ENERGY-TRANSFER ASSAYS OF RAT RENAL CORTICAL ENDOSOMAL FUSION - EVIDENCE FOR SUPERFUSION

The complex of components necessary to allow endosomal fusion includes both membrane-bound receptors and several soluble proteins. Although these factors have been isolated from cultured cell lines, and endosom al fusion has been reconstituted in vitro for vesicular systems from y east to synaptosomes, there is a paucity of data from mammalian system s. To investigate fusion in rat renal cortical endosomes, we began by developing a fusion assay. As the immunoglobulin and avidin-based prob es almost universally employed in fusion assays are excluded by the gl omerular ultrafiltration barrier, it was necessary to begin by finding ultrafilterable probes which could serve as a fusion assay. We labele d the apical endosomal pathway of the renal proximal tubule by intrave nous infusion of ultrafilterable fluorescent dextrans. Energy transfer from entrapped fluorescein-dextran to rhodamine-dextran had a narrow concentration dependence but allowed fluorometric assay of endosomal f usion. The ''spectroscopic ruler'' property of energy transfer, whereb y it will only occur at < 60 Angstrom, makes fusion measurements unequ ivocal. The energy transfer efficiency of fluorometric (48 +/- 1%) and flow cytometry (57 +/- 1%) assays were close to the theoretical optim um (57%). Energy transfer is detected as a decrease in fluorescence of the fluorescein donor and an increase in fluorescence of the rhodamin e acceptor. Our endosomal fusion assay was utilized to determine the o ptimal conditions for fusion of rat renal cortical light endosomes and heavy endosomes. Independent measurements of fluorescein-dextran and rhodamine-dextran on an endosome-by-endosome basis using dual-beam two -color flow cytometry demonstrated that each fusion event involves mul tiple endosomes rather than a single pair of endosomes. Electron micro scopy analysis demonstrated that the average vesicle diameter was five times larger in the fused heavy endosomal fractions compared with con trol fractions without fusion. Hence, fusion of mammalian renal cortic al endosomes reconstituted in vitro is consistent with multiple fusion events dubbed superfusion.