Cell Staining: Fluorescent Labelling of Endocytic Compartments

Abstract

Endocytic compartments are acidic organelles involved in the transport of extracellular solutes and ligands from the plasma membrane to early, then to late endosomes and eventually to lysosomes. Endocytic compartments can be fluorescently labelled by fluid phase endocytosis, internalized specific ligands, resident proteins and pH‐sensitive probes.

Keywords: endocytosis; endosome; lysosome; membrane traffic; green fluorescent protein

Figure 1.

Schematic view of the endocytic pathway. Ligands and solutes are internalized from the external medium and reach the early endosomes after 5–10 min. Materials can be recycled to the plasma membrane through the recycling endosomes or subsequently transferred to late endosomes (15–30 min) and eventually to lysosomes (40–60 min) for degradation. Stationary endocytic organelles can be identified by specific markers: (1) EEA1, Rab5, the transferrin receptor for early endosomes; (2) Rab4 and the transferrin receptor for the recycling compartment; (3) Rab7, the cation‐independent mannose 6‐phosphate receptor (CI‐MPR) for late endosomes; (4) lysosomal hydrolases such as cathepsin D and lysosomal membrane glycoproteins (LAMP1, LAMP2, CD63), vacuolar ATPase for lysosomes. A lipid, the lysobisphosphatidic acid (LBPA), has been described to be enriched in late endocytic compartments (Kobayashi et al., ).

Figure 2.

Examples of fluorescent labelling of endocytic compartments. (a) Fluorescent labelling of early and late endosomes. HeLa cells were allowed to internalize fluorescein isothiocyanate (FITC)‐transferrin (green), fixed/permeabilized and then incubated with an anti‐Rab7 antibody revealed by a Texas red‐conjugated secondary antibody (red). The dotted line delimits the nucleus (N). (b) LAMP1‐GFP is recruited on vacuoles surrounding intracellular bacteria (arrowheads). Live HeLa cells expressing LAMP1‐GFP were infected with Salmonella typhimurium and directly observed under an epifluorescence microscope. (c) In vitro reconstitution of early endosome fusion. Isolated early endosomes containing FITC‐BSA (green) or rhodamine‐BSA (red) were mixed in a fusion‐competent assay. The yellow colour results from the mixing of contents of both fluorescent probes. The scale bars represent (a) 10 μm, (b) 5 μm and (c) 1 μm.

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Further Reading

Gruenberg J and Maxfield FR (1995) Membrane transport in the endocytic pathway. Current Opinion in Cell Biology 7(4): 552–563.

Ellenberg J, Lippincott‐Schwartz J and Presley JF (1999) Dual‐colour imaging with GFP variants. Trends in Cell Biology 9(2): 52–56.

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Wilson T and Hastings JW (1998) Bioluminescence. Annual Review of Cell and Developmental Biology 14: 197–230.

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How to Cite close
Gorvel, Jean‐Pierre, and Méresse, Stéphane(Apr 2001) Cell Staining: Fluorescent Labelling of Endocytic Compartments. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002630]