Regulation of membrane trafficking and subcellular organization of endocytic compartments revealed with FM1-43 in resting and activated human T cells.
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 approximately 10-fold higher in activated than in resting T cells. Elevation of cytosolic free Ca2+ 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.
Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.
Alla F
AF
Fomina
Thomas J
TJ
Deerinck
Mark H
MH
Ellisman
Michael D
MD
Cahalan
D000199:Actins / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects / Q000502:physiology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D008854:Microscopy, Electron; D015388:Organelles / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D013601:T-Lymphocytes / Q000378:metabolism* / Q000648:ultrastructure; D013696:Temperature; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure
D000199:Actins; D004791:Enzyme Inhibitors; C073804:FM1 43; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D002118:Calcium