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Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells.

Rodríguez A, Webster P, Ortego J, Andrews NW - J. Cell Biol. (1997)

Bottom Line: Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+-containing buffers to streptolysin O-permeabilized cells induced exocytosis of approximately 10% of the total lysosomes of NRK cells.The process was also detected in other cell types such as epithelial cells and myoblasts.Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca2+, such as neutrophil azurophil granules, mast cell-specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca2+ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types. Here we demonstrate that elevation in the intracellular free Ca2+ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme beta-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+-containing buffers to streptolysin O-permeabilized cells induced exocytosis of approximately 10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells. These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca2+-regulated exocytosis.

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Exocytosis of BSA–gold-loaded lysosomes is triggered by [Ca2+]i elevation. NRK cells loaded with 5 nm BSA–gold complexes  for 4 h followed by a chase of 2 h were incubated with PBS (A and B) or 10 μM ionomycin (C–H) for 2.5 (C and D) or 10 min (E–H).  Transmission EM sections show gold-loaded vesicles (arrows), observed in close proximity to the plasma membrane in ionomycintreated cells (C, E, and F). Exocytosed BSA–gold was also detected after exposure to ionomycin (D and F). Labeling with antibodies  against lgp120 (10 nm gold) was detected on BSA–gold-containing vesicles (G and H). Small arrows indicate anti-lgp120 labeling on lysosomes; arrowheads indicate anti-lgp120 labeling on the plasma membrane after ionomycin treatment. Bars, 1 μM.
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Figure 2: Exocytosis of BSA–gold-loaded lysosomes is triggered by [Ca2+]i elevation. NRK cells loaded with 5 nm BSA–gold complexes for 4 h followed by a chase of 2 h were incubated with PBS (A and B) or 10 μM ionomycin (C–H) for 2.5 (C and D) or 10 min (E–H). Transmission EM sections show gold-loaded vesicles (arrows), observed in close proximity to the plasma membrane in ionomycintreated cells (C, E, and F). Exocytosed BSA–gold was also detected after exposure to ionomycin (D and F). Labeling with antibodies against lgp120 (10 nm gold) was detected on BSA–gold-containing vesicles (G and H). Small arrows indicate anti-lgp120 labeling on lysosomes; arrowheads indicate anti-lgp120 labeling on the plasma membrane after ionomycin treatment. Bars, 1 μM.

Mentions: Another fluid-phase tracer, BSA–gold, was used for specific visualization of lysosomes by EM. NRK cell lysosomes were loaded with BSA–gold, and then treated with 10 μM ionomycin for different periods of time. In PBStreated control NRK cells, a population of vesicles containing BSA–gold and with the characteristic morphology and dimensions of lysosomes (Holtzman, 1989) was observed, mostly clustered in the perinuclear area (Fig. 2, A and B). In contrast, cells exposed to ionomycin showed a more dispersed distribution of gold-loaded vesicles, with accumulation in the proximity of the plasma membrane (Fig. 2, C–F). Images that strongly suggested exocytic events, with extracellular release of the gold complexes and other membranous lysosomal contents, were observed at both 2.5 and 10 min after exposure to ionomycin (Fig. 2, D and F). No similar images were detected in control cells (Fig. 2, A and B).


Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells.

Rodríguez A, Webster P, Ortego J, Andrews NW - J. Cell Biol. (1997)

Exocytosis of BSA–gold-loaded lysosomes is triggered by [Ca2+]i elevation. NRK cells loaded with 5 nm BSA–gold complexes  for 4 h followed by a chase of 2 h were incubated with PBS (A and B) or 10 μM ionomycin (C–H) for 2.5 (C and D) or 10 min (E–H).  Transmission EM sections show gold-loaded vesicles (arrows), observed in close proximity to the plasma membrane in ionomycintreated cells (C, E, and F). Exocytosed BSA–gold was also detected after exposure to ionomycin (D and F). Labeling with antibodies  against lgp120 (10 nm gold) was detected on BSA–gold-containing vesicles (G and H). Small arrows indicate anti-lgp120 labeling on lysosomes; arrowheads indicate anti-lgp120 labeling on the plasma membrane after ionomycin treatment. Bars, 1 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139854&req=5

Figure 2: Exocytosis of BSA–gold-loaded lysosomes is triggered by [Ca2+]i elevation. NRK cells loaded with 5 nm BSA–gold complexes for 4 h followed by a chase of 2 h were incubated with PBS (A and B) or 10 μM ionomycin (C–H) for 2.5 (C and D) or 10 min (E–H). Transmission EM sections show gold-loaded vesicles (arrows), observed in close proximity to the plasma membrane in ionomycintreated cells (C, E, and F). Exocytosed BSA–gold was also detected after exposure to ionomycin (D and F). Labeling with antibodies against lgp120 (10 nm gold) was detected on BSA–gold-containing vesicles (G and H). Small arrows indicate anti-lgp120 labeling on lysosomes; arrowheads indicate anti-lgp120 labeling on the plasma membrane after ionomycin treatment. Bars, 1 μM.
Mentions: Another fluid-phase tracer, BSA–gold, was used for specific visualization of lysosomes by EM. NRK cell lysosomes were loaded with BSA–gold, and then treated with 10 μM ionomycin for different periods of time. In PBStreated control NRK cells, a population of vesicles containing BSA–gold and with the characteristic morphology and dimensions of lysosomes (Holtzman, 1989) was observed, mostly clustered in the perinuclear area (Fig. 2, A and B). In contrast, cells exposed to ionomycin showed a more dispersed distribution of gold-loaded vesicles, with accumulation in the proximity of the plasma membrane (Fig. 2, C–F). Images that strongly suggested exocytic events, with extracellular release of the gold complexes and other membranous lysosomal contents, were observed at both 2.5 and 10 min after exposure to ionomycin (Fig. 2, D and F). No similar images were detected in control cells (Fig. 2, A and B).

Bottom Line: Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+-containing buffers to streptolysin O-permeabilized cells induced exocytosis of approximately 10% of the total lysosomes of NRK cells.The process was also detected in other cell types such as epithelial cells and myoblasts.Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca2+, such as neutrophil azurophil granules, mast cell-specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca2+ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types. Here we demonstrate that elevation in the intracellular free Ca2+ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme beta-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+-containing buffers to streptolysin O-permeabilized cells induced exocytosis of approximately 10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells. These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca2+-regulated exocytosis.

Show MeSH
Related in: MedlinePlus