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Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.

Mullock BM, Bright NA, Fearon CW, Gray SR, Luzio JP - J. Cell Biol. (1998)

Bottom Line: Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein.We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes.The consequences of this fusion to the maintenance and function of lysosomes are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom.

ABSTRACT
Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37 degrees C, late endosome-lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

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Immunoelectron  microscopy identifying avidin (8-nm gold) and cathepsin L (15-nm gold) immunoreactivity in organelles  from late endosome, lysosome, and hybrid fractions.  (a) A late endosome peak  fraction from the preparative  1–22% Ficoll gradient showing an endosomal structure  containing Av-ASF. (b) Hybrid organelles formed after  the content mixing assay incubation accumulated at the  20% Nycodenz/20% Ficoll  interface of the step gradient  (see Fig. 2). These membrane-bounded organelles  from the interface peak fractions were immunolabeled  with antibodies to both cathepsin L and avidin. (c) Electron-dense lysosomes that could be immunolabeled with anti–cathepsin L antibodies accumulated at the 45%/20% Nycodenz interface of the  step gradient after the content mixing assay. Bar, 200 nm.
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Figure 4: Immunoelectron microscopy identifying avidin (8-nm gold) and cathepsin L (15-nm gold) immunoreactivity in organelles from late endosome, lysosome, and hybrid fractions. (a) A late endosome peak fraction from the preparative 1–22% Ficoll gradient showing an endosomal structure containing Av-ASF. (b) Hybrid organelles formed after the content mixing assay incubation accumulated at the 20% Nycodenz/20% Ficoll interface of the step gradient (see Fig. 2). These membrane-bounded organelles from the interface peak fractions were immunolabeled with antibodies to both cathepsin L and avidin. (c) Electron-dense lysosomes that could be immunolabeled with anti–cathepsin L antibodies accumulated at the 45%/20% Nycodenz interface of the step gradient after the content mixing assay. Bar, 200 nm.

Mentions: Representative micrographs demonstrating the morphology of the hybrid organelles and of lysosome and late endosome preparations are shown in Fig. 4 (8-nm gold particles label immunoreactive avidin and 15-nm gold particles label cathepsin L). Material from the lysosomal peak at the 45%/20% Nycodenz interface contained mostly electron-dense structures labeled with cathepsin L, but little avidin (Fig. 4 c). Quantitation of avidin- and cathepsin L–labeled structures in the late endosome preparation used in the fusion assay showed that many endosomes were heavily loaded with avidin but contained very little cathepsin L (Fig. 3). However, since a significant proportion of cathepsin L–positive, avidin-negative structures were also observed in the endosomal preparation, late endosomes were incubated in the content mixing assay in the absence of lysosomes at 37°C for 10 min. No membrane could be recovered from the Nycodenz/Ficoll interface of a step gradient after such an incubation (data not shown).


Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.

Mullock BM, Bright NA, Fearon CW, Gray SR, Luzio JP - J. Cell Biol. (1998)

Immunoelectron  microscopy identifying avidin (8-nm gold) and cathepsin L (15-nm gold) immunoreactivity in organelles  from late endosome, lysosome, and hybrid fractions.  (a) A late endosome peak  fraction from the preparative  1–22% Ficoll gradient showing an endosomal structure  containing Av-ASF. (b) Hybrid organelles formed after  the content mixing assay incubation accumulated at the  20% Nycodenz/20% Ficoll  interface of the step gradient  (see Fig. 2). These membrane-bounded organelles  from the interface peak fractions were immunolabeled  with antibodies to both cathepsin L and avidin. (c) Electron-dense lysosomes that could be immunolabeled with anti–cathepsin L antibodies accumulated at the 45%/20% Nycodenz interface of the  step gradient after the content mixing assay. Bar, 200 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140175&req=5

Figure 4: Immunoelectron microscopy identifying avidin (8-nm gold) and cathepsin L (15-nm gold) immunoreactivity in organelles from late endosome, lysosome, and hybrid fractions. (a) A late endosome peak fraction from the preparative 1–22% Ficoll gradient showing an endosomal structure containing Av-ASF. (b) Hybrid organelles formed after the content mixing assay incubation accumulated at the 20% Nycodenz/20% Ficoll interface of the step gradient (see Fig. 2). These membrane-bounded organelles from the interface peak fractions were immunolabeled with antibodies to both cathepsin L and avidin. (c) Electron-dense lysosomes that could be immunolabeled with anti–cathepsin L antibodies accumulated at the 45%/20% Nycodenz interface of the step gradient after the content mixing assay. Bar, 200 nm.
Mentions: Representative micrographs demonstrating the morphology of the hybrid organelles and of lysosome and late endosome preparations are shown in Fig. 4 (8-nm gold particles label immunoreactive avidin and 15-nm gold particles label cathepsin L). Material from the lysosomal peak at the 45%/20% Nycodenz interface contained mostly electron-dense structures labeled with cathepsin L, but little avidin (Fig. 4 c). Quantitation of avidin- and cathepsin L–labeled structures in the late endosome preparation used in the fusion assay showed that many endosomes were heavily loaded with avidin but contained very little cathepsin L (Fig. 3). However, since a significant proportion of cathepsin L–positive, avidin-negative structures were also observed in the endosomal preparation, late endosomes were incubated in the content mixing assay in the absence of lysosomes at 37°C for 10 min. No membrane could be recovered from the Nycodenz/Ficoll interface of a step gradient after such an incubation (data not shown).

Bottom Line: Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein.We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes.The consequences of this fusion to the maintenance and function of lysosomes are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom.

ABSTRACT
Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37 degrees C, late endosome-lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

Show MeSH