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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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Characterization of Ca2+-induced lysosome exocytosis  in permeabilized cells. Release of β-hexosaminidase from SLOpermeabilized NRK cells was measured after addition of 0 or 1  μM Ca2+ buffers. (a) Kinetics of release of β-hexosaminidase  (black circles) and LDH (white circles) from NRK cells after addition of 1 μM Ca2+ at 37°C. (b) Sensitivity of β-hexosaminidase  release to microtubule depolymerization. Cells were treated with  10 μM colchicine for 1 h before addition of the Ca2+ buffers for 5  min at 37°C. (c) Temperature dependence of β-hexosaminidase  release. Cells were incubated for 5 min at 37°C to allow SLO pore  formation, and then the secretion assay was performed at 4° or  37°C for 5 min. (d) Sensitivity of β-hexosaminidase release to  ATP depletion. After permeabilization, cells were incubated in  the presence of 2 mM MgATP or an ATP-depleting system (5  mM glucose, 150 U/ml hexokinase) for 15 min at 37°C, before  performing the 5-min secretion assay. In all experiments, the  amount of enzyme released is expressed as a percentage of the  total enzyme content of control cells. The data represent the average of triplicate determinations ±SD.
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Figure 9: Characterization of Ca2+-induced lysosome exocytosis in permeabilized cells. Release of β-hexosaminidase from SLOpermeabilized NRK cells was measured after addition of 0 or 1 μM Ca2+ buffers. (a) Kinetics of release of β-hexosaminidase (black circles) and LDH (white circles) from NRK cells after addition of 1 μM Ca2+ at 37°C. (b) Sensitivity of β-hexosaminidase release to microtubule depolymerization. Cells were treated with 10 μM colchicine for 1 h before addition of the Ca2+ buffers for 5 min at 37°C. (c) Temperature dependence of β-hexosaminidase release. Cells were incubated for 5 min at 37°C to allow SLO pore formation, and then the secretion assay was performed at 4° or 37°C for 5 min. (d) Sensitivity of β-hexosaminidase release to ATP depletion. After permeabilization, cells were incubated in the presence of 2 mM MgATP or an ATP-depleting system (5 mM glucose, 150 U/ml hexokinase) for 15 min at 37°C, before performing the 5-min secretion assay. In all experiments, the amount of enzyme released is expressed as a percentage of the total enzyme content of control cells. The data represent the average of triplicate determinations ±SD.

Mentions: The kinetics of Ca2+-induced lysosomal secretion in NRK cells, as measured by release of β-hexosaminidase, showed a rapid increase in the first 5 min that did not significantly change with longer incubation times (Fig. 9 a). Conversely, there was a continuous increase in the extracellular accumulation of LDH, due to cytoplasmic leakage from the permeabilized cells. As mentioned before, however, the degree of cytosol loss that occurred during the first 5 min after permeabilization is not sufficient to account for the sharp decrease in β-hexosaminidase secretion; as shown in Fig. 8 a, transferrin recycling, a process known to depend on cytosolic factors (Podbilewicz and Mellman., 1990; Galli et al., 1994), proceeds in a linear fashion for at least 20 min, under the same conditions.


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

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

Characterization of Ca2+-induced lysosome exocytosis  in permeabilized cells. Release of β-hexosaminidase from SLOpermeabilized NRK cells was measured after addition of 0 or 1  μM Ca2+ buffers. (a) Kinetics of release of β-hexosaminidase  (black circles) and LDH (white circles) from NRK cells after addition of 1 μM Ca2+ at 37°C. (b) Sensitivity of β-hexosaminidase  release to microtubule depolymerization. Cells were treated with  10 μM colchicine for 1 h before addition of the Ca2+ buffers for 5  min at 37°C. (c) Temperature dependence of β-hexosaminidase  release. Cells were incubated for 5 min at 37°C to allow SLO pore  formation, and then the secretion assay was performed at 4° or  37°C for 5 min. (d) Sensitivity of β-hexosaminidase release to  ATP depletion. After permeabilization, cells were incubated in  the presence of 2 mM MgATP or an ATP-depleting system (5  mM glucose, 150 U/ml hexokinase) for 15 min at 37°C, before  performing the 5-min secretion assay. In all experiments, the  amount of enzyme released is expressed as a percentage of the  total enzyme content of control cells. The data represent the average of triplicate determinations ±SD.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Characterization of Ca2+-induced lysosome exocytosis in permeabilized cells. Release of β-hexosaminidase from SLOpermeabilized NRK cells was measured after addition of 0 or 1 μM Ca2+ buffers. (a) Kinetics of release of β-hexosaminidase (black circles) and LDH (white circles) from NRK cells after addition of 1 μM Ca2+ at 37°C. (b) Sensitivity of β-hexosaminidase release to microtubule depolymerization. Cells were treated with 10 μM colchicine for 1 h before addition of the Ca2+ buffers for 5 min at 37°C. (c) Temperature dependence of β-hexosaminidase release. Cells were incubated for 5 min at 37°C to allow SLO pore formation, and then the secretion assay was performed at 4° or 37°C for 5 min. (d) Sensitivity of β-hexosaminidase release to ATP depletion. After permeabilization, cells were incubated in the presence of 2 mM MgATP or an ATP-depleting system (5 mM glucose, 150 U/ml hexokinase) for 15 min at 37°C, before performing the 5-min secretion assay. In all experiments, the amount of enzyme released is expressed as a percentage of the total enzyme content of control cells. The data represent the average of triplicate determinations ±SD.
Mentions: The kinetics of Ca2+-induced lysosomal secretion in NRK cells, as measured by release of β-hexosaminidase, showed a rapid increase in the first 5 min that did not significantly change with longer incubation times (Fig. 9 a). Conversely, there was a continuous increase in the extracellular accumulation of LDH, due to cytoplasmic leakage from the permeabilized cells. As mentioned before, however, the degree of cytosol loss that occurred during the first 5 min after permeabilization is not sufficient to account for the sharp decrease in β-hexosaminidase secretion; as shown in Fig. 8 a, transferrin recycling, a process known to depend on cytosolic factors (Podbilewicz and Mellman., 1990; Galli et al., 1994), proceeds in a linear fashion for at least 20 min, under the same conditions.

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