Comparative behavior of lysosomes and the pre-lysosome compartment (PLC) in in vivo cell fusion experiments

Interspecies cell fusion was used to compare protein intermixing within the mannose 6-phosphate receptor (MPR)-enriched pre-lysosome compartment (PLC) and within the MPR-negative lysosomal compartment. Both compartments were positive for lysosomal glycoprotein (lgp) membrane markers but were morphologically distinct. In most experiments, rat-mouse cell syncytia were formed by u.v.-inactivated Sindbis virus-mediated fusion. By immunogold electron microscopy of syncytia, extensive intermixing of species-specific lysosomal membrane proteins was observed in both lysosomes and PLC. At 3 h post cell fusion, multiple-label immunogold studies showed that 82% of the lysosome-like structures positive for the rat lysosomal membrane protein LIMP-I were also positive for the mouse lysosomal membrane protein mLAMP-1. By immunofluorescence, LIMP-I and mLAMP-1 co-localized with a t1/2 of 30 min after cell fusion; although the lgp-positive organelle populations had evidently interchanged their proteins, the lysosomal structures remained small, punctate bodies distributed throughout the syncytoplasm as observed in single cells. In contrast, the initially separate units of the PLC congregated with a t1/2 of 1 h to form large, pre-lysosome complexes associated with individual nuclear clusters. At the electron-microscope level, gold markers endocytized by the rat and mouse parent cells in a 1 h uptake followed by a 16-20 h chase co-localized in these extended PLC complexes, as did the membrane markers mLAMP-1 and LIMP-I. The density of labeling for rat MPR in the extended PLCs was markedly decreased, consistent with membrane fusions and dilution of the antigen upon congregation of the PLC compartments from the donor cells. The extended PLC complex behaved as a late endocytic compartment, as shown by co-localization of the MPR and rhodamine-dextran following a 10 min dextran uptake and a 50 min chase. These differences in behavior between lysosomes and the PLC in rat-mouse cell syncytia suggest that the pathway(s) of protein intermixing with respect to the two organelles may be different.