Limits...
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.

Show MeSH

Related in: MedlinePlus

Ca2+-dependent  release of β-hexosaminidase,  lucifer yellow, and 3H-dextran from permeabilized  NRK cells. SLO-permeabilized cells were incubated for  5 min at 37°C in permeabilization buffer containing different Ca2+ concentrations.  (a) β-hexosaminidase activity released. (b) Lucifer yellow released from previously  loaded cells. (c) 3H-Dextran  released from previously loaded cells. Values are expressed as a percentage of the total content of either β-hexosaminidase, lucifer yellow, or 3H-dextran in control cells. The data represent the average of triplicate determinations ±SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139854&req=5

Figure 6: Ca2+-dependent release of β-hexosaminidase, lucifer yellow, and 3H-dextran from permeabilized NRK cells. SLO-permeabilized cells were incubated for 5 min at 37°C in permeabilization buffer containing different Ca2+ concentrations. (a) β-hexosaminidase activity released. (b) Lucifer yellow released from previously loaded cells. (c) 3H-Dextran released from previously loaded cells. Values are expressed as a percentage of the total content of either β-hexosaminidase, lucifer yellow, or 3H-dextran in control cells. The data represent the average of triplicate determinations ±SD.

Mentions: To determine the optimal Ca+ concentration inducing exocytosis of lysosomes in the permeabilized cell system, we performed the β-hexosaminidase, lucifer yellow, and 3H-dextran release assays in buffers with Ca2+ concentrations ranging from 0–5 μM. Optimal release of lysosomal markers, reaching 7–10-fold above the levels observed in the absence of Ca2+, was found to require 1–5 μM Ca2+, although exocytosis was already detectable at 0.1 μM Ca2+ (Fig. 6, a–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)

Ca2+-dependent  release of β-hexosaminidase,  lucifer yellow, and 3H-dextran from permeabilized  NRK cells. SLO-permeabilized cells were incubated for  5 min at 37°C in permeabilization buffer containing different Ca2+ concentrations.  (a) β-hexosaminidase activity released. (b) Lucifer yellow released from previously  loaded cells. (c) 3H-Dextran  released from previously loaded cells. Values are expressed as a percentage of the total content of either β-hexosaminidase, lucifer yellow, or 3H-dextran in 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 6: Ca2+-dependent release of β-hexosaminidase, lucifer yellow, and 3H-dextran from permeabilized NRK cells. SLO-permeabilized cells were incubated for 5 min at 37°C in permeabilization buffer containing different Ca2+ concentrations. (a) β-hexosaminidase activity released. (b) Lucifer yellow released from previously loaded cells. (c) 3H-Dextran released from previously loaded cells. Values are expressed as a percentage of the total content of either β-hexosaminidase, lucifer yellow, or 3H-dextran in control cells. The data represent the average of triplicate determinations ±SD.
Mentions: To determine the optimal Ca+ concentration inducing exocytosis of lysosomes in the permeabilized cell system, we performed the β-hexosaminidase, lucifer yellow, and 3H-dextran release assays in buffers with Ca2+ concentrations ranging from 0–5 μM. Optimal release of lysosomal markers, reaching 7–10-fold above the levels observed in the absence of Ca2+, was found to require 1–5 μM Ca2+, although exocytosis was already detectable at 0.1 μM Ca2+ (Fig. 6, a–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