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Figurę (Chapitre 1) : IIypoxia (riggers furin relocalization lo the basal plasma membranę

HT- 1080-eGFPfur cells were seeded on collagen type IV-coated slides and culturcd in normoxic or hypoxic chambers for 4 h. (A) Micrographs of furin (eGFP), TGN (TGN46; red) and merged images. The associated graph shows the percentage of furin with TGN as described under Materials and Methods. (B) Micrographs of furin (eGFP), plasma membranes (Dii: red) and merged images (binary mask-overlay). The associated graph shows furin with the basal plasma membranę. The index was calculated as described under Materials and Methods. (C) Three-dimensional reconstruction of a normoxic or hypoxic celi showing furin relocalization to the plasma membranę. Large central images show the basal plasma membranę of the cells, inset squares represent the middle section of the cells and horizontal and vertical Z-axis are shown for each celi. Column, mean; bar, SE; BPM, basal plasma membranę; ** P = 0.003. *** P < 0.0001; scalę bars correspond to 5 pm.

To examine the influence of hypoxia on furin localization, furinGFP HT-1080 cells were exposed to standard (21 %) or Iow (1%) oxygen concentration. Furin localization was observed and quantitated by confocal microscopy. In contrast to furin localization to the TGN in normoxia, cxposure of the cells to 1% O: for 4 h, resulted in a significant decrease in colocalization of furin and TGN-46, together with a dispersion of smali furin-containing vesicles throughoul the cytoplasm (Fig. 1A). Co-staining with the celi membranę labeling reagent Dii, revealed an inereased redistribution of furin to the basal plasma membrano (Fig. IB). A similar redistribution was observed in the case of untagged furin (supplementary Fig. IB). To determine whether the relocalization of furin was polarized toward the plasma membranę, an image projection was produced by stacking each of the individual ^-sections of the two tluorescent probes into one image, resulting in a three-dimensional (3D) representation and an overlay of both furin and celi membranę staining. Furin in hypoxic cells was redistributed mainly toward the basal plasma membranę (bottom of the celi), with fewer staining present at the top of the celi (Fig. 1C). These changcs in furin redistribution were not due to celi alterations such as decrease in celi viability and/or generał impairments in celi trafficking mechanisms. Indeed, HT-1080 cells exhibit no significant inerease in celi death as determined by trypan blue staining (data not shown). Moreover, re-exposition of the celi culture resulted in a rapid (30 min) relocalization of furin back to perinuclear TGN-46 positive location (Fig. 2A,B). Also, Western biot analysis of furin overexpressing celi lysates showed no changes within the rclative proportion of the proform and the maturę form of furin (supplementary Fig.2). Becausc the multistep proccss