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Fig. 8. Enlargeosome exocytosis in DCs exposed to the tracers or ionomycin. Panels A-D show cell images obtained from DCs permeabilized after fixation. The enlargeosome marker, d/A (red), which appears in small cytoplasmic puncta of resting DCs (A), is redistributed to the cell surface after exposure to Bds, as shown in the same cells: (B) redistribution of d/A (red); (C) the Bds taken up in 10 minutes incubation (green); (D) the merged image, which reveals the negativity for d/A of the intracellular Bd-rich puncta and the lack of coincidence of the Bds and the d/A also at the cell surface. Panels E-F and H-M show merged, red-green images of cells that were not permeabilized before immunolabelling. In these preparations, d/A (red), an enlargeosome lumenal membrane protein, is labelled only after exocytosis, at the external surface of the plasma membrane. No surface d/A signal is appreciable in resting DCs (E), whereas after Bd application (green, 10 minutes) a strong surface d/A signal becomes appreciable (F). Panel G shows the time-course of the d/A surface labelling in DCs fixed from 1 to 20 minutes after application of Bds or Dex, revealed by FACS analysis and expressed as arbitrary units. Panel H shows that the surface redistribution of d/A induced by 10 minutes of exposure to Bds was not decreased by DC pretreatment with Vac1 (quantification in ten fields), whereas it was blocked by pretreatment with Wort (panel I) and preloading with BAPTA (panel J), Panels K-M show that the d/A surface redistribution induced by IONO (K) was unaffected by pretreatment of the cells with Wort (L) or Vac1 (M). Bar, 10 µm (D), also valid for A-C. Bar, 10 µm (F) and H-M.
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