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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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Ionomycin induces exocytosis of β-hexosaminidase and  lysosomal fluid-phase tracers but not LDH from intact NRK fibroblasts. (a and b) NRK cells were incubated at 37°C with either  PBS (open circles) or 10 μM ionomycin in PBS (black circles). At  the indicated time points, the incubation buffer was collected and  assayed for β-hexosaminidase and LDH activity. The amount of  enzyme released at each point is expressed as a percentage of the  total content of enzyme in control cells. (c and d) Lysosomes of  NRK cells were loaded with lucifer yellow or 3H-dextran by fluidphase endocytosis followed by a 2-h chase, before treatment with  PBS or 10 μM ionomycin for 10 min. The incubation buffer was  collected, lucifer yellow was detected by reading the fluorescence, and 3H-dextran was detected by scintillation counting. The  amount of lucifer yellow or 3H-dextran released in each sample is  expressed as a percentage of the total amount of tracer present in  control cells. The data represent the average of triplicate determinations ±SD.
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Figure 1: Ionomycin induces exocytosis of β-hexosaminidase and lysosomal fluid-phase tracers but not LDH from intact NRK fibroblasts. (a and b) NRK cells were incubated at 37°C with either PBS (open circles) or 10 μM ionomycin in PBS (black circles). At the indicated time points, the incubation buffer was collected and assayed for β-hexosaminidase and LDH activity. The amount of enzyme released at each point is expressed as a percentage of the total content of enzyme in control cells. (c and d) Lysosomes of NRK cells were loaded with lucifer yellow or 3H-dextran by fluidphase endocytosis followed by a 2-h chase, before treatment with PBS or 10 μM ionomycin for 10 min. The incubation buffer was collected, lucifer yellow was detected by reading the fluorescence, and 3H-dextran was detected by scintillation counting. The amount of lucifer yellow or 3H-dextran released in each sample is expressed as a percentage of the total amount of tracer present in control cells. The data represent the average of triplicate determinations ±SD.

Mentions: To test if an increase in [Ca2+]i would stimulate exocytosis of lysosomes, the calcium ionophore ionomycin was added to NRK fibroblasts in a Ca2+-containing buffer (PBS with 1 mM CaCl2). Cells were incubated at 37°C with 10 μM ionomycin, and release of the lysosomal enzyme β-hexosaminidase was measured in the incubation buffer. This enzyme is frequently used as a marker for lysosomes, since 90% of the total β-hexosaminidase in NRK cells is located in this compartment (Griffiths et al., 1990). A continuous release of β-hexosaminidase, typically reaching ∼10% of the total enzyme content of the cells after 10 min, was specifically triggered by ionomycin (Fig. 1 a).


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

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

Ionomycin induces exocytosis of β-hexosaminidase and  lysosomal fluid-phase tracers but not LDH from intact NRK fibroblasts. (a and b) NRK cells were incubated at 37°C with either  PBS (open circles) or 10 μM ionomycin in PBS (black circles). At  the indicated time points, the incubation buffer was collected and  assayed for β-hexosaminidase and LDH activity. The amount of  enzyme released at each point is expressed as a percentage of the  total content of enzyme in control cells. (c and d) Lysosomes of  NRK cells were loaded with lucifer yellow or 3H-dextran by fluidphase endocytosis followed by a 2-h chase, before treatment with  PBS or 10 μM ionomycin for 10 min. The incubation buffer was  collected, lucifer yellow was detected by reading the fluorescence, and 3H-dextran was detected by scintillation counting. The  amount of lucifer yellow or 3H-dextran released in each sample is  expressed as a percentage of the total amount of tracer present in  control cells. The data represent the average of triplicate determinations ±SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139854&req=5

Figure 1: Ionomycin induces exocytosis of β-hexosaminidase and lysosomal fluid-phase tracers but not LDH from intact NRK fibroblasts. (a and b) NRK cells were incubated at 37°C with either PBS (open circles) or 10 μM ionomycin in PBS (black circles). At the indicated time points, the incubation buffer was collected and assayed for β-hexosaminidase and LDH activity. The amount of enzyme released at each point is expressed as a percentage of the total content of enzyme in control cells. (c and d) Lysosomes of NRK cells were loaded with lucifer yellow or 3H-dextran by fluidphase endocytosis followed by a 2-h chase, before treatment with PBS or 10 μM ionomycin for 10 min. The incubation buffer was collected, lucifer yellow was detected by reading the fluorescence, and 3H-dextran was detected by scintillation counting. The amount of lucifer yellow or 3H-dextran released in each sample is expressed as a percentage of the total amount of tracer present in control cells. The data represent the average of triplicate determinations ±SD.
Mentions: To test if an increase in [Ca2+]i would stimulate exocytosis of lysosomes, the calcium ionophore ionomycin was added to NRK fibroblasts in a Ca2+-containing buffer (PBS with 1 mM CaCl2). Cells were incubated at 37°C with 10 μM ionomycin, and release of the lysosomal enzyme β-hexosaminidase was measured in the incubation buffer. This enzyme is frequently used as a marker for lysosomes, since 90% of the total β-hexosaminidase in NRK cells is located in this compartment (Griffiths et al., 1990). A continuous release of β-hexosaminidase, typically reaching ∼10% of the total enzyme content of the cells after 10 min, was specifically triggered by ionomycin (Fig. 1 a).

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