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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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Effect of elevated  [Ca2+]i on exocytosis of  transferrin receptor–containing endosomes. (a) Kinetics  of release of preinternalized  125I-transferrin from SLOpermeabilized NRK cells in  the absence of Ca2+. (b) Release of preinternalized 125Itransferrin from SLO-permeabilized NRK cells at different Ca2+ concentrations. Values are expressed as a  percentage of the total 125Itransferrin present in each sample (cell associated plus released). The data represent the average of triplicate determinations ±SD. (c)  FACS® analysis of NRK cells in suspension treated for 5 min with PBS (black line) or 10 mM ionomycin in PBS (gray line). Cells were  incubated with FITC-transferrin for 30 min at 4°C before fixation.
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Figure 8: Effect of elevated [Ca2+]i on exocytosis of transferrin receptor–containing endosomes. (a) Kinetics of release of preinternalized 125I-transferrin from SLOpermeabilized NRK cells in the absence of Ca2+. (b) Release of preinternalized 125Itransferrin from SLO-permeabilized NRK cells at different Ca2+ concentrations. Values are expressed as a percentage of the total 125Itransferrin present in each sample (cell associated plus released). The data represent the average of triplicate determinations ±SD. (c) FACS® analysis of NRK cells in suspension treated for 5 min with PBS (black line) or 10 mM ionomycin in PBS (gray line). Cells were incubated with FITC-transferrin for 30 min at 4°C before fixation.

Mentions: To verify if the elevation in [Ca2+]i triggering secretion of lysosomes also affected the exocytosis of other elements of the endocytic pathway, we measured the release of previously internalized 125I-labeled transferrin using the same SLO-permeabilized cell system. As described previously (Galli et al., 1994), permeabilized cells released 125I-transferrin linearly for ∼20 min after permeabilization (Fig. 8 a). Confirming previous reports that describe a small stimulation in the exocytosis of transferrin receptor–containing vesicles upon treatment with ionophores or Ca2+-mobilizing agonists (Buys et al., 1984; Wiley and Kaplan, 1984), there was a minor increase (1.5-fold) in the extracellular release of transferrin at increasing Ca2+ concentrations (Fig. 8 b). The release of apotransferrin into the medium after recycling to the cell surface allows the use of externally added, labeled transferrin to estimate the number of receptors on the surface at a given time. Using this method, we found that the number of receptors available for binding of FITC-transferrin on the surface of NRK cells was also slightly increased after exposure to 10 μM ionomycin (Fig. 8 c).


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

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

Effect of elevated  [Ca2+]i on exocytosis of  transferrin receptor–containing endosomes. (a) Kinetics  of release of preinternalized  125I-transferrin from SLOpermeabilized NRK cells in  the absence of Ca2+. (b) Release of preinternalized 125Itransferrin from SLO-permeabilized NRK cells at different Ca2+ concentrations. Values are expressed as a  percentage of the total 125Itransferrin present in each sample (cell associated plus released). The data represent the average of triplicate determinations ±SD. (c)  FACS® analysis of NRK cells in suspension treated for 5 min with PBS (black line) or 10 mM ionomycin in PBS (gray line). Cells were  incubated with FITC-transferrin for 30 min at 4°C before fixation.
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Related In: Results  -  Collection

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Figure 8: Effect of elevated [Ca2+]i on exocytosis of transferrin receptor–containing endosomes. (a) Kinetics of release of preinternalized 125I-transferrin from SLOpermeabilized NRK cells in the absence of Ca2+. (b) Release of preinternalized 125Itransferrin from SLO-permeabilized NRK cells at different Ca2+ concentrations. Values are expressed as a percentage of the total 125Itransferrin present in each sample (cell associated plus released). The data represent the average of triplicate determinations ±SD. (c) FACS® analysis of NRK cells in suspension treated for 5 min with PBS (black line) or 10 mM ionomycin in PBS (gray line). Cells were incubated with FITC-transferrin for 30 min at 4°C before fixation.
Mentions: To verify if the elevation in [Ca2+]i triggering secretion of lysosomes also affected the exocytosis of other elements of the endocytic pathway, we measured the release of previously internalized 125I-labeled transferrin using the same SLO-permeabilized cell system. As described previously (Galli et al., 1994), permeabilized cells released 125I-transferrin linearly for ∼20 min after permeabilization (Fig. 8 a). Confirming previous reports that describe a small stimulation in the exocytosis of transferrin receptor–containing vesicles upon treatment with ionophores or Ca2+-mobilizing agonists (Buys et al., 1984; Wiley and Kaplan, 1984), there was a minor increase (1.5-fold) in the extracellular release of transferrin at increasing Ca2+ concentrations (Fig. 8 b). The release of apotransferrin into the medium after recycling to the cell surface allows the use of externally added, labeled transferrin to estimate the number of receptors on the surface at a given time. Using this method, we found that the number of receptors available for binding of FITC-transferrin on the surface of NRK cells was also slightly increased after exposure to 10 μM ionomycin (Fig. 8 c).

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