) Quantification of pooled data from all TIRF experiments on single vesicle secretion. Data points show secretion frequency in control, 5 μM ionomycin-, or 500 ng/mL latrunculin B-treated CCL21-mCherry or CCL21ΔC-mCherry expressing single LECs as indicated in the figure. The dot blot graph shows mean ± SD secretion frequency in pooled data of single cells from 6 independent experiments, cell number (n) is indicated in the figure.

(E) Data points show CCL21-mCherry vesicle speed, displacement, and duration in the TIRF imaging plane (basolateral membrane) in control and 13 latrunculin B (500 ng/mL)-treated single LECs, which displayed highest CCL21 secretion (see Figure 3D latrunculin B column). The dot blot graph shows mean ± SD of vesicle tracks (51–513/cell) in pooled data of single cells (n = 12 for control and 13 for latrunculin B) of three independent experiments. See Movie S3.