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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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Release of β-hexosaminidase and LDH from intact and  SLO-permeabilized NRK cells. Intact cells (black columns) and  SLO-permeabilized cells (white columns), were incubated in permeabilization buffer containing either 0, 1, or 5 μM Ca2+ for 5  min at 37°C. Supernatants were assayed for (a) β-hexosaminidase  and (b) LDH activity. The amount of enzyme released in each  sample 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 5: Release of β-hexosaminidase and LDH from intact and SLO-permeabilized NRK cells. Intact cells (black columns) and SLO-permeabilized cells (white columns), were incubated in permeabilization buffer containing either 0, 1, or 5 μM Ca2+ for 5 min at 37°C. Supernatants were assayed for (a) β-hexosaminidase and (b) LDH activity. The amount of enzyme released in each sample is expressed as a percentage of the total enzyme content of control cells. The data represent the average of triplicate determinations ±SD.

Mentions: Initially, to determine the efficiency of the permeabilization procedure under our experimental conditions, SLOtreated NRK cells were stained with propidium iodide, a fluorescent nuclear stain that does not penetrate intact cells. Virtually all cells were found to be permeabilized, when observed under a fluorescence microscope (not shown). The release of β-hexosaminidase was then measured in permeabilized and nonpermeabilized cells, in the absence or presence of 1 and 5 μM Ca2+. As shown in Fig. 5 a, in a 5-min assay, an approximate fivefold increase in the release of β-hexosaminidase from permeabilized cells was triggered by addition of 1 or 5 μM Ca2+.


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

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

Release of β-hexosaminidase and LDH from intact and  SLO-permeabilized NRK cells. Intact cells (black columns) and  SLO-permeabilized cells (white columns), were incubated in permeabilization buffer containing either 0, 1, or 5 μM Ca2+ for 5  min at 37°C. Supernatants were assayed for (a) β-hexosaminidase  and (b) LDH activity. The amount of enzyme released in each  sample 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 5: Release of β-hexosaminidase and LDH from intact and SLO-permeabilized NRK cells. Intact cells (black columns) and SLO-permeabilized cells (white columns), were incubated in permeabilization buffer containing either 0, 1, or 5 μM Ca2+ for 5 min at 37°C. Supernatants were assayed for (a) β-hexosaminidase and (b) LDH activity. The amount of enzyme released in each sample is expressed as a percentage of the total enzyme content of control cells. The data represent the average of triplicate determinations ±SD.
Mentions: Initially, to determine the efficiency of the permeabilization procedure under our experimental conditions, SLOtreated NRK cells were stained with propidium iodide, a fluorescent nuclear stain that does not penetrate intact cells. Virtually all cells were found to be permeabilized, when observed under a fluorescence microscope (not shown). The release of β-hexosaminidase was then measured in permeabilized and nonpermeabilized cells, in the absence or presence of 1 and 5 μM Ca2+. As shown in Fig. 5 a, in a 5-min assay, an approximate fivefold increase in the release of β-hexosaminidase from permeabilized cells was triggered by addition of 1 or 5 μM Ca2+.

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