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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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Elevation in [Ca2+]i induces secretion of the 31-kD lysosomally processed form of cathepsin D. Rabbit anti–cathepsin  D antibodies were used to probe a Western blot containing a lysate and concentrated supernatants of IMR-90 human fibroblasts. (Lane 1) total lysate; (lane 2) concentrated supernatant of  cells treated with PBS for 5 min; (lane 3) concentrated supernatant of cells treated with 10 μM ionomycin for 5 min; (lanes 4 and  5) concentrated supernatants of SLO-permeabilized cells incubated for 5 min in buffers containing 0 or 1 μM Ca2+, respectively. Different ECL exposures were performed for each treatment to allow visualization of the secreted mature 31-kD  cathepsin D band.
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Figure 7: Elevation in [Ca2+]i induces secretion of the 31-kD lysosomally processed form of cathepsin D. Rabbit anti–cathepsin D antibodies were used to probe a Western blot containing a lysate and concentrated supernatants of IMR-90 human fibroblasts. (Lane 1) total lysate; (lane 2) concentrated supernatant of cells treated with PBS for 5 min; (lane 3) concentrated supernatant of cells treated with 10 μM ionomycin for 5 min; (lanes 4 and 5) concentrated supernatants of SLO-permeabilized cells incubated for 5 min in buffers containing 0 or 1 μM Ca2+, respectively. Different ECL exposures were performed for each treatment to allow visualization of the secreted mature 31-kD cathepsin D band.

Mentions: To confirm the lysosomal origin of the Ca2+-triggered exocytic products, we examined the lysosomal protease cathepsin D released into the supernatant of cells with elevated [Ca2+]i. Cathepsin D is processed into a mature polypeptide of 31 kD only after being transported to lysosomes (Gieselmann et al., 1983). Elevation of [Ca2+]i in the human fibroblast cell line IMR-90, by treatment with ionomycin or by addition of 1 μM Ca2+ buffer to SLO-permeabilized cells, induced the appearance in the supernatant of the 31-kD mature form of cathepsin D (Fig. 7, lanes 3 and 5). A band of identical migration was also detected in whole cell extracts (Fig. 7, lane 1). The intermediate (47 kD) and precursor (53 kD) forms of the protein (Gieselmann et al., 1983) were detected in the supernatant at similar levels in each condition, probably reflecting the constitutive extracellular release of unprocessed cathepsin D that can occur before reuptake and targeting to lysosomes (von Figura and Hasilik, 1986; Kornfeld and Mellman, 1989). The higher molecular mass bands also appearing in Fig. 7 are due to nonspecific cross-reaction of the antibodies.


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

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

Elevation in [Ca2+]i induces secretion of the 31-kD lysosomally processed form of cathepsin D. Rabbit anti–cathepsin  D antibodies were used to probe a Western blot containing a lysate and concentrated supernatants of IMR-90 human fibroblasts. (Lane 1) total lysate; (lane 2) concentrated supernatant of  cells treated with PBS for 5 min; (lane 3) concentrated supernatant of cells treated with 10 μM ionomycin for 5 min; (lanes 4 and  5) concentrated supernatants of SLO-permeabilized cells incubated for 5 min in buffers containing 0 or 1 μM Ca2+, respectively. Different ECL exposures were performed for each treatment to allow visualization of the secreted mature 31-kD  cathepsin D band.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Elevation in [Ca2+]i induces secretion of the 31-kD lysosomally processed form of cathepsin D. Rabbit anti–cathepsin D antibodies were used to probe a Western blot containing a lysate and concentrated supernatants of IMR-90 human fibroblasts. (Lane 1) total lysate; (lane 2) concentrated supernatant of cells treated with PBS for 5 min; (lane 3) concentrated supernatant of cells treated with 10 μM ionomycin for 5 min; (lanes 4 and 5) concentrated supernatants of SLO-permeabilized cells incubated for 5 min in buffers containing 0 or 1 μM Ca2+, respectively. Different ECL exposures were performed for each treatment to allow visualization of the secreted mature 31-kD cathepsin D band.
Mentions: To confirm the lysosomal origin of the Ca2+-triggered exocytic products, we examined the lysosomal protease cathepsin D released into the supernatant of cells with elevated [Ca2+]i. Cathepsin D is processed into a mature polypeptide of 31 kD only after being transported to lysosomes (Gieselmann et al., 1983). Elevation of [Ca2+]i in the human fibroblast cell line IMR-90, by treatment with ionomycin or by addition of 1 μM Ca2+ buffer to SLO-permeabilized cells, induced the appearance in the supernatant of the 31-kD mature form of cathepsin D (Fig. 7, lanes 3 and 5). A band of identical migration was also detected in whole cell extracts (Fig. 7, lane 1). The intermediate (47 kD) and precursor (53 kD) forms of the protein (Gieselmann et al., 1983) were detected in the supernatant at similar levels in each condition, probably reflecting the constitutive extracellular release of unprocessed cathepsin D that can occur before reuptake and targeting to lysosomes (von Figura and Hasilik, 1986; Kornfeld and Mellman, 1989). The higher molecular mass bands also appearing in Fig. 7 are due to nonspecific cross-reaction of the antibodies.

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