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Diversity of raft-like domains in late endosomes.

Sobo K, Chevallier J, Parton RG, Gruenberg J, van der Goot FG - PLoS ONE (2007)

Bottom Line: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking.Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.Implications of these findings for late endosomal functions are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.

ABSTRACT

Background: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains.

Methodology/principal findings: Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.

Conclusions/significance: In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed.

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Related in: MedlinePlus

Immunoelectron microscopic localization of flotillin-1 and GPI-anchored proteins on multivesicular endosomes.Cultured C2C12 cells were fixed in paraformaldehyde and processed for frozen sectioning. Sections were labeled with antibodies to flotillin-1 and 15 nm protein A-gold and then overlaid with aerolysin-biotin followed by 10 nm anti-biotin-gold. Aerolysin labeling for GPI-anchored proteins is mainly within the internal membranes of the late endosomes. In contrast, flotillin-1 labeling (large arrowheads) is predominantly associated with the limiting membrane (small arrowheads). Bars, 100 nm.
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pone-0000391-g005: Immunoelectron microscopic localization of flotillin-1 and GPI-anchored proteins on multivesicular endosomes.Cultured C2C12 cells were fixed in paraformaldehyde and processed for frozen sectioning. Sections were labeled with antibodies to flotillin-1 and 15 nm protein A-gold and then overlaid with aerolysin-biotin followed by 10 nm anti-biotin-gold. Aerolysin labeling for GPI-anchored proteins is mainly within the internal membranes of the late endosomes. In contrast, flotillin-1 labeling (large arrowheads) is predominantly associated with the limiting membrane (small arrowheads). Bars, 100 nm.

Mentions: The segregation between GPI-anchored proteins and flotillin-1 was confirmed by electron microscopy using C2C12 cells (Fig. 5, the flotillin-1 antibodies showed negligible labeling by immunoelectron microscopy on BHK cells). For quantifications, frozen sections were double labeled for GPI-anchored proteins (using aerolysin) and flotillin-1. Well-preserved multivesicular late endosomes were examined at random and gold particles (nā€Š=ā€Š450) were assigned to the limiting membrane or to internal membranes. For 85% of late endosomes, flotillin-1 labeling was higher on limiting membranes with a ratio of 4.8 to 1. On 15% of late endosomes, flotillin-1 was however more abundant on internal membranes leading to an over all ratio of labeling on limiting vs. internal membranes of 3.1 .to 1. The distribution of GPI-anchored proteins was the reverse with a ratio of limiting to internal membranes of 0.3 to 1 for 80% of late endosomes. Again 20% of late endosomes behaved differently showing a higher GPI labeling on the limiting membrane leading to an over all labeling ratio of 0.44 to 1 of limiting to internal membranes.


Diversity of raft-like domains in late endosomes.

Sobo K, Chevallier J, Parton RG, Gruenberg J, van der Goot FG - PLoS ONE (2007)

Immunoelectron microscopic localization of flotillin-1 and GPI-anchored proteins on multivesicular endosomes.Cultured C2C12 cells were fixed in paraformaldehyde and processed for frozen sectioning. Sections were labeled with antibodies to flotillin-1 and 15 nm protein A-gold and then overlaid with aerolysin-biotin followed by 10 nm anti-biotin-gold. Aerolysin labeling for GPI-anchored proteins is mainly within the internal membranes of the late endosomes. In contrast, flotillin-1 labeling (large arrowheads) is predominantly associated with the limiting membrane (small arrowheads). Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1851096&req=5

pone-0000391-g005: Immunoelectron microscopic localization of flotillin-1 and GPI-anchored proteins on multivesicular endosomes.Cultured C2C12 cells were fixed in paraformaldehyde and processed for frozen sectioning. Sections were labeled with antibodies to flotillin-1 and 15 nm protein A-gold and then overlaid with aerolysin-biotin followed by 10 nm anti-biotin-gold. Aerolysin labeling for GPI-anchored proteins is mainly within the internal membranes of the late endosomes. In contrast, flotillin-1 labeling (large arrowheads) is predominantly associated with the limiting membrane (small arrowheads). Bars, 100 nm.
Mentions: The segregation between GPI-anchored proteins and flotillin-1 was confirmed by electron microscopy using C2C12 cells (Fig. 5, the flotillin-1 antibodies showed negligible labeling by immunoelectron microscopy on BHK cells). For quantifications, frozen sections were double labeled for GPI-anchored proteins (using aerolysin) and flotillin-1. Well-preserved multivesicular late endosomes were examined at random and gold particles (nā€Š=ā€Š450) were assigned to the limiting membrane or to internal membranes. For 85% of late endosomes, flotillin-1 labeling was higher on limiting membranes with a ratio of 4.8 to 1. On 15% of late endosomes, flotillin-1 was however more abundant on internal membranes leading to an over all ratio of labeling on limiting vs. internal membranes of 3.1 .to 1. The distribution of GPI-anchored proteins was the reverse with a ratio of limiting to internal membranes of 0.3 to 1 for 80% of late endosomes. Again 20% of late endosomes behaved differently showing a higher GPI labeling on the limiting membrane leading to an over all labeling ratio of 0.44 to 1 of limiting to internal membranes.

Bottom Line: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking.Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.Implications of these findings for late endosomal functions are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.

ABSTRACT

Background: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains.

Methodology/principal findings: Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.

Conclusions/significance: In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed.

Show MeSH
Related in: MedlinePlus