DENSE CORE LYSOSOMES CAN FUSE WITH LATE ENDOSOMES AND ARE RE-FORMED FROM THE RESULTANT HYBRID ORGANELLES

Electron microscopy was used to evaluate the function and formation of dense core lysosomes. Lysosomes were preloaded with bovine serum albu min (BSA)-gold conjugates by fluid phase endocytosis using a pulse-cha se protocol, The gold particles present in dense core lysosomes and la te endosomes were flocculated, consistent with proteolytic degradation of the BSA. A second pulse of BSA-gold also accumulated in the pre-le aded dense core lysosomes at 37 degrees C, but accumulation was revers ibly blocked by incubation at 20 degrees C. Time course experiments in dicated that mixing of the two BSA-gold conjugates initially occurred upon fusion of mannose 6-phosphate receptor-positive/lysosomal glycopr otein-positive late endosomes with dense core lysosomes. Treatment for 5 hours with wortmannin, a phosphatidyl inositide 3-kinase inhibitor, caused a reduction in number of dense core lysosomes preloaded with B SA-gold and prevented a second pulse of BSA-gold accumulating in them. After wortmannin treatment the tno BSA-gold conjugates were mixed in swollen late endosomal structures, Incubation of NRK cells with 0.03 M sucrose resulted in the formation of swollen sucrosomes which were mo rphologically distinct from preloaded dense core lysosomes and were id entified as late endosomes and hybrid endosome-lysosome structures, Su bsequent endocytosis of invertase resulted in digestion of the sucrose and re-formation of dense core lysosomes. These observations suggest that dense core lysosomes are biologically active storage granules of lysosomal proteases which can fuse with late endosomes and be re-forme d from the resultant hybrid organelles prior to subsequent cycles of f usion and re-formation.