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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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Ca2+ agonists induce a low level of β-hexosaminidase  release and appearance of lgp120 in the plasma membrane. (a)  NRK cells were incubated with different concentrations of bombesin for 5 min, and the incubation buffer was assayed for β-hexosaminidase activity. The amount of enzyme released at each  point is expressed as a percentage of the total content of enzyme  in control cells. The data represent the average of triplicate determinations ± SD. (b) Same as a, except that cells were incubated  for 5 min with PBS, TSF, or heat-inactivated TSF. (c) lgp120 surface staining 5 min after exposure to PBS; (d) lgp120 surface  staining of NRK cells 5 min after exposure to 10 μM bombesin.  Bar, 5 μM.
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Figure 4: Ca2+ agonists induce a low level of β-hexosaminidase release and appearance of lgp120 in the plasma membrane. (a) NRK cells were incubated with different concentrations of bombesin for 5 min, and the incubation buffer was assayed for β-hexosaminidase activity. The amount of enzyme released at each point is expressed as a percentage of the total content of enzyme in control cells. The data represent the average of triplicate determinations ± SD. (b) Same as a, except that cells were incubated for 5 min with PBS, TSF, or heat-inactivated TSF. (c) lgp120 surface staining 5 min after exposure to PBS; (d) lgp120 surface staining of NRK cells 5 min after exposure to 10 μM bombesin. Bar, 5 μM.

Mentions: The results described above indicated that ionomycinmediated influx of Ca2+ from the extracellular medium induced fusion of lysosomes with the plasma membrane. To verify if Ca2+ mobilized from intracellular stores by receptor-mediated agonists had the same effect, release of β-hexosaminidase was measured in the supernatant of cells treated with several concentrations of the signaling peptide bombesin. As shown in Fig. 4 a, bombesin also induced lysosomal exocytosis in a dose-dependent way, but to a much lesser extent than ionomycin (a 1.5-fold maximum increase). When tested for induction of translocation to the plasma membrane of the lysosomal protein lgp120, bombesin was found to induce levels detectable by immunofluorescence (Fig. 4, c and d).


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

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

Ca2+ agonists induce a low level of β-hexosaminidase  release and appearance of lgp120 in the plasma membrane. (a)  NRK cells were incubated with different concentrations of bombesin for 5 min, and the incubation buffer was assayed for β-hexosaminidase activity. The amount of enzyme released at each  point is expressed as a percentage of the total content of enzyme  in control cells. The data represent the average of triplicate determinations ± SD. (b) Same as a, except that cells were incubated  for 5 min with PBS, TSF, or heat-inactivated TSF. (c) lgp120 surface staining 5 min after exposure to PBS; (d) lgp120 surface  staining of NRK cells 5 min after exposure to 10 μM bombesin.  Bar, 5 μM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Ca2+ agonists induce a low level of β-hexosaminidase release and appearance of lgp120 in the plasma membrane. (a) NRK cells were incubated with different concentrations of bombesin for 5 min, and the incubation buffer was assayed for β-hexosaminidase activity. The amount of enzyme released at each point is expressed as a percentage of the total content of enzyme in control cells. The data represent the average of triplicate determinations ± SD. (b) Same as a, except that cells were incubated for 5 min with PBS, TSF, or heat-inactivated TSF. (c) lgp120 surface staining 5 min after exposure to PBS; (d) lgp120 surface staining of NRK cells 5 min after exposure to 10 μM bombesin. Bar, 5 μM.
Mentions: The results described above indicated that ionomycinmediated influx of Ca2+ from the extracellular medium induced fusion of lysosomes with the plasma membrane. To verify if Ca2+ mobilized from intracellular stores by receptor-mediated agonists had the same effect, release of β-hexosaminidase was measured in the supernatant of cells treated with several concentrations of the signaling peptide bombesin. As shown in Fig. 4 a, bombesin also induced lysosomal exocytosis in a dose-dependent way, but to a much lesser extent than ionomycin (a 1.5-fold maximum increase). When tested for induction of translocation to the plasma membrane of the lysosomal protein lgp120, bombesin was found to induce levels detectable by immunofluorescence (Fig. 4, c and d).

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