The relationship between lumenal and limiting membranes in swollen late endocytic compartments formed after wortmannin treatment or sucrose accumulation

Immunofluorescence and electron microscopy were used to evaluate the format ion of swollen endosomes in NRK cells after treatment with wortmannin or su crose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen lat e endocytic vacuoles, positive for lysosomal glycoproteins or cation-indepe ndent mannose 6-phosphate receptors, but those induced by wortmannin were c haracterised by time-dependent accumulation of lumenal vesicles, whereas th ose induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic a cid, remained unchanged and was present within the lumen of the swollen vac uoles. Consumption of plasma membrane and peripheral early endosomes, and t he appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is su fficient to account for the observed phenotype of cells treated with wortma nnin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an ex planation for the paucity of lumenal vesicles in swollen sucrosomes. Our da ta suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with t he failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis.