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Mast Cell-specific Gangliosides and FcRI Follow the Same Endocytic Pathway From Lipid Rafts in RBL-2H3 Cells

Constance Oliver, Akira Fujimura, Adriana Maria Mariano Silveira e Souza, Rodrigo Orlandini de Castro, Reuben P. Siraganian and Maria Celia Jamur

Department of Cell and Molecular Biology and Pathogenic Bioagents, Faculdade de Medicina de Ribeirão Preto–Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil (CO,AMMSS,ROC,MCJ); Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland (AF,RPS); and First Department of Oral Anatomy, School of Dentistry, Iwate Medical University, Morioka, Japan (AF)

Correspondence to: Constance Oliver, PhD, Department of Cell and Molecular Biology and Pathogenic Bioagents, Faculdade de Medicina de Ribeirão Preto–Universidade de São Paulo, Ribeirão Preto, SP, Brazil. E-mail: coliver{at}fmrp.usp.br

Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcRI 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 FcRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcRI 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 FcRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 55:315–325, 2007)

Key Words: mast cells • FcRI • lipid rafts • endocytosis • gangliosides • electron microscopy

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