CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR ACTIVATION STIMULATES ENDOSOME FUSION IN-VIVO

Previous studies have suggested a role for cystic fibrosis transmembra ne conductance regulator (CFTR) in the regulation of intracellular ves icular trafficking, A quantitative fluorescence method was used to tes t the hypothesis that CFTR expression and activation affects endosome- endosome fusion in intact cells, Endosomes from CFTR-expressing and co ntrol (vector-transfected) Swiss 3T3 fibroblasts were labeled by inter nalization with ifluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (Bo dipy)-avidin, a fluid-phase marker whose fluorescence increases approx imate to 8-fold upon biotin binding. Cells were washed, chased, and th en labeled with biotin-albumin or biotin-transferrin, The fraction of Bodipy-avidin-labeled endosomes that fused with biotin-containing endo somes (f(fusion)) was quantified by ratio imaging microfluorimetry, En dosome fusion in unstimulated CFTR-expressing cells was similar to tha t in control cells, However, in CFTR-expressing cells activated by for skolin, f(fusion) was increased bg 1.30 +/- 0.18- and 2.65 +/- 0.17-fo ld for a 0 and 10 min chase time between avidin and biotin-albumin pul ses; f(fusion) also increased (1.32 +/- 0.11-fold) when biotin-transfe rrin replaced biotin-albumin, The stimulation of endosome fusion was n ot due to differences in rates of endocytosis or endosomal acidificati on. Endosome fusion was not stimulated by forskolin in Cl--depleted CF TR-expressing cells, suggesting that the increase in endosome fusion i s due to the CFTR chloride channel activity. These results provide evi dence that CFTR is involved in the regulation of endosome fusion and, thus, a possible basis for the cellular defects associated with cystic fibrosis.