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

Exp Cell Res. 2003 Nov 15;291(1):150-66. doi: 10.1016/s0014-4827(03)00372-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / drug effects
  • Actins / metabolism
  • Calcium / metabolism
  • Cell Compartmentation / drug effects
  • Cell Compartmentation / physiology*
  • Cell Membrane / metabolism*
  • Cell Membrane / ultrastructure
  • Cells, Cultured
  • Endocytosis / drug effects
  • Endocytosis / physiology*
  • Enzyme Inhibitors / pharmacology
  • 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
  • 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

Substances

  • Actins
  • Enzyme Inhibitors
  • FM1 43
  • Pyridinium Compounds
  • Quaternary Ammonium Compounds
  • Calcium