IMAGING OF ENDOSOME FUSION IN BHK FIBROBLASTS BASED ON A NOVEL FLUOROMETRIC AVIDIN-BIOTIN BINDING ASSAY

A fluorescence assay of in vivo endosome fusion was developed and appl ied to define the kinetics of endosome fusion in baby hamster kidney ( BHK) fibroblasts. The assay is based on an similar to 10-fold enhancem ent of the green fluorescence of BODIPY-avidin upon biotin binding. Th e BODIPY-avidin fluorescence enhancement occurred in <25 ms, was pH-in dependent, and involved a BODIPY-tryptophan interaction. For endocytos is in vivo, BHK fibroblasts were pulse-labeled with BODIPY-avidin toge ther with a red (rhodamine) fluorescent fusion-independent chromophore (TMR). After specified chase times in a nonfluorescent medium, a seco nd cohort of endosomes was pulse-labeled with biotin-conjugated albumi n, dextran, or transferrin. Fusion of biotin-containing endosomes with avidin-containing endosomes was quantified by ratio imaging of BODIPY -to-TMR fluorescence in individual endosomes, using imaging methods de veloped for endosome pH studies. Analysis of BODIPY-to-TMR ratio distr ibutions in avidin-labeled endosomes exposed to zero and maximum bioti n indicated >90% sensitivity for detection of endosome fusion, in avid in pulse (10 min) -chase-biotin albumin pulse (10 min) studies, both f used and unfused endosomes were identified; the fractions of avidin-la beled endosomes that fused with biotin-labeled endosomes were 0.48, 0. 21, 0.16, and 0.07 for 0-, 5-, 10-, and 20-min chase times. Fitting of fusion data to a mathematical model of in vivo endosome fusion requir ed the existence of an intermediate fusion compartment. Pulse-chase st udies performed with biotin-transferrin to label the early/recycling e ndosomes indicated that after a 10-min chase, avidin-labeled endosomes reached a compartment that was inaccessible to biotin-transferrin. Th e assay was also applied to determine whether endosome fusion was infl uenced by temperature, pH (bafilomycin A1), second messengers (cAMP ag onists, phorbol 12-myristate 13-acetate, staurosporine), and growth-re lated factors (platelet-derived growth factor, genistein). The results establish a sensitive fluorescence assay to quantify the fusion of ve sicular compartments in living cells.