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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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Diagrammatic representation of the  direct fusion of late endosomes with lysosomes to form a hybrid organelle of intermediate density. Binding of late endosomes to  lysosomes may be mediated by filamentous  structures that have been observed by electron microscopy. The formation of the hybrid  organelle requires NSF and SNAPs and is inhibited by GDI, although the exact step at  which they function and their order of interaction are unknown. Previous experiments  with NRK cells (Bright et al., 1997) suggest  that dense-core lysosomes may reform from  hybrid organelles. This requires recondensation of contents and probably retrieval of  membrane to an unknown location, possibly  to an earlier site in the endocytic pathway. cat  L, cathepsin L-positive structures.
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Figure 8: Diagrammatic representation of the direct fusion of late endosomes with lysosomes to form a hybrid organelle of intermediate density. Binding of late endosomes to lysosomes may be mediated by filamentous structures that have been observed by electron microscopy. The formation of the hybrid organelle requires NSF and SNAPs and is inhibited by GDI, although the exact step at which they function and their order of interaction are unknown. Previous experiments with NRK cells (Bright et al., 1997) suggest that dense-core lysosomes may reform from hybrid organelles. This requires recondensation of contents and probably retrieval of membrane to an unknown location, possibly to an earlier site in the endocytic pathway. cat L, cathepsin L-positive structures.

Mentions: In the present study we have demonstrated the direct fusion of preexisting dense lysosomes with late endosomes. By morphological examination of NRK cells that had endocytosed colloidal gold–albumin conjugates, we have previously obtained evidence that dense-core lysosomes fuse with late endosomes in living cells to form hybrid compartments (Bright et al., 1997). In both that and other studies (van Deurs et al., 1995; Futter et al., 1996), fine striations were sometimes observed between bound endosomes and lysosomes. These may contribute to the binding involved in the interaction of late endosomes with lysosomes. The formation of a hybrid organelle as a result of lysosome– endosome fusion, which has now been observed both in a cell-free system and in vivo (Bright et al., 1997), suggests the presence of a membrane recovery system to avoid the build up of swollen hybrid structures within the cell. This is likely to be a very dynamic process allowing rapid retrieval of membrane proteins present in endosomes but not lysosomes and ensuring that individual hybrid organelles exist only for a short period of time unless further fusion events take place. It is also of interest that in the present cell-free system, a nonhydrolyzable GTP analogue added to the preincubation of late endosomes caused inhibition of subsequent content mixing between endosomes and lysosomes, suggesting that vesicles were being formed from late endosomes but were not destined for fusion with lysosomes. There is accumulating evidence that coat proteins exist on endosomes and lysosomes and could play a role in membrane retrieval (Seaman et al., 1993; Whitney et al., 1995; Aniento et al., 1996; Stoorvogel et al., 1996; Traub et al., 1996). A diagrammatic representation of the processes involved in endosome–lysosome fusion and subsequent recovery from the hybrid organelle is shown in Fig. 8. In vivo, the endosome–lysosome hybrid almost certainly recondenses to a dense lysosome by a combination of membrane retrieval and digestive processes (Bright et al., 1997). The ability to separate the hybrids from both late endosomes and dense lysosomes should enable us to examine the condensation process, as well as to search for the proteins involved in controlling the fusion reaction.


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)

Diagrammatic representation of the  direct fusion of late endosomes with lysosomes to form a hybrid organelle of intermediate density. Binding of late endosomes to  lysosomes may be mediated by filamentous  structures that have been observed by electron microscopy. The formation of the hybrid  organelle requires NSF and SNAPs and is inhibited by GDI, although the exact step at  which they function and their order of interaction are unknown. Previous experiments  with NRK cells (Bright et al., 1997) suggest  that dense-core lysosomes may reform from  hybrid organelles. This requires recondensation of contents and probably retrieval of  membrane to an unknown location, possibly  to an earlier site in the endocytic pathway. cat  L, cathepsin L-positive structures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140175&req=5

Figure 8: Diagrammatic representation of the direct fusion of late endosomes with lysosomes to form a hybrid organelle of intermediate density. Binding of late endosomes to lysosomes may be mediated by filamentous structures that have been observed by electron microscopy. The formation of the hybrid organelle requires NSF and SNAPs and is inhibited by GDI, although the exact step at which they function and their order of interaction are unknown. Previous experiments with NRK cells (Bright et al., 1997) suggest that dense-core lysosomes may reform from hybrid organelles. This requires recondensation of contents and probably retrieval of membrane to an unknown location, possibly to an earlier site in the endocytic pathway. cat L, cathepsin L-positive structures.
Mentions: In the present study we have demonstrated the direct fusion of preexisting dense lysosomes with late endosomes. By morphological examination of NRK cells that had endocytosed colloidal gold–albumin conjugates, we have previously obtained evidence that dense-core lysosomes fuse with late endosomes in living cells to form hybrid compartments (Bright et al., 1997). In both that and other studies (van Deurs et al., 1995; Futter et al., 1996), fine striations were sometimes observed between bound endosomes and lysosomes. These may contribute to the binding involved in the interaction of late endosomes with lysosomes. The formation of a hybrid organelle as a result of lysosome– endosome fusion, which has now been observed both in a cell-free system and in vivo (Bright et al., 1997), suggests the presence of a membrane recovery system to avoid the build up of swollen hybrid structures within the cell. This is likely to be a very dynamic process allowing rapid retrieval of membrane proteins present in endosomes but not lysosomes and ensuring that individual hybrid organelles exist only for a short period of time unless further fusion events take place. It is also of interest that in the present cell-free system, a nonhydrolyzable GTP analogue added to the preincubation of late endosomes caused inhibition of subsequent content mixing between endosomes and lysosomes, suggesting that vesicles were being formed from late endosomes but were not destined for fusion with lysosomes. There is accumulating evidence that coat proteins exist on endosomes and lysosomes and could play a role in membrane retrieval (Seaman et al., 1993; Whitney et al., 1995; Aniento et al., 1996; Stoorvogel et al., 1996; Traub et al., 1996). A diagrammatic representation of the processes involved in endosome–lysosome fusion and subsequent recovery from the hybrid organelle is shown in Fig. 8. In vivo, the endosome–lysosome hybrid almost certainly recondenses to a dense lysosome by a combination of membrane retrieval and digestive processes (Bright et al., 1997). The ability to separate the hybrids from both late endosomes and dense lysosomes should enable us to examine the condensation process, as well as to search for the proteins involved in controlling the fusion reaction.

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