MULTIVESICULAR BODIES IN HEP-2 CELLS ARE MATURING ENDOSOMES

Conventional fluorescence microscopy of fixed HEp-2 cells as well as v ideo microscopy of living cells incubated with transferrin-Texas Red ( Tf-TxR) for < 60 min revealed distinct punctuate endosomal structures. Quantitative ultrastructural analysis using horseradish peroxidase (H RP) and cationized gold as tracers showed that spherical multivesicula r bodies (MVBs) were the predominant endocytic compartments in HEp-2 c ells and that MVBs within 60 to 90 min matured into lysosomes still co ntaining internal vesicles. The number of labeled MVBs increased conti nuously from 2.5 min to 30 min of tracer incubation. However, when the cells were pulsed for 5 min followed by 10 or 25 min chases, the numb er of labeled MVBs corresponded to that obtained after 5 min of contin uous incubation. The diameter of labeled MVBs was largely constant wit h time, but the number of internal MVB vesicles increased. Thus, early or newly formed MVBs contained few internal vesicles, whereas late MV Bs, that is to say, MVBs that have existed for some period of time, co ntained numerous internal vesicles, and finally a mixture of membranou s material or myelin figures and vesicles. It is thus in principle pos sible to distinguish between early and late MVBs in HEp-2 cells on the basis of morphology. However, the difference in number of internal ve sicles applies only to the entire MVB population; after only 2.5 to 5 min of incubation, MVBs with numerous internal vesicles could also be reached by internalized tracer. Concomitant with the gradual changes i n morphology, the MVBs also showed a characteristic change in content of marker proteins as detected by immunogold labeling on ultracryosect ions. Hence, early MVBs with relatively few internal vesicles and typi cally reached by internalized tracers within 5 min contained transferr in receptors (TfRs). By contrast, MVBs with many internal vesicles and labeled after 60 min of incubation contained mannose-phosphate recept ors (MPRs), and the MVBs with distinct membranous material or myelin f igures in addition to the internal vesicles were enriched in the lysos ome membrane protein lamp-1. Thus, there seems to be a gradual maturat ion of MVBs in HEp-2 cells.