HOMOTYPIC FUSION BETWEEN AGGREGATED LYSOSOMES TRIGGERED BY ELEVATED [CA2+](I) IN FIBROBLASTS

Previous studies demonstrated that microinjection of antibodies to the cytoplasmic domain of the lysosomal glycoprotein lgp120 induces aggre gation of lysosomes in NRK cells. Here we show that the antibody-clust ered vesicles do not co-localize with MPR and beta-COP-containing orga nelles, confirming their lysosomal nature, Observations by transmissio n and high voltage electron microscopy indicated that, although tightl y apposed to each other, aggregated lysosomes remained as separate ves icles, with an average diameter of 0.3-0.4 mu m. However, when cells m icroinjected with antibody were exposed to the Ca2+ ionophore ionomyci n, large vesicles were formed within the lysosome clusters, suggesting the occurrence of lysosome-lysosome fusion, Stereological measurement s of lysosome diameters on confocal and transmission electron could ha ve originated from the fusion of 3 up to 15 individual lysosomes, To v erify if agents that mobilize Ca2+ from intracellular stores had the s ame effect, anti-lgp-120-microinjected cells were treated with thapsig argin, and with the receptor-mediated agonists bombesin and thrombin, Thapsigargin also induced the formation of large lgp120-containing ves icles, detected by both confocal and transmission electron microscopy, Analysis of antibody-clustered lysosomes in streptolysin O-permeabili zed cells indicated that an intracellular free Ca2+ concentration of 1 mu M was sufficient to trigger formation of large lysosomes.