Regulation of membrane trafficking and subcellular organization of endocytic compartments revealed with FM1-43 in resting and activated human T cells

• Published in: Experimental cell research 2003-11, Vol.291 (1), p.150-166
• Main Authors: Fomina, Alla F, Deerinck, Thomas J, Ellisman, Mark H, Cahalan, Michael D
• Format: Article
• Language: English
• Subjects: Actins - drug effects; Actins - metabolism; Calcium; Calcium - metabolism; Cell Compartmentation - drug effects; Cell Compartmentation - physiology; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cells, Cultured; Cytoskeleton; Endocytosis; Endocytosis - drug effects; Endocytosis - physiology; Endosomes; Enzyme Inhibitors - pharmacology; Exosomes; Humans; Kinetics; Lymphocyte Activation - drug effects; Lymphocyte Activation - physiology; Lysosomes - drug effects; Lysosomes - metabolism; Membrane Microdomains - drug effects; Membrane Microdomains - metabolism; Microscopy, Electron; Organelles - metabolism; Photoconversion; Protein Transport - drug effects; Protein Transport - physiology; Pyridinium Compounds - pharmacokinetics; Quaternary Ammonium Compounds - pharmacokinetics; T-Lymphocytes - metabolism; T-Lymphocytes - ultrastructure; Temperature; Transport Vesicles - metabolism; Transport Vesicles - ultrastructure
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/S0014-4827(03)00372-0

FM1-43, a fluorescent styryl dye that penetrates into and stains membranes, was used to investigate kinetics of constitutive endocytosis and to visualize the fate of endocytic organelles in resting and activated human T lymphocytes. The rate of dye accumulation was strongly temperature dependent and ∼10-fold higher in activated than in resting T cells. Elevation of cytosolic free Ca 2+ concentration with thapsigargin or ionomycin further accelerated the rate of FM1-43 accumulation associated with cytosolic actin polymerization. Direct modulation of actin polymerization affected membrane trafficking. Actin condensation beneath the plasma membrane with calyculin A abolished FM1-43 internalization, whereas actin depolymerization with cytochalasin D had no effect. Photoconversion of DAB by FM1-43 revealed altered endocytic compartment targeting associated with T cell activation. Internalized cargo was carried to lysosome-like compartments in resting T cells and to multivesicular bodies (MVB) in activated T cells. Externalization of exosomes from MVB occurred commonly in activated but not in resting T cells. T cell exosomes contained raft-associated CD3 proteins, GM1 glycosphingolipids, and phosphatidylserine at the outer membrane leaflet. The present study demonstrates the utility of FM1-43 as a marker of membrane trafficking in T cells and reveals possible mechanisms of its modulation during T cell activation.