Mast cell-specific gangliosides and FcepsilonRI follow the same endocytic pathway from lipid rafts in RBL-2H3 cells.
Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Oliver
Constance
C
Department of Cell and Molecular Biology and Pathogenic Bioagents, Faculdade de Medicina de Ribeirão Preto-Universidade de São Paulo, Ribeirão Preto, SP, Brazil. coliver@fmrp.usp.br
Fujimura
Akira
A
Silveira E Souza
Adriana Maria Mariano
AM
Orlandini de Castro
Rodrigo
R
Siraganian
Reuben P
RP
Jamur
Maria Celia
MC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D045744:Cell Line, Tumor; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology*; D005732:Gangliosides / Q000276:immunology / Q000378:metabolism*; D017979:Gold Colloid / Q000737:chemistry / Q000276:immunology; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008407:Mast Cells / Q000737:chemistry; D021962:Membrane Microdomains / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D016253:Microscopy, Immunoelectron; D017455:Receptors, IgE / Q000378:metabolism*; D015398:Signal Transduction
D000911:Antibodies, Monoclonal; D005732:Gangliosides; D017979:Gold Colloid; D017455:Receptors, IgE; D006735:Horseradish Peroxidase