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

Alla F Fomina, Thomas J. Deerinck, Mark H. Ellisman, Michael D. Cahalan

Research output: Contribution to journalArticle

61 Scopus citations

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 ∼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.

Original languageEnglish (US)
Pages (from-to)150-166
Number of pages17
JournalExperimental Cell Research
Volume291
Issue number1
DOIs
StatePublished - Nov 15 2003

Keywords

  • Calcium
  • Cytoskeleton
  • Endocytosis
  • Endosomes
  • Exosomes
  • Photoconversion

ASJC Scopus subject areas

  • Cell Biology

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