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 aggregation of lysosomes in NRK cells. Here we show that the antibody-clustered vesicles do not co-localize with MPR and ss-COP-containing organelles, confirming their lysosomal nature. Observations by transmission and high voltage electron microscopy indicated that, although tightly apposed to each other, aggregated lysosomes remained as separate vesicles, with an average diameter of 0.3-0.4 micron. However, when cells microinjected with antibody were exposed to the Ca2+ ionophore ionomycin, large vesicles were formed within the lysosome clusters, suggesting the occurrence of lysosome-lysosome fusion. Stereological measurements of lysosome diameters on confocal and transmission electron microscopy indicated that the large lgp120-positive vesicles could have originated from the fusion of 3 up to 15 individual lysosomes. To verify if agents that mobilize Ca2+ from intracellular stores had the same effect, anti-lgp120-microinjected cells were treated with thapsigargin, and with the receptor-mediated agonists bombesin and thrombin. Thapsigargin also induced the formation of large lgp120-containing vesicles, detected by both confocal and transmission electron microscopy. Analysis of antibody-clustered lysosomes in streptolysin O-permeabilized cells indicated that an intracellular free Ca2+ concentration of 1 microM was sufficient to trigger formation of large lysosomes.
Bakker
A C
AC
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Webster
P
P
Jacob
W A
WA
Andrews
N W
NW
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008845:Microinjections; D008854:Microscopy, Electron; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D013301:Streptolysins
D000906:Antibodies; D015703:Antigens, CD; D001426:Bacterial Proteins; D007476:Ionophores; D052118:Lysosomal-Associated Membrane Protein 1; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D013301:Streptolysins; C025964:streptolysin O; D015759:Ionomycin; D002118:Calcium