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Figurę (Chapitre 1)3. Furin internalization in hypoxic cells involves both clathrin and lipid rafts

Cells were seeded on collagen type lV-coated slides, cultured in normoxic or hypoxic chambers for 4 h. (A) Micrographs of furin (eGFP), clathrin (red) and merged images. (B) Micrographs of furin (eGFP), caveolin-l (red) and merged images. (C) Co-immunoprecipitation of furin with clathrin and cavcolin-l in HT-1080 cells ineubated in norrnoxia or hypoxia. (D) Graph showing the percentage of furin with the basal plasma membranę. Cells were seeded on collagen type IV-coated slides, left either untreated (control; CTL) or pretreated with MpCD (300 pM) or chlorpromazine (5 pM) prior to culture in normoxic or hypoxic chambers for 4 h. Cells were then labeled with Dii processed for confocal microscopy and fluorogram analysis were performed as described under Materials and Methods. Column, mcan; bar, SE; BPM, basal plasma membranę; * P < 0.04; scalę bars correspond to 5 pm.

The inerease in cell-surface furin involves a Rab4 recycling pathway

Internalized proteins can be recycled back to the plasma membranę by two main GTPase associated pathways. Thesc involve a short and rapid loop pathway from early endosomes to the plasma membranę via Rab4, and a Rabl 1-dependent long-loop pathway from the perinuclear recycling endosomes to the plasma membranę. To study the potential involvement of these pathways in hypoxia-induced cell-surface furin expression, HT-1080 cells were transfectcd with wild type Rab4 or Rabll GTPases and ineubated under normoxic or hypoxic conditions. Results indicated that furin colocalized with both Rab4 and Rabll at perinuclear areas in normoxic and hypoxic cells. Moreover, significant colocalization between furin and Rab4, but not Rabl 1, was observed at the periphery of hypoxic cells (Fig. 5A,B). To define the implication of these recycling pathways in cell-surface furin expression, HT-1080 cells were transfectcd with dominant negative forms of either Rab4 (Rab4N123I, which is unable to bind to guanosine nucleotides (Cormont et al., 1996) or Rabl 1 (Rabl 1S25N, which preferentially binds guanosine diphosphate (Ullrich et al., 1996), followed by quantification of basal plasma membrane-associated furin. Overexpression of Rab4WT inereased the amounts of plasma membrane-associated furin in normoxic cells, mimicking the effect of hypoxia on furin relocalization (Fig.5C). Conversely, overexpression of Rab4N123I significantly decreased the amounts of plasma membrane-associated furin in hypoxic cells. In contrast, overexpression of RabllWT or RabllS25N had no impact on plasma membranę associated furin accumulation in