regulation of membrane trafﬁcking and subcellular organization …crt.biomol.uci.edu/pub/pdf/2003...

Regulation of membrane trafficking and subcellular organizationof endocytic compartments revealed with FM1-43 in resting

and activated human T cells

Alla F. Fomina,a,1 Thomas J. Deerinck,b Mark H. Ellisman,b and Michael D. Cahalana,*a Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA

b National Center for Microscopy and Imaging Research, Center for Research in Biological Structure and the Department of Neurosciences,University of California, San Diego, La Jolla, CA 92093, USA

Received 21 March 2003, revised version received 18 June 2003

Abstract

FM1-43, a fluorescent styryl dye that penetrates into and stains membranes, was used to investigate kinetics of constitutive endocytosisand to visualize the fate of endocytic organelles in resting and activated human T lymphocytes. The rate of dye accumulation was stronglytemperature dependent and �10-fold higher in activated than in resting T cells. Elevation of cytosolic free Ca2� concentration withthapsigargin or ionomycin further accelerated the rate of FM1-43 accumulation associated with cytosolic actin polymerization. Directmodulation of actin polymerization affected membrane trafficking. Actin condensation beneath the plasma membrane with calyculin Aabolished FM1-43 internalization, whereas actin depolymerization with cytochalasin D had no effect. Photoconversion of DAB by FM1-43revealed altered endocytic compartment targeting associated with T cell activation. Internalized cargo was carried to lysosome-likecompartments in resting T cells and to multivesicular bodies (MVB) in activated T cells. Externalization of exosomes from MVB occurredcommonly in activated but not in resting T cells. T cell exosomes contained raft-associated CD3 proteins, GM1 glycosphingolipids, andphosphatidylserine at the outer membrane leaflet. The present study demonstrates the utility of FM1-43 as a marker of membrane traffickingin T cells and reveals possible mechanisms of its modulation during T cell activation.© 2003 Elsevier Inc. All rights reserved.

Keywords: Endocytosis; Endosomes; Exosomes; Calcium; Photoconversion; Cytoskeleton

Introduction

Membrane trafficking plays an important role in regulat-ing the surface expression of membrane proteins in T lym-phocytes and provides an important mechanism to modulateimmune responses. For example, T cell receptors (TCR)undergo continuous recycling [1–3], resulting in a steady-state level of receptor expression at the plasma membrane(PM). Receptor ligation by antigen/MHC complexes re-duces TCR expression on the T cell surface [4–7], either by

preventing recycling [2] or by shifting the equilibrium be-tween recycling/degradation pathways in favor of lysosomaldegradation [7,8]. Long-lasting down-regulation of TCRexpression is thought to be critical for termination of theimmune response [9,10]. Some viruses, including HIV, alterreceptor internalization/recycling mechanisms and shift theequilibrium in favor of receptor degradation to evade im-mune surveillance [11].

Despite the importance of constitutive membrane traf-ficking, the origin and fate of endosomal compartmentshave not been studied extensively in T lymphocytes, andkinetics of endocytosis and mechanisms of its regulation inT cells remain undefined. Lymphocyte activation by anti-gen/MHC induces T cell proliferation, lymphokine secre-tion, and changes in expression of membrane proteins.

* Corresponding author. Fax: �1-949-824-3143.E-mail address: [email protected] (M.D. Cahalan).1 Present address: Department of Human Physiology, University of

California, Davis, Davis, CA 95616.

R

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Experimental Cell Research 291 (2003) 150–166 www.elsevier.com/locate/yexcr

0014-4827/$ – see front matter © 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0014-4827(03)00372-0

However, effects of lymphocyte activation on membranetrafficking remain unclear. Several biochemically distinctcompartments for membrane uptake and turnover have beenidentified in other cell types, including primary endocyticvesicles, early endosomes (EE), late endosomes (LE), andlysosomes [12]. Hematopoietic cells possess LE that mayserve as a secretory compartment [13]. Vesiculated LE, alsoknown as multivesicular bodies (MVB), can fuse with theplasma membrane to release vesicles, termed exosomes[14–16]. Selected membrane and cytosolic proteins are re-leased with exosomes into the extracellular space. Exo-somes produced by dendritic cells enhance antigen presen-tation and have been shown to induce potent antitumorimmune responses in mice [17]. Human PHA/TCR-acti-vated T cells, T cell clones, and Jurkat T cells have beenfound to release microvesicles that contain MHC II mole-cules and several other PM and endosomal proteins into theculture medium [14,18,19], suggesting that externalizationof exosomes may also occur in T lymphocytes. Difficultiesin elucidating the mechanisms of forming and releasingvesicle-associated proteins have been compounded in Tcells because the fate of endocytic vesicles and the dynam-ics of transport intermediates remain uncertain. Structuralrelationships among endocytic vesicles, intracellular com-partments, and externalized material have not been estab-lished.

To address the issue of endocytic compartment biogen-esis in T cells it is necessary to elucidate the dynamicinteractions between membrane compartments. Substantialinsight into the dynamics of synaptic vesicle cycling hasbeen made using the styryl dye FM1-43 [20,21]. FM1-43 isan amphipathic molecule that intercalates spontaneouslyinto the outer leaflet of cell membranes without diffusingacross the membrane. Within the lipid environment,FM1-43 exhibits 50- to 100-fold increased fluorescenceintensity than the dye in aqueous solution. FM1-43 can beinternalized by vesicular endocytosis, and an increase inintracellular fluorescence can therefore be used as an indexof endocytosis. Moreover, intense illumination of FM1-43in fixed specimens in the presence of oxygen and diamino-benzidine (DAB) catalyzes the polymerization of DAB intoan insoluble osmiophilic reaction product that can be visu-alized by electron microscopy [22]. The photoconversionprocedure allows for identification of endocytic compart-ments in living cells by real-time fluorescence monitoring,followed by a snapshot with EM resolution, thereby permit-ting correlation of fluorescence measurements with the ul-trastructure [23]. Here, we introduce the use of the FM1-43to investigate real-time membrane trafficking dynamics andmechanisms of its regulation in human T lymphocytes.Photoconversion of FM1-43 allows definition of structuralintermediates of endocytic compartments, tracked from thePM to early and late endosomes and back to the PM withEM resolution.

Materials and methods

Cell culture and chemicals

Unless otherwise indicated, all chemicals were fromSigma (St. Louis, MO). Normal human peripheral bloodmononuclear cells were isolated using Accuspin System-Histoplaque-1077 density gradient separation tubes. CD3�

cells were purified using human T cell enrichment columns(R&D Systems, Minneapolis, MN) and cultured in mediacontaining 88% RPMI (Fisher Scientific, Pittsburgh, PA),1% MEM, 1% Na-pyruvate, 1% 1-glutamine, and 0.035�L/L �-mercaptoethanol supplemented with 10% FCS(Omega Scientific, Tarzana, CA). Unless otherwise indi-cated, T cells were stimulated in vitro with phytohemagglu-tinin P (PHA, 40 �g/ml, Difco, Detroit, MI) for 48–96 h toactivate T cell proliferation and lymphokine secretion. Insome experiments, T cells were activated with ionomycin(200 nM) � PMA (10 nM). Cells were placed on a poly-L-lysine-coated coverslip 10 min before the experiment.Apoptosis/necrosis of adherent cells was assessed by an-nexin V and propidium iodide staining (Vibrant ApoptosisAssay kit #2, Molecular Probes, Eugene, OR). Ionomycin,thapsigargin Calyculin A, and Cytochalasin D were fromCalbiochem (La Jolla, CA). FM1-43 and FM4-64 (Molec-ular Probes, Eugene, OR) were used at 5 �M final concen-tration in Tyrode solution.

Fluorescence measurements

A coverslip with adherent cells was mounted onto therecording chamber on the stage of a Zeiss Axiovert 10microscope. Cells were continuously superfused with exter-nal solutions of different composition. Most experimentswere done in modified Tyrode solution containing (in mM)160 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 5 Hepes, 10 glucose,pH � 7.3 adjusted with NaOH. For some experiments, Ca2�

and Mg2� were omitted from the solution and 5 mM EGTAwas added (Ca2�-free Tyrode solution). Fluorescence im-ages were acquired with a Biorad MRC 600 laser scanningconfocal imaging system using a 63x/1.25 n.a. oil immer-sion Zeiss objective. For FM1-43 and Alexa Fluor 488-conjugated annexin V and cholera-toxin B (MolecularProbes), 488 nm excitation and 515 nm emission filters wereused. For FM4-64 fluorescence, 514 nm excitation and 600nm emission filters were used. The pinhole was opened to20% of its maximal diameter. For kinetic analysis all imageswere taken at identical acquisition settings. Temperaturecontrol was achieved by heated/cooled water circulationaround the objective, and by continuous superfusion of therecording chamber with solution of the desired temperature.Perfusion solutions were delivered through a temperature-controlled multitube pre-heater (Cell Micro Controls, Vir-ginia Beach, VA) placed �100 �m from the field of view.

151A.F. Fomina et al. / Experimental Cell Research 291 (2003) 150–166

Kinetic analysis of FM1-43 accumulation

Off-line analysis was performed with Scion Image soft-ware (Scion Corporation, Frederick, MD). The total fluo-rescence intensity was measured in unprocessed imagesfrom the manually defined area within each individual cell(shown in Fig. 1). Plasma membrane and bright peripheralfluorescent patches stained immediately after dye applica-tion were excluded from consideration. Total fluorescenceintensity was normalized to the area and plotted againsttime. The value obtained from the first image acquired 30 safter application of FM1-43 was taken as a backgroundfluorescence and was subtracted from all measurements.The rate constants (r) were estimated by fitting the experi-mentally derived time courses of FM1-43 accumulationwith the first-order exponential function: [FM1-43]in(t) �[FM1-43]max � (1�e�rt), where [FM1-43]in(t) is a meanfluorescence intensity at a given time; [FM1-43]max is amean fluorescence intensity of maximally loaded dye; r isthe rate constant; and t is the time. Given the assumptionsthat dye did not change fluorescence intensity within theendocytic compartment and that dye did not recycle back tothe extracellular space to a significant degree during theshort incubation period, the r value approximates the rate ofconstitutive endocytosis. Because the cytosolic region isenlarged in activated T cells it is reasonable to expect that alarger area within the plane of focus could account for someof the increase in fluorescence intensity. From thin-sectionelectron microscope images, the ratio of nucleus to total celldiameter was 0.7 � 0.02 and 0.63 � 0.02 (n � 16) forresting and activated cells, respectively. From this, we es-timate that the increase in cytosolic area (outside of thenucleus) in activated compared to resting T cells wouldaccount for a 20%–25% increase in mean fluorescence in-tensity measured from confocal planes, but cannot accountfor the observed 10-fold increase. The relative change in therate constants for a 10 °C change in temperature, the Q10,was calculated by: Q10 � (r2/r1)10/(T

2�T1), where, r1 is therate constant of FM1-43 accumulation at lower temperatureT1 and r2 is the rate constant of FM1-43 accumulation athigher temperature T2. Arrhenius activation energies weredetermined from: Ea � (RT1T2/(T2�T1))xln(r2/r1), where Ris the gas constant and T1 and T2 are temperatures indegrees Kelvin. In practice, experimental data were trans-ferred into plots and Q10 and Ea determined from the slopesof linear regression fits of experimental data (Microcal Or-igin 6.0, Microcal Software, Inc., Northampton, MA). Allplots represent mean value � s.e.m.

Photoconversion of DAB and EM imaging

Photoconversion experiments were performed as de-scribed [24]. Briefly, living cells were loaded with FM1-43for different times and conditions as specified in Results.Cells were washed and then fixed with 2% glutaraldehyde(Electron Microscopy Sciences, Ft. Washington, PA) in

sodium cacodylate buffer (0.1 M, pH 7.4, Ted Pella Inc.,Redding, CA) for 20 min, rinsed in buffer, and treated for 5min in KCn (20 mM), aminotriazole (5 mM), glycine inbuffer (50 mM) to reduce nonspecific background. Forphotoconversion, diaminobenzidine (DAB, 1 mg/ml) in oxy-genated sodium cacodylate (0.1 M) was added to the cham-ber. DAB solution was refreshed every 3 min and cells wereilluminated by a 75 W xenon lamp through a Zeiss 63x/1.25n.a. objective using a 488 nm filter for 10 to 15 min until abrownish reaction product appeared in place of the greenfluorescence. Cells were then washed in buffer and post-fixed in 1% osmium tetroxide (Electron Microscopy Sci-ences) for 30 min. Cells were rinsed in distilled water,dehydrated in ethanol, embedded in Durcupan resin, andpolymerized at 60 °C for 48 h. To distinguish any nonspe-cific reaction between intracellular components and DAB,we performed photoconversion of DAB in cells that werenever exposed to FM1-43. Except for mitochondria, nononspecific staining of internal organelles was induced byillumination in the absence of FM1-43 even after 1.5 hexposure to light. The nonspecific staining of the mitochon-dria was easily recognizable and was excluded from con-sideration. Photoconversion of cells bathed with FM1-43-containing solution for �30 s before fixation revealed a thinring of electron-dense reaction product at the PM due to dyepartitioning into the lipid bilayer. All other images pre-sented in the results were taken from cells that were fixedafter FM1-43 was briefly washed from the extracellularsolution and therefore contained only remaining traces ofthe photoconversion reaction product at the PM. Morpho-metric measurement of intracellular organelles was per-formed in thin section images using Scion Image software.

Immunohistochemistry

Adherent cells were fixed with 4% paraformaldelhyde in2XPBS (Fisher Scientific, Pittsburgh, PA) for 30 min, thenwashed three times with 2X PBS for 15 min. Cells werepermeablilized in a saponin solution (0.075% w/v saponin,1% bovine serum albumin, 1% goat serum in 2X PBS) for1 h. For F-actin staining, permeablilized cells were incu-bated with 2 U/ml Oregon Green-conjugated phalloidin(Molecular Probes) in 2X PBS for 20 min at room temper-ature, washed three times with 2X PBS for 15 min, and thenmounted on slides with Slow Fade glycerol buffer (Molec-ular Probes). For TCR staining, permeablilized cells wereincubated with primary mouse anti-human CD3� antibodies(UCHT1, BD Biosciences Pharmingen, San Diego, CA)diluted 1:500 for 45 min at room temperature, washed threetimes with 2X PBS containing 2% of goat serum, and thenincubated with rabbit anti-mouse Alexa Fluor 488 second-ary antibodies (Molecular Probes) for 30 min at room tem-perature. Cells were washed three times with 2X PBS for 15min and then mounted on slides. For IL2 staining, primaryrat anti-human IL2 antibodies (BD Biosciences Pharmin-

152 A.F. Fomina et al. / Experimental Cell Research 291 (2003) 150–166

gen, San Diego, CA) were used as primary, and chickenanti-rat Alexa Fluor 488 antibodies (Molecular Probes)were used as secondary. Only background fluorescence wasdetected when permeabilized cells were stained with sec-ondary antibodies only. For the IL2 assay, cells were incu-bated with Golgi Stop solution (BD Biosciences, FranklinLakes, NJ) for 6 h before fixation.

Assessment of mitochondrial transmembrane potential(��m)

��m was measured using 3,3�-dihexyloxacarbocynineiodide (DiOC6(3), Molecular Probes) at a final concentra-tion of 40 nM in PBS (stock 1 mM in methanol). Adherentcells were incubated with DiOC6(3) for 15 min at 37 °C,

Fig. 1. FM1-43 accumulation in resting and activated T cells is temperature dependent. T cells were exposed to 5 �M FM1-43 in Tyrode solution for 21 minwhile confocal images were acquired every 7 min. Resting (A–F) and activated (G–O) T cells exposed to FM1-43 for 30 s (top panels), 21 min (middlepanels), and following 1 min wash (lower panels) at temperatures indicated above each column. Note the lack of nuclear staining and dye accumulation withinthe cytosol after washout. Scale bars are 10 �m. (P) Mean fluorescence intensity in resting and activated T cells plotted against time (left and middle panels).Intracellular fluorescence for each cell was measured within a circled area to exclude membrane fluorescence and then divided by the total measurement areato yield the mean fluorescence intensity. Bright peripheral fluorescence patches were excluded from analysis. Each time course is an average of fourexperiments from different cultures. Smooth lines are exponential fits of experimental data. At 34 °C, the rates of endocytosis were 0.07 min�1 in activatedT cells, compared to 0.008 min�1 in resting T cells. Arrhenius plot (right panel) of rate constants (r) of FM1-43 accumulation in activated T cells. T is theabsolute temperature. The activation energies (Ea) were calculated from the slopes of linear regression fits within the temperature ranges of 14–24 °C and24–34 °C. If not shown, the error bars are smaller than the symbol size.


followed by confocal imaging analysis (excitation 488 nm;emission 515 nm). As a positive control, the mitochondrialuncoupling agent carbonyl cyanide m-chlorophenyl-hydra-zone (CCCP) was applied for 10 min to the same cells atroom temperature, or apoptosis was induced by incubationof cells with 1 �M staurosporine (Calbiochem, San Diego,CA) for 6 h at 37 °C.

Results

Accelerated endocytosis in activated T cells

To visualize endocytosis in resting and activated humanT lymphocytes, we exposed cells to FM1-43 for varyingtimes and monitored fluorescence by confocal microscopy.FM1-43 rapidly stained the plasma membrane (PM) of bothresting and PHA-activated T cells as a result of dye parti-tioning into the lipid bilayer (Fig. 1 top panels). In resting Tcells, the ring of staining indicated a uniform distribution ofdye within the PM. In activated T cells, brightly fluorescentpatches averaging 2.4 � 1.4 �m in size (n � 271) appearedat the cell periphery immediately after addition of FM1-43to the extracellular solution, simultaneously with PM stain-ing (Fig. 1G, J, and M). Surface membrane staining andperipheral patches of FM1-43 fluorescence did not changeafter several minutes of exposure to FM1-43 (Fig. 1, middlepanels). Upon washout of FM1-43, PM staining and bright-ness of the peripheral patches was substantially reduced, butperipheral patches remained visible for several minutes(Fig. 1, lower panels). The bright peripheral patches, foundonly in activated T cells, consisted of clusters of externalvesicles adherent to the PM, as shown below by electronmicroscopy (EM). After several minutes of exposure, inaddition to staining the cell surface, FM1-43 accumulatedinside the cell but was excluded from the nucleus. Afterwashout, dye was retained within the cells (Fig. 1, lowerpanels). Intracellular accumulation was especially apparentin activated T cells at or above room temperature. Similarresults were obtained with T cells activated by ionomycin �phorbol myristate acetate (PMA, data not shown).

The accumulation of dye within T cells may reflectconstitutive endocytosis. Consistent with this mechanism,we found that rates of dye uptake were strongly temperaturedependent. In resting T cells, intracellular FM1-43 accumu-lation was not observed after 20 min of incubation at 24 °C(Fig. 1A–C), but patches of FM1-43 fluorescence appearedwithin the cytosol at 34 °C (Fig. 1D–F). Activated T cellsaccumulated substantially more dye than resting T cells at

24 °C (Fig. 1J–L), and dye accumulation was stronglyenhanced at 34 °C (Fig. 1M–O) but completely blocked at14 °C (Fig. 1G–I). To assess endocytosis quantitatively, wefitted the time course of intracellular FM1-43 fluorescenceaccumulation in resting and activated T cells at differenttemperatures with first-order exponential functions (Fig.1P). The rate constant (r) of dye uptake was strongly de-pendent upon temperature and was approximately one orderof magnitude larger at any given temperature in activated Tcells, consistent with a strong enhancement of the rate ofendocytosis, compared to resting T cells (Fig. 1P, left andmiddle panels). An Arrhenius plot of the temperature de-pendence of r in activated T cells (Fig. 1P, right panel)deviates from a straight line with an inflection point in thevicinity of 24 °C, indicating that the temperature depen-dence is stronger at lower temperatures. The energies ofactivation (Ea) calculated from the slopes of linear regres-sion fits are 153 kJ/mol (Q10 � 7.8) and 76 kJ/mol (Q10 �3.9) in the lower (14–24 °C) and high (24–34 °C) temper-ature ranges, respectively. The nonlinear Arrhenius plotmay indicate that lipids undergo a phase transition [25] andthat membrane fluidity is a rate-limiting factor for consti-tutive endocytosis in T cells.

Rate of endocytosis increased by elevated [Ca2�]i

Ca2� plays an important role in shaping T cell responses[26]. In addition, Ca2� regulates endocytosis in neuronalcells and hematopoeitic cells [27,28]. Therefore, we ex-plored the effects of Ca2� on rates of FM 1-43 dye accu-mulation, using either ionomycin, a Ca2� ionophore, orthapsigargin, an inhibitor of the SERCA pump Ca2�-ATP-ase, to elevate cytosolic free Ca2� concentration ([Ca2�]i).Both ionomycin (Fig. 2A–C) and thapsigargin (Fig. 2D–F)significantly accelerated the rate of FM1-43 accumulation inactivated T cells, in comparison to control cells (Fig. 1J–Land Fig. 2J). Ionomycin also increased the rate of FM1-43accumulation in resting T cells but to a lesser extent than inactivated T cells (n � 4, data not shown). Removal ofexternal Ca2� did not affect the basal rate of FM1-43internalization (Fig. 2G–I, J), indicating that extracellularCa2� influx is not required to maintain the basal rate ofmembrane internalization. Consistent with the previousfinding by Zweifach [29], �10% of cells displayed annexinV staining around the entire plasma membrane (not shown)and increased FM1-43 surface fluorescence, presumablydue to phosphatidylserine (PS) scrambling. These cells wereexcluded from analysis. Our data demonstrate that the rateof endocytosis in human T cells is accelerated by elevated

Fig. 2. Elevation of [Ca2�]i accelerates the rate of FM1-43 accumulation in activated T cells. Activated T cells exposed to FM1-43 for 30 s (A, D, G), and21 min (B, E, H) in the presence of 0.5 �M ionomycin (B, C); 1 �M thapsigargin (E, F); and in Ca2�-free Tyrode solution (G–I). All experiments were doneat 24 °C. Scale bars are 10 �m. (J) Time courses of FM1-43 accumulation in normal Tyrode solution (closed circles); in Ca2�-free Tyrode solution (opencircles); in the presence of 0.1 �M (closed squares) and 0.5 �M (closed triangles) ionomycin; and in the presence of 1 �M thapsigargin (open squares). Eachtime course is an average of six experiments from different cultures.


[Ca2�]i although basal endocytosis does not require Ca2�

influx.

Effects of cytoskeletal modulators

Engagement of T cell receptors during antigen presenta-tion results in dramatic changes in cytoskeleton organiza-tion [30]. We tested whether changes in actin polymeriza-tion affect the rate of membrane trafficking in T cells. Innormal Tyrode solution, F-actin was distributed uniformlynear the membrane as revealed with phalloidin staining(Fig. 3A). Elevation of [Ca2�]i with thapsigargin producedactin polymerization in the cytosol (Fig. 3B) and, as shownabove (Fig. 2), was accompanied by an increased rate ofmembrane internalization. Incubation of T cells with cy-tochalasin D (Cyto D), a membrane permeable inhibitor ofactin polymerization [31], induced formation of numerousprocesses and PM shedding. In these cells, tight aggregatesof polymerized actin were located in cell extremities (Fig.3C), and some patches were found within the cytosol. Thepolymerized actin layer that appeared to be resistant to CytoD was observed in cell processes and beneath the PM. Thesefindings are compatible with observations of cytoskeletalrearrangement in other cell types [31,32]. Monitoring ofFM1-43 fluorescence within the cytosol revealed that treat-ment with Cyto D did not significantly affect the rate ofFM1-43 accumulation (Fig. 3F–I, N) in activated T cells. Incontrast, treatment with calyculin A (Caly A), a serine/threonine phosphatase (PP1 and PP2A) inhibitor, abolisheduptake of FM1-43 (Fig. 3J, K, N). Caly A induced theformation of a dense actin ring beneath the plasma mem-brane (Fig. 3D), consistent with effects described in othercell types [32–36]. Caly A-treated cells acquired a typicalkidney-like shape and appeared to be curved around a singletightly condensed cytosolic actin bundle. To explorewhether the inhibition of vesicular trafficking by Caly Awas due to modification of the cytoskeleton, Caly A wasapplied together with Cyto D. Coapplication of Caly A andCyto D produced an intermediate effect on polymerizedactin distribution; the dense near-membrane actin ring waspresent, but actin patches within the cytosol were also ob-served (Fig. 3E). FM1-43 accumulation was partially res-

cued in these cells (Fig. 3L–N). We conclude that actinpolymerization can affect the vesicular trafficking in T cellsin different ways. Polymerized actin in the cytosol couldsupport vesicle transport and facilitate membrane internal-ization upon elevation of [Ca2�]i. In contrast, tight conden-sation of actin beneath the PM creates a barrier that inhibitsmembrane trafficking. The limited amount of Cyto D-resis-tant actin is probably sufficient to maintain constitutivevesicular trafficking in Cyto D-treated cells.

Changes in trafficking compartments revealed by EM

Photoconversion of DAB by FM1-43 allows the subcel-lular origin of FM1-43 fluorescence to be visualized withEM resolution [22]. When cells were incubated withFM1-43 for 20 min at room temperature, followed by wash-ing to remove dye from the PM, the electron-dense reactionproduct was found within a pool of vesiculo-tubular com-partments located within the organelle-enriched pole of bothresting (Fig. 4A–C) and activated (Fig. 4D–I) T cells. Somevesicles with reaction product appeared to pinch inwardfrom the PM (Fig. 4C) and probably represent primaryendocytic vesicles. The average size of FM1-43-positivevesicular compartments within the cytosol was 72 � 2 nm(n � 312); long tubular structures with similar diameter (68� 4 nm) were 234 � 20 nm in length (n � 73). Some of thetubular structures appeared to be formed by clusters of smallvesicles (Fig. 4B, E, H). The appearance of FM1-43-posi-tive compartments in resting and activated T cells wassimilar to early endosomes (EE) described in other celltypes [12,37].

Many FM1-43-positive vesiculo-tubular structures wereobserved in continuity with intermediate-sized (153 � 28nm, n � 14) vacuoles located in the perinuclear region ofactivated T cells (Fig. 4F and I). These vacuoles oftencontained a small amount of reaction product and displayedearly signs of inward vesiculation suggesting that they arelate endosomes (LE) targeted by EE. Within single cells,FM1-43-positive intermediate-sized LE coexisted withlarge vesiculated vacuoles (388 � 30 nm, n � 18) thatcontained no dye (FM1-43�), even though FM1-43-positiveEE were located in close proximity to the FM1-43� vacu-oles (Fig. 4E and F). These results suggested that EE targeta specific pool of perinuclear LE.

Fig. 3. Cytoskeleton polymerization may affect the rate of FM1-43 accumulation in activated T cells. Activated T cells were stained with Oregon Greenphalloidin after fixation. Before fixation, cells were incubated in normal Tyrode solution (A), in the presence of 1 �M thapsigargin (5 min, B), 10 �Mcytochalasin D (20 min, C), 200 nM calyculin A (20 min, D), or both 10 �M cytochalasin D and calyculin A (20 min, E). Pretreatments with cytochalasinD and calyculin A were done at 37 °C for 20 min whereas thapsigargin was applied at 24 °C. In parallel experiments, activated T cells were exposed toFM1-43 for 30 s (F, H, J, L), or 21 min (G, I, K, M) in normal Tyrode solution (F, G); after pretreatment with 10 �M cytochalasin D (H, I); or 200 nMcalyculin A (J, K); or both 10 �M cytochalasin D and 200 nM calyculin A (L, M). Note that in panels J and K a bright FM1-43 fluorescence spot that appearsto be located in the cytosol (cell on the left) is in the extracellular space, because staining emerged immediately after dye application. This area was excludedfrom the analysis. The time courses of FM1-43 accumulation were acquired at 24 °C. Scale bars are 10 �m. (N) Time courses of FM1-43 accumulation innormal Tyrode solution (closed circles); in the presence of 10 �M cytochalasin D (open triangles); in the presence of 200 nM calyculin A (open squares);and in the presence of both 10 �M cytochalasin D and 200 nM calyculin A (closed triangles). Each time course is an average of six experiments from differentcultures.


In order to visualize late endocytic compartments, restingand activated T cells were incubated with FM1-43 for 1.5 hat 37 °C. In resting T cells the photoconversion reactionproduct was found in the EE adjacent to perinuclear vacu-oles (Fig. 5A and B) and inside peripheral vacuoles that

displayed no internal vesiculation but appeared as homoge-neously filled compartments (Fig. 5A and C) typical oflysosomes [38,39]. Strikingly different FM1-43-positive LEcontaining numerous vesicles (mulivesicular bodies, MVB)were observed in activated T cells after 1.5 h of incubation

Fig. 4. Photoconversion of DAB by FM1-43 reveals similar early endocytic compartments in resting and activated T cells. Electron micrographs showingrepresentative images obtained from resting (A–C) and activated (D–I) T cells exposed to FM1-43 for 20 min at 24 °C. Note that (A) shows two parts ofa single nucleus (N) separated by a cleft. The photoconversion reaction product (dark staining) appeared within the cytosol of resting (A–C) and activated(D–I) T cells. After 5 min of incubation with FM1-43 at room temperature, an electron-dense reaction product was found only around the PM (not shown).(B) Enlargement of the boxed area in (A), and (C) showing examples of EE compartments in resting T cells. The arrow indicates a vesicle that appears topinch off from the PM and may represent a primary endocytic vesicle. Staining around the PM is due to residual FM1-43 in the PM after washout. (D andG) A pool of EE (dark staining) stretching from the PM toward the nucleus. (E and F) and (H and I) Enlargements of the boxed areas (1) and (2) in (D) and(G). Note tubular structures that appear to be formed by several small vesicles (arrows in (E) and (H)). Note fusion of FM1-43-positive vesicles with smallperinuclear MVB (arrowheads in (F) and I), whereas large MVB (* in E and F) remain FM1-43�. N, nucleus. Scale bars are 0.5 �m.


with FM1-43 (Fig. 5D–G). These FM1-43-positive MVBwere found anywhere in the cytosol, and were larger (367 �24 nm, n � 18) than perinuclear FM1-43-positive LE. Inaddition, large MVB containing a small amount of photo-conversion product were also observed within a single cell(Fig. 5F). Interestingly, small vesicles/tubules were found incontinuity with a large FM1-43-positive MVB (Fig. 5G),suggesting that directed targeting continues until the MVBreaches a critical size or state. Alternatively, vesicles maybud off from the mature MVB, as was suggested for den-dritic cells [40]. Thus, our data indicate that resting andactivated T cells employ similar structural intermediatesthat operate from the PM to perinuclear LE. In activated Tcells the perinuclear LE enlarge, vesiculate, and drift awayfrom the nucleus within 1.5 h. In contrast, LE in resting Tcells appear to mature to lysosomes within the same periodof time.

In many cell types, MVB undergo maturation intolysosomes as a standard pathway for degradation of in-ternalized membrane proteins [12]. However, in activatedT cells we observed fusion of the limiting membrane ofFM1-43-positive MVB with the PM (Fig. 6A and B),resulting in release of vesicles enclosed in the MVB, asecretory process known as exosome externalization

[16]. Externalized vesicles were 120 � 10 nm (n � 121)in diameter, a size typical for exosomes. Fusion of FM1-43-positive MVB with the PM and externalization ofexosomes was observed as early as 1.5 h after dye wasallowed to internalize. Extracellular vesiculated mem-brane aggregates composed of �110 –160 nm vesiclesadherent to the plasma membrane were present in themajority of activated T cells (Fig. 6C and D). The exter-nal vesicles were often found in areas of cell-to cellcontact, sometimes within the extensive net of lamellopo-dia stretching from one cell to another (Fig. 6E and F).Interestingly, exosomes retained FM1-43 even after PMstaining was significantly reduced by extensive washing.Because clusters of externalized exosomes tend to retaindye, we conclude that they correspond to large peripheralpatches of FM1-43 fluorescence in activated T cells(Figs. 1 and 2).

Taken together, our data demonstrate that human T cellsconstantly internalize their PM by endocytosis into EE thatthen carry their cargo into lysosome-like LE in resting Tcells and into MVB in activated T cells. In activated T cells,but not resting T cells, the MVB can externalize exosomesinto the extracellular space by means of fusion of the lim-iting membrane of MVB with the PM.

Fig. 5. Photoconversion of DAB by FM1-43 reveals different late endocytic compartments in resting and activated T cells. Representative images obtainedfrom resting (A–C) and activated (D–G) T cells exposed to FM1-43 for 1.5 h at 37 °C. (B) Enlargement of the boxed area (1) in (A). Note smallFM1-43-positive vesicles docked to the perinuclear vacuole. (C) Enlargement of the boxed area (2) in (A). Note that FM1-43-positive endosomes in restingT cells display no internal vesiculation. (E and F) Enlargements of boxed areas (1) and (2) in (D). Note that FM1-43-positive endosomes in activated T cellsin (E) have a multivesicular structure. (F) A large MVB weakly stained with FM1-43. (G) EE continue to target large FM1-43-positive MVB. N, nucleus.Scale bars are 0.5 �m.


Colocalization of exosomes with CD3�, cholera toxin B,and annexin V staining

In order to assess whether FM1-43 follows the samepathway of internalization as surface receptors, we per-formed double staining of endocytic compartments withFM4-64 (a red-shifted variant of FM1-43) and Alexa Fluor488-conjugated anti-CD3�. Fig. 7A–C demonstrates in ac-tivated T cells that internalized anti-CD3�, visible as punc-tate staining in the cytosol, colocalizes with FM4-64 fluo-rescence. Although colocalization is restricted to a fewintracellular compartments, this observation strongly sug-gests that CD3� is internalized to the same endosomes asFM4-64. On the cell periphery, bright spots of FM4-64fluorescence originated from the clusters of exosomes alsocolocalized with anti-CD3� immunofluorescence. Colocal-ization of exosome clusters with anti-CD3� immunofluo-rescence suggests that CD3� downregulation may followthe pathway of exosome externalization (Figs. 5 and 6). Tofurther characterize the molecular composition of exo-somes, we stained surface membranes with Alexa Fluorcon-jugated cholera toxin B (CT-B), a marker of lipid rafts [41,42], or annexin V, a marker of phosphatidylserine (PS). Fig.7D–F, demonstrates that CT-B staining was confined tosmall areas that colocalized with bright fluorescence patchesof FM4-64, indicating that exosomes bear raft-associatedGM1 glycosphingolipid. In addition, annexin V stainingwas restricted to the same areas (Fig. 7G–I), indicatingexposure of PS on the outer surface of exosomes.

Because transverse redistribution of plasma membranePS is an early indicator of apoptosis [43], we investigatedwhether exosome externalization is a characteristic of via-ble, nonapoptotic cells. We found that 81% of adherentactivated T cells (363 out of 445 cells from four donors)expressed exosomes positively stained by annexin V; noneof these cells were stained with PI, indicating that the cellswere not necrotic. Surface PS exposure by apoptotic cells isassociated with loss of mitochondrial transmembrane poten-tials (��m) [44]. However, we found that cells bearingexosomes at their surface maintained a high ��m that couldbe dissipated by CCCP, the mitochondrial uncoupler, or bytreatment with staurosporine, an agent that induces apopto-sis (Fig. 8). In addition, the majority (67%) of activated Tcells were producing IL2 (102 out of 162 cells tested), asassessed by immunohistochemical staining (not shown).Only 27 out of 445 cells (4.5%) were uniformly stained withannexin V, of which seven displayed nuclear PI staining.

Thus, the majority of adherent activated T cells displayedhigh mitochondrial potentials and were competent to pro-duce cytokines. These data demonstrate that viable, acti-vated T cells release exosomes that bear properties of raftmembranes and expose PS locally at their outer membraneleaflet.

Discussion

Dynamic monitoring of membrane trafficking

In this study, we show that FM1-43 is rapidly and con-stitutively internalized by vesicular endocytosis in human Tcells and, therefore, can be utilized for unraveling funda-mental mechanisms that regulate membrane trafficking.Real-time monitoring of FM1-43 fluorescence indicated thatthe rate of constitutive membrane trafficking was �10-foldhigher in activated T cells compared to resting. Fast anddynamic regulation of membrane protein expression mayhelp to shape specific responses in activated T cells. Fur-thermore, elevation of [Ca2�]i significantly acceleratedmembrane internalization. Ca2� signaling was shown pre-viously to be enhanced in activated T cells, compared toresting T cells [45,46]. Therefore, augmented Ca2� signal-ing may enhance membrane traffic when activated T cellsare restimulated by antigen. Extracellular Ca2� influx wasnot required for basal endocytosis in T cells, in good agree-ment with previous reports in neuronal cells [47,48], and inrat peritoneal mast cells in which high-affinity Ca2� siteswere implicated in membrane uptake [27].

Cytoskeletal elements are involved in different steps ofthe endocytic process, including membrane invaginationand fission, as well as vesicle movement in the cytoplasm[49,50]. We found that [Ca2�]i elevation in human T cellsproduced actin polymerization and accelerated FM1-43 ac-cumulation, thus implying the involvement of the actincytoskeleton in regulation of endocytosis. However, actindisruption with Cyto D had no effect on the rate of FM1-43accumulation, consistent with results in several other mam-malian cells [51–53]. Increased mobility of endocytic ves-icles after treatment with agents that sequester monomericactin could be due to removal of a diffusional barrier [54].It is also possible that a cortical layer of actin underlying thePM in Cyto D-treated cells (Fig. 3C) could still supportvesicle formation and movement away from the PM. On theother hand, strong condensation of cortical actin beneath thePM with Caly A abolished FM1-43 accumulation, consis-

Fig. 6. Photoconversion reveals exosome externalization by activated T cells. (A) Fusion of a MVB with the PM and exosome externalization in activatedT cells. Cells were loaded with FM1-43 for 1 h and 20 min at 37 °C, washed for 5 min in Tyrode solution, fixed, and subjected to photoconversion. (B)Enlargement of the boxed area in (A). (C and D) Clusters of external membranes adherent to the PM of activated T cell. Cells were exposed to FM1-43 for20 min, and dye was then washed out. Note that many vesicles display cup-shaped morphology typical of exosomes. (E) Photoconversion reaction productis found trapped within external vesicles in regions of cell–cell contact. (F) Enlargement of the boxed area in (E) Note that EE (dark staining) and MVB(*) are located in the vicinity of cell–cell contact. N, nucleus; m, mitochondria. Scale bars are 0.5 �m.


tent with the diffusional barrier hypothesis. Thus, our dataconfirm that vesicular trafficking in T cells can be fine-tunedby spatially restricted cytoskeleton modification.

Rates of endocytosis in T cells were strongly temper-ature dependent, consistent with studies of receptor-mediated and fluid-phase endocytosis in other cell types[55–58]. The Arrhenius plot of endocytic rates in Fig. 1deviates from a straight line at �24 °C and reveals valuesof activation energy similar to those measured for cleav-

age of the GPI-anchored protein, 5�-nucleotidase,from the surface of porcine lymphocyte PM [59]. Abiphasic Arrhenius plot is common for many membrane-bound enzymes and may result from a lipid phase tran-sition from a gel to a fluid liquid crystalline phase, fromlipid phase separation within the membrane [60,25], orfrom other factors such as a change in activation entropyor changes in membrane components other than lipids[59].

Fig. 7. FM4-64 fluorescence colocalizes with CD3�, cholera toxin-B staining and annexin V staining in activated T cells. (A–C) Anti-CD3� and FM4-64 infixed cells. Activated T cells were first incubated with FM4-64 for 30 min at 37 °C. Ionomycin (0.1 �M) and PMA (50 nM) were added to promoteinternalization of TCR/CD3 complexes [3]. After FM4-64 was thoroughly washed out, cells were fixed and costained with primary anti-CD3� antibody andAlexa Fluor 488-conjugated secondary antibody. (A, green) Distribution of anti-CD3�-Alexa Fluor 488 fluorescence, acquired with 515 nm band passemission filter. Note PM distribution of CD3� in two cells and complete internalization of CD3� in one cell. (B, red) FM4–64 fluorescence acquired with600 nm long pass emission filter. (C) Superimposed images from (A) and (B); yellow indicates colocalization of FM4–64 fluorescence with anti-CD3�

fluorescence. Note colocalization within endosomes (arrows) and within extracellular exosome clusters (arrowheads) (D–I) Living T cells were stained for15 min at room temperature with CT-B-Alexa Fluor 488 (D, green) or annexin V-Alexa Fluor 488 (G, green) and then images were acquired with 515 nmband pass emission filter. Cells were then briefly (30 s) exposed to FM4-64 and fluorescence acquired with 600 nm long pass emission filter (E and H, red).(F and I) Superimposed images from (D) and (E), and (G) and (H), correspondingly. Note that surface CT-B and annexin V staining colocalize with exosomeclusters revealed with FM4–64. Scale bars are 10 �m.


Endocytic compartments and exosome externalization

The ability of FM1-43 to convert DAB into an elec-tron-dense product provides a unique opportunity to re-late real-time fluorescence monitoring of endocytosis inliving cells to the ultrastructural compartments within thecell. T cells employ structurally different primary endo-cytic vesicles to mediate membrane receptor trafficking.TCR and transferrin receptor are internalized via clath-rin-coated pits [61,62], whereas IL2 R, CD4, CD8, CD19,and CD20 are internalized primarily via uncoated vesi-cles [63,64]. The size of FM1-43-positive intracellularvesicles (50 –100 nm) is consistent with either coated oruncoated endocytic vesicles found in T lymphocytes [64].Colocalization with anti-CD3� within the cell indicatesthat the dye follows the clathrin-coated vesicle pathway,among other endocytic pathways. Therefore, FM1-43 ap-pears to be a powerful tool for investigation of T cellmembrane dynamics and mechanisms that regulate mem-brane receptor internalization.

Tracking the fate of membrane-associated FM1-43within the cell revealed a significant change in the organi-zation of endocytic compartments as T cells become acti-vated. Early endosomes (EE) in resting and activated T cellsdisplayed similar morphology to those in other cell types[12], but late endosomes appear to take different paths whenT cells are activated. In resting T cells, FM1-43 was foundin lysosome-like vacuoles that lacked internal membranes,whereas activated T cells readily accumulated the dyewithin MVB. Our results indicate that fusion of MVB withthe PM of activated (but not resting) human T cells resultsin externalization of exosomes. This pathway may allowcertain proteins to avoid degradation and initiate signaltransduction in neighboring cells. The mechanisms that reg-ulate the fate of MVB within the activated T cell remain tobe determined.

Clusters of exosomes at the surface of activated T cellswere revealed as bright fluorescence patches of FM1-43/FM4-64 that appeared simultaneously with PM stainingimmediately after dye application. Exosome clusterswere usually located in cell– cell contacts and differed inmembrane composition from the PM by enrichment ofraft-associated GM1 and by exposure of PS on the outermembrane leaflet (Fig. 7). The presence of PS at the outersurface of T cell exosomes is consistent with platelet-derived exosomes [65]. FM1-43 can change its spectralproperties depending on the lipid environment [29,66], aproperty that may account for the bright fluorescencefrom exosomes in the presence of dye and the prolongedfluorescence from exosome clusters after dye washout(Fig. 1). Exosome clusters observed by electron micros-copy were composed of single vesicles 100 –200 nm indiameter. Previous studies showed that similar-sized mi-crovesicles at the surface of activated T cells, T cellclones, and Jurkat T cells bear biologically active pro-teins including TCR, adhesion molecules CD2 andLFA-1, MHC class I and class II, and the chemokinereceptor CXCR4 [18]. Our colocalization study revealedthe presence of raft-associated CD3� (a TCR component)in exosomes, in addition to GM1 glycosphingolipid, in-dicating that exosomes are formed by raft membranes. Inlymphocytes that lack caveolae [41], raft domains wereimplicated in GPI-anchored protein endocytosis [67]. Wehypothesize that the composition of internalized raft do-mains may be preserved when processed via LE com-partments and then externalized by exosomes. It is pos-sible that MVB also serve as vehicles for externalizationof newly synthesized and recycled raft components. Ex-ternalization of exosomes formed by raft membranesfrom endocytic compartments may also participate in theformation of larger, micron-sized raft aggregates at thesurface of T cells after CT-B or TCR cross-linking [68].

The functional role of exosomes in the immune re-sponse remains controversial [16]. Recently, costimula-tory rafts that appear to originate from cytoplasmic ves-icles were implicated as important amplifiers of the later

Fig. 8. Exosomes at the surface of viable activated T cells. (A) FM4–64surface fluorescence (red) obtained from living cells after brief (30 s)exposure to FM4–64. (B) Same cells stained with DiOC6 (3) (green). (C)Same cells after incubation with 10 �M CCCP for 15 min at roomtemperature. (D) Histograms of DiOC6 (3) intensity measured from un-treated activated T cells (control, top panel); from activated T cells treatedwith 10 �M CCCP for 15 min at room temperature (middle panel); andfrom activated T cells preincubated with 1 �M staurosporine for 6 h at 37°C to induce apoptosis (lower panel). Fluorescence intensities were mea-sured from randomly selected individual adherent T cells. Scale bars are 10�m.


phases of TCR-mediated stimulation [69, 70]. We sug-gest that specific types of raft aggregates can be deliveredby exosome externalization and may play a role in pro-viding costimulatory signals for activated T cells. More-over, it is established that exposure of PS on apoptoticcells triggers phagocytosis by macrophages and DCs[71,72] in turn resulting in presentation of antigens thatwere expressed by the phagocytosed cells [73]. We spec-ulate that exosomes released by nonapoptotic T cells canalso be phagocytosed, resulting in enhanced antigen pre-sentation by professional APC. In addition, Fas andAPO2/TRAIL death ligands have been found in mi-crovesicles released by Jurkat and PHA-activated humanT cells [14,19] and may suppress the immune response.Finally, exosome externalization can result in permanentTCR down-regulation, similar to down-regulation of thetransferrin receptor during reticulocyte maturation[74,75]. T cell exosomes carrying membrane receptorsand exposed PS may underlie long-range cell-to-cell in-teractions by phagocytosis or transendocytosis.

Acknowledgments

This work was supported by NIH grants NS14609 andGM41514 (M.D.C.), NIH/NCRR grant P41RR04050(M.H.E.), a Scientist Development Grant 0030275N fromthe American Heart Association (A.F.F.), and by PublicHealth Service Research grant M01 RR00827 from theNational Center for Research Resources (UCI General Clin-ical Research Center). The authors thank Craig Walsh andNicolas Blanchard for insightful comments. TatianaKrasieva, Luette Forrest, and Olga Safrina provided valu-able experimental assistance.

References

[1] M.S. Krangel, Endocytosis and recycling of the T3-T cell receptorcomplex, The role of T3 phosphorylation, J. Exp. Med. 165 (1987)1141–1159.

[2] H. Liu, M. Rhodes, D.L. Wiest, D.A. Vignali, On the dynamics ofTCR:CD3 complex cell surface expression and downmodulation,Immunity 13 (2000) 665–675.

[3] Y. Minami, L.E. Samelson, R.D. Klausner, Internalization and cy-cling of the T cell antigen receptor, Role of protein kinase C, J. Biol.Chem. 262 (1987) 13342–13347.

[4] B. Hemmer, I. Stefanova, M. Vergelli, R.N. Germain, R. Martin,Relationships among TCR ligand potency, thresholds for effectorfunction elicitation, and the quality of early signaling events in humanT cells, J. Immunol. 160 (1998) 5807–5814.

[5] Y. Itoh, B. Hemmer, R. Martin, R.N. Germain, Serial TCR engage-ment and down-modulation by peptide: MHC molecule ligands: re-lationship to the quality of individual TCR signaling events, J. Im-munol. 162 (1999) 2073–2080.

[6] S. Valitutti, S. Muller, M. Cella, E. Padovan, A. Lanzavecchia, Serialtriggering of many T-cell receptors by a few peptide-MHC com-plexes, Nature 375 (1995) 148–151.

[7] S. Valitutti, S. Muller, M. Salio, A. Lanzavecchia, Degradation of Tcell receptor (TCR)-CD3-zeta complexes after antigenic stimulation,J. Exp. Med. 185 (1997) 1859–1864.

[8] U. D’Oro, M.S. Vacchio, A.M. Weissman, J.D. Ashwell, Activationof the Lck tyrosine kinase targets cell surface T cell antigen receptorsfor lysosomal degradation, Immunity 7 (1997) 619–628.

[9] Z. Cai, H. Kishimoto, A. Brunmark, M.R. Jackson, P.A. Peterson, J.Sprent, Requirements for peptide-induced T cell receptor downregu-lation on naive CD8� T cells, J. Exp. Med. 185 (1997) 641–651.

[10] D.M. La Face, C. Couture, K. Anderson, G. Shih, J. Alexander, A.Sette, T. Mustelin, A. Altman, H.M. Grey, Differential T cell signal-ing induced by antagonist peptide-MHC complexes and the associ-ated phenotypic responses, J. Immunol. 158 (1997) 2057–2064.

[11] P.A. Roche, Intracellular protein traffic in lymphocytes: “How do Iget THERE from HERE”? Immunity 11 (1999) 391–398.

[12] I. Mellman, Endocytosis and molecular sorting, Annu. Rev. Cell Dev.Biol. 12 (1996) 575–625.

[13] E.J. Blott, G.M. Griffiths, Secretory lysosomes, Nat. Rev. Mol. CellBiol. 3 (2002) 122–131.

[14] K. Denzer, M.J. Kleijmeer, H.F. Heijnen, W. Stoorvogel, H.J. Geuze,Exosome: from internal vesicle of the multivesicular body to inter-cellular signaling device, J. Cell Sci. 113 (2000) 3365–3374.

[15] W. Stoorvogel, M.J. Kleijmeer, H.J. Geuze, G. Raposo, The biogen-esis and functions of exosomes, Traffic 3 (2002) 321–330.

[16] C. Thery, L. Zitvogel, S. Amigorena, Exosomes: composition, bio-genesis and function, Nat. Rev. Immunol. 2 (2002) 569–579.

[17] L. Zitvogel, A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza,P. Ricciardi-Castagnoli, G. Raposo, S. Amigorena, Eradication ofestablished murine tumors using a novel cell-free vaccine: dendriticcell-derived exosomes, Nat. Med. 4 (1998) 594–600.

[18] N. Blanchard, D. Lankar, F. Faure, A. Regnault, C. Dumont, G.Raposo, C. Hivroz, TCR activation of human T cells induces theproduction of exosomes bearing the TCR/CD3/zeta complex, J. Im-munol. 168 (2002) 3235–3241.

[19] I. Monleon, M.J. Martinez-Lorenzo, L. Monteagudo, P. Lasierra, M.Taules, M. Iturralde, A. Pineiro, L. Larrad, M.A. Alava, J. Naval, A.Anel, Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during acti-vation-induced death of human T cells, J. Immunol. 167 (2001)6736–6744.

[20] W.J. Betz, F. Mao, C.B. Smith, Imaging exocytosis and endocytosis,Curr. Opin. Neurobiol. 6 (1996) 365–371.

[21] A.J. Cochilla, J.K. Angleson, W.J. Betz, Monitoring secretory mem-brane with FM1-43 fluorescence, Annu. Rev. Neurosci. 22 (1999)1–10.

[22] A.W. Henkel, L.L. Simpson, R.M. Ridge, W.J. Betz, Synaptic vesiclemovements monitored by fluorescence recovery after photobleachingin nerve terminals stained with FM1-43, J. Neurosci. 16 (1996)3960–3967.

[23] N. Harata, J.L. Pyle, A.M. Aravanis, M. Mozhayeva, E.T. Kavalali,R.W. Tsien, Limited numbers of recycling vesicles in small CNSnerve terminals: implications for neural signaling and vesicular cy-cling, Trends Neurosci. 24 (2001) 637–643.

[24] G. Gaietta, T.J. Deerinck, S.R. Adams, J. Bouwer, O. Tour, D.W.Laird, G.E. Sosinsky, R.Y. Tsien, M.H. Ellisman, Multicolor andelectron microscopic imaging of connexin trafficking, Science 296(2002) 503–507.

[25] H. Sandermann, Regulation of membrane enzymes by lipids, Bio-chim. Biophys. Acta 515 (1978) 209–237.

[26] G.R. Crabtree, Generic signals and specific outcomes: signalingthrough Ca2�, calcineurin, and NF-AT, Cell 96 (1999) 611–614.

[27] W. Almers, E. Neher, Gradual and stepwise changes in the membranecapacitance of rat peritoneal mast cells, J. Physiol. (London) 386(1987) 205–217.


[28] P. De Camilli, K. Takei, Molecular mechanisms in synaptic vesicleendocytosis and recycling, Neuron 16 (1996) 481–486.

[29] A. Zweifach, FM1-43 reports plasma membrane phospholipid scram-bling in T-lymphocytes, Biochem. J. 349 (2000) 255–260.

[30] O. Acuto, D. Cantrell, T cell activation and the cytoskeleton, Annu.Rev. Immunol. 18 (2000) 165–184.

[31] J.A. Cooper, Effects of cytochalasin and phalloidin on actin, J. CellBiol. 105 (1987) 1473–1478.

[32] R.L. Patterson, D.B. van Rossum, D.L. Gill, Store-operated Ca2�

entry: evidence for a secretion-like coupling model, Cell 98 (1999)487–499.

[33] P. Kreienbuhl, H. Keller, V. Niggli, Protein phosphatase inhibitorsokadaic acid and calyculin A alter cell shape and F-actin distributionand inhibit stimulus-dependent increases in cytoskeletal actin of hu-man neutrophils, Blood 80 (1992) 2911–2919.

[34] G.P. Downey, A. Takai, R. Zamel, S. Grinstein, C.K. Chan, Okadaicacid-induced actin assembly in neutrophils: role of protein phospha-tases, J. Cell Physiol. 155 (1993) 505–519.

[35] N. Hosoya, M. Mitsui, F. Yazama, H. Ishihara, H. Ozaki, H. Karaki,D.J. Hartshorne, H. Mohri, Changes in the cytoskeletal structure ofcultured smooth muscle cells induced by calyculin-A, J. Cell Sci. 105(1993) 883–890.

[36] N. Shinoki, M. Sakon, J. Kambayashi, M. Ikeda, E. Oiki, M.Okuyama, K. Fujitani, Y. Yano, T. Kawasaki, M. Monden, Involve-ment of protein phosphatase-1 in cytoskeletal organization of culturedendothelial cells, J. Cell Biochem. 59 (1995) 368–375.

[37] M.S. Robinson, C. Watts, M. Zerial, Membrane dynamics in endo-cytosis, Cell 84 (1996) 13–21.

[38] J.P. Luzio, B.M. Mullock, P.R. Pryor, M.R. Lindsay, D.E. James,R.C. Piper, Relationship between endosomes and lysosomes, Bio-chem. Soc. Trans. 29 (2001) 476–480.

[39] J.P. Luzio, B.A. Rous, N.A. Bright, P.R. Pryor, B.M. Mullock, R.C.Piper, Lysosome-endosome fusion and lysosome biogenesis, J. CellSci. 113 (2000) 1515–1524.

[40] J. Murk, W. Stoorvogel, M. Kleijmeer, H. Geuze, The plasticity ofmultivesicular bodies and the regulation of antigen presentation, Se-min. Cell Dev. Biol. 13 (2002) 303–311.

[41] A.M. Fra, E. Williamson, K. Simons, R.G. Parton, Detergent-insol-uble glycolipid microdomains in lymphocytes in the absence ofcaveolae, J. Biol. Chem. 269 (1994) 30745–30748.

[42] T. Harder, P. Scheiffele, P. Verkade, K. Simons, Lipid domain struc-ture of the plasma membrane revealed by patching of membranecomponents, J. Cell Biol. 141 (1998) 929–942.

[43] M.C. Stuart, J.G. Damoiseaux, P.M. Frederik, J.W. Arends, C.P.Reutelingsperger, Surface exposure of phosphatidylserine during ap-optosis of rat thymocytes precedes nuclear changes, Eur. J. Cell Biol.76 (1998) 77–83.

[44] G. Kroemer, N. Zamzami, S.A. Susin, Mitochondrial control of ap-optosis, Immunol. Today 18 (1997) 44–51.

[45] S.D. Hess, M. Oortgiesen, M.D. Cahalan, Calcium oscillations inhuman T and natural killer cells depend upon membrane potential andcalcium influx, J. Immunol. 150 (1993) 2620–2633.

[46] A.F. Fomina, C.M. Fanger, J.A. Kozak, M.D. Cahalan, Single chan-nel properties and regulated expression of Ca2� release-activatedCa2� (CRAC) channels in human T cells, J. Cell Biol. 150 (2000)1435–1444.

[47] T.A. Ryan, H. Reuter, B. Wendland, F.E. Schweizer, R.W. Tsien, S.J.Smith, The kinetics of synaptic vesicle recycling measured at singlepresynaptic boutons, Neuron 11 (1993) 713–724.

[48] T.A. Ryan, S.J. Smith, H. Reuter, The timing of synaptic vesicleendocytosis, Proc. Natl. Acad. Sci. USA 93 (1996) 5567–5571.

[49] B. Qualmann, M.M. Kessels, R.B. Kelly, Molecular links betweenendocytosis and the actin cytoskeleton, J. Cell Biol. 150 (2000)F111–F116.

[50] L. Lanzetti, P.P. Di Fiore, G. Scita, Pathways linking endocytosis andactin cytoskeleton in mammalian cells, Exp. Cell Res. 271 (2001)45–56.

[51] T.A. Gottlieb, I.E. Ivanov, M. Adesnik, D.D. Sabatini, Actin micro-filaments play a critical role in endocytosis at the apical but not thebasolateral surface of polarized epithelial cells, J. Cell Biol. 120(1993) 695–710.

[52] L.M. Fujimoto, R. Roth, J.E. Heuser, S.L. Schmid, Actin assemblyplays a variable, but not obligatory role in receptor-mediated endo-cytosis in mammalian cells, Traffic 1 (2000) 161–171.

[53] M.R. Jackman, W. Shurety, J.A. Ellis, J.P. Luzio, Inhibition of apicalbut not basolateral endocytosis of ricin and folate in Caco-2 cells bycytochalasin D, J. Cell Sci. 107 (1994) 2547–2556.

[54] I. Gaidarov, F. Santini, R.A. Warren, J.H. Keen, Spatial control ofcoated-pit dynamics in living cells, Nat. Cell Biol. 1 (1999) 1–7.

[55] D. Illinger, P. Poindron, J.G. Kuhry, Fluid phase endocytosis inves-tigated by fluorescence with trimethylamino-diphenylhexatriene inL929 cells; the influence of temperature and of cytoskeleton depoly-merizing drugs, Biol. Cell. 73 (1991) 131–138.

[56] Z. Mamdouh, M.C. Giocondi, R. Laprade, C. Le Grimellec, Temper-ature dependence of endocytosis in renal epithelial cells in culture,Biochim. Biophys. Acta 1282 (1996) 171–173.

[57] H. Tomoda, Y. Kishimoto, Y.C. Lee, Temperature effect on endocy-tosis and exocytosis by rabbit alveolar macrophages, J. Biol. Chem.264 (1989) 15445–15450.

[58] P.H. Weigel, J.A. Oka, Temperature dependence of endocytosis me-diated by the asialoglycoprotein receptor in isolated rat hepatocytes.Evidence for two potentially rate-limiting steps, J. Biol. Chem. 256(1981) 2615–2617.

[59] M.T. Lehto, F.J. Sharom, PI-specific phospholipase C cleavage of areconstituted GPI-anchored protein: modulation by the lipid bilayer,Biochemistry 41 (2002) 1398–1408.

[60] J. Kumamoto, J.K. Raison, J.M. Lyons, Temperature “breaks” inArrhenius plots: a thermodynamic consequence of a phase change, J.Theor. Biol. 31 (1971) 47–51.

[61] J. Dietrich, X. Hou, A.M. Wegener, C. Geisler, CD3 gamma containsa phosphoserine-dependent di-leucine motif involved in down-regu-lation of the T cell receptor, EMBO J. 13 (1994) 2156–2166.

[62] F. Luton, V. Legendre, J.P. Gorvel, A.M. Schmitt-Verhulst, C. Boyer,Tyrosine and serine protein kinase activities associated with ligand-induced internalized TCR/CD3 complexes, J. Immunol. 158 (1997)3140–3147.

[63] C. Lamaze, A. Dujeancourt, T. Baba, C.G. Lo, A. Benmerah, A.Dautry-Varsat, Interleukin 2 receptors and detergent-resistant mem-brane domains define a clathrin-independent endocytic pathway, Mol.Cell. 7 (2001) 661–671.

[64] O. Burkhardt, H.J. Merker, Immunoelectron microscopic investiga-tions of patching, capping, endocytotic and shedding processes in Tand B lymphocytes, Ann. Anat. 184 (2002) 45–53.

[65] H.F. Heijnen, A.E. Schiel, R. Fijnheer, H.J. Geuze, J.J. Sixma, Ac-tivated platelets release two types of membrane vesicles: mi-crovesicles by surface shedding and exosomes derived from exocy-tosis of multivesicular bodies and alpha-granules, Blood 94 (1999)3791–3799.

[66] U. Schote, J. Seelig, Interaction of the neuronal marker dye FM1-43with lipid membranes. Thermodynamics and lipid ordering, Biochim.Biophys. Acta 1415 (1998) 135–146.

[67] M. Deckert, M. Ticchioni, A. Bernard, Endocytosis of GPI-anchoredproteins in human lymphocytes: role of glycolipid-based domains,actin cytoskeleton, and protein kinases, J. Cell Biol. 133 (1996)791–799.

[68] P.W. Janes, S.C. Ley, A.I. Magee, Aggregation of lipid rafts accom-panies signaling via the T cell antigen receptor, J. Cell Biol. 147(1999) 447–461.

[69] L. Tuosto, I. Parolini, S. Schroder, M. Sargiacomo, A. Lanzavecchia,A. Viola, Organization of plasma membrane functional rafts upon Tcell activation, Eur. J. Immunol. 31 (2001) 345–349.


[70] V. Horejsi, Membrane rafts in immunoreceptor signaling: newdoubts, new proofs? Trends Immunol. 23 (2002) 562–564.

[71] P.M. Henson, D.L. Bratton, V.A. Fadok, The phosphatidylserinereceptor: a crucial molecular switch? Nat. Rev. Mol. Cell Biol. 2(2001) 627–633.

[72] R.A. Schlegel, M.K. Callahan, P. Williamson, The central role ofphosphatidylserine in the phagocytosis of apoptotic thymocytes, Ann.NY Acad. Sci. 926 (2000) 217–225.

[73] M.L. Albert, S.F. Pearce, L.M. Francisco, B. Sauter, P. Roy, R.L.Silverstein, N. Bhardwaj, Immature dendritic cells phagocytose apo-

ptotic cells via alphavbeta5 and CD36, and cross-present antigens tocytotoxic T lymphocytes, J. Exp. Med. 188 (1998) 1359–1368.

[74] R.M. Johnstone, M. Adam, J.R. Hammond, L. Orr, C. Turbide,Vesicle formation during reticulocyte maturation. Association ofplasma membrane activities with released vesicles (exosomes),J. Biol. Chem. 262 (1987) 9412–9420.

[75] R.M. Johnstone, A. Mathew, A.B. Mason, K. Teng, Exosome forma-tion during maturation of mammalian and avian reticulocytes: evi-dence that exosome release is a major route for externalization ofobsolete membrane proteins, J. Cell Physiol. 147 (1991) 27–36.


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