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

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 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.

The Plant Defense Elicitor Cryptogein Stimulates Clathrin-Mediated Endocytosis Correlated with Reactive Oxygen Species Production in Bright Yellow-2 Tobacco Cells

“…However, both indicate very early stimulation of endocytosis upon elicitor treatment. Moreover, these results can be related to the work of Fomina et al (2003), who used FM1-43 dye to show accelerated endocytosis in phytohemagglutininactivated lymphocyte T cells compared to resting cells and argued that the dye follows the CCV pathway within cells. Finally, very recently in Arabidopsis and tobacco BY-2 cells, genetic interference using the C-terminal clathrin heavy chain (so-called hub domain), which prevents clathrin cage formation, has been shown to inhibit FM4-64 uptake proving for a large part its clathrin dependence (Dhonukshe et al, 2007;Tahara et al, 2007).…”

Exosomes as intercellular signalosomes and pharmacological effectors

“…It has been argued however that many works reporting internalization actually monitored the fate of exosome aggregates, since exosomes spontaneously aggregate in-vitro. However exosomes exit the cell as aggregates in activated T cells [80], and natural transfer of exosomes between two different type of cells in co-culture by macropinocytosis has been recently reported [81]. In hematopoietic-derived cell lines (K562, MT4) exosomes were shown to enter cells via phagocytosis and not by macropinocytosis or by internalization pathways involving caveolae or clathrin-coated vesicles [76].…”

“…This observation indicates that the MVB compartment features a dual function of being a producer of exosomes but also a receiver of exogenous exosomes. In each case a different subset of MVB would be involved, since exosome release is sectorized in the cell [80] and appearsnot involve all the MVBs. In that respect two distincts MVB pathways, one for lysosomal targeting and the other for exosome secretion have been characterized [17].…”

Therapeutic and diagnostic applications of extracellular vesicles

“…The elimination after IV injection occurs via hepatic and renal routes [117] in which MPS-associated macrophages seem to play a key role [118]. It is conceivable that this recognition is in part mediated by the exposure of PS at the external side of EV (subtypes) [120,121]. It is of note that in these studies tumor-or HEK 293T-derived EVs have been used.…”