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Vol. 20, Issue 2, 600-615, January 15, 2009
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*Faculty of Life Sciences, The University of Manchester, M13 9PT Manchester, United Kingdom; Department of Biochemistry, Structural and Molecular Biology, Jozef Stefan Institute, 1000 Ljubljana, Slovenia; Section of Cell Aging and Degeneration, Department of Drug Research and Evaluation, Istituto Superiore Sanità, 00161 Rome, Italy; and Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
Submitted September 11, 2008;
Revised October 30, 2008;
Accepted November 14, 2008
Monitoring Editor: Marcos Gonzalez-Gaitan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Fas-ligand (FasL) (CD95L). The extrinsic pathway has been traditionally envisaged as a linear sequence of events emanating from the complex formed by ligated receptors with adaptor proteins and associated enzymes (Peter and Krammer, 2003
). However, it has been increasingly appreciated that death receptors such as Fas/CD95 undergo rapid internalization after triggering (Algeciras-Schimnich et al., 2002; Eramo et al., 2004; Lee et al., 2006), in a process that is essentially equivalent to the well known internalization of other surface receptors such as that for epidermal growth factor (Di Fiore and De Camilli, 2001). The complexes formed by activated death receptors seem to maintain signaling after internalization, in part using the early traffic of endocytic vesicles as a platform for intracellular activation of caspases (Lee et al., 2006; cf. Di Fiore and De Camilli, 2001). This situation seems to apply, in particular, to type I cells, which do not require mitochondria for caspase activation (Peter and Krammer, 2003).
Most cells of our body require mitochondria for cell execution along the extrinsic pathway and are usually defined of type II (Green et al., 2003; Peter and Krammer, 2003). They include mature T lymphocytes and related lines such as Jurkat, which show distinctive differences from type I cells in trafficking internalized Fas/CD95 (Algeciras-Schimnich et al., 2002; Eramo et al., 2004; Siegel et al., 2004; Lee et al., 2006). In these cells, Fas stimulation increases the uptake of the membrane probe N-(3-triethylammonium propyl)-4-(dibutilamino)styrylpyrodinum dibromide (Kawasaki et al., 2000; Matarrese et al., 2008) and of pynocytic markers (Kenis et al., 2004; Matarrese et al., 2008). In addition, it induces an unusual intermix of endosomes with mitochondria and other organelles (Ouasti et al., 2007; Matarrese et al., 2008).
The dynamics of endocytic organelles forms part of membrane traffic, which follows diverse portals of endocytosis (Conner and Schmid, 2003; Mayor and Pagano, 2007). After Fas triggering, the clathrin-dependent pathway of endocytosis is likely to be stimulated early to favor receptor internalization (Lee et al., 2006). However, Fas signaling also promotes a progressive block of clathrin-dependent endocytosis via caspase-mediated cleavage of components of this pathway (Austin et al., 2006). Hence, it has remained unclear to what extent Fas-enhanced endocytosis may contribute to the propagation of death signaling (Siegel et al., 2004; Austin et al., 2006; Reihner and Häussinger, 2008; Chaigne-Delalande et al., 2008). Moreover, the specific portal(s) engaged by Fas signaling have not been identified.
We have noted an intriguing similarity between the Fas-induced changes in membrane traffic and those previously documented in the activation of T cells. Fas signaling enhances exocytosis after the initial wave of increased endocytosis (Ouasti et al., 2007; Reihner and Häussinger, 2008), whereas T cell activation increases both endocytosis and exocytosis, which become polarized at different sides of the cell (Krummel and Macara, 2006). Given this similarity and the complexity of the diverse routes of membrane traffic, we undertook a systematic study of every endocytic system known to be present in T cells (Table 1). We have clarified that Fas enhances Rho GTPase-dependent routes polarizing membranes toward the Golgi region.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Cell Culture
Different batches of the of human acute T cell leukemia line Jurkat J6.1 were obtained from European Tissue Collection (Salisbury, United Kingdom). Likewise the related CEM line and a caspase-8–deficient line (provided by Dr. M. McFairlane, MRC Toxicology Unit, Leicester, United Kingdom), cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere with 5% (vol/vol) CO2 at 37°C.
Immunocytochemistry
Cells resuspended in modified Ringer buffer (RB; 145 mM NaCl, 4.5 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 5 mM K-HEPES, pH 7.4, and 10 mM glucose) were loaded with diverse fluorescent probes for 20–30 min, washed, and incubated at 
2 x 106/ml with recombinant FasL or CH-11 (routinely at a final concentration of 0.5 µg/ml) before plating onto coverslips coated with poly-lysine. After an adhesion period of 10–15 min at 37°C, cells were transferred on ice for 5 min (to reduce cellular movement) and then washed with phosphate-buffered saline (PBS) twice before fixation with 4% (wt/vol) paraformaldehyde in PBS. For immunodetection of internal proteins, cells were permeabilized with 0.5% Triton X-100 or 0.1% saponin, blocked with appropriate serum, incubated with monoclonal antibodies for 30–60 min, washed again, and then incubated for 45–60 min with secondary anti-mouse antibodies conjugated with AlexaFluor 488 or Rhodamine X (Ouasti et al., 2007). For surface staining, fixed cells were treated with diluted solutions of fluorescently-labeled monoclonal antibodies (optimized for each individual application) containing bovine serum albumin to minimize background binding (Naslavsky et al., 2004). Nonspecific fluorescence staining was evaluated using corresponding isotypic immunoglobulin-conjugates (Elward et al., 2005).
Fluorescence and Time-Lapse Microscopy
Fluorescence imaging was carried out in wide-field conventional microscopes (either Zeiss [Carl Zeiss, Jena, Germany] or Olympus [Olympus, Tokyo, Japan]) and with the DeltaVision RT system, in which images were acquired at 20°C with an automated Olympus IX71 microscope and oil-immersed objectives. Using software Rx. 3.4.3 (Applied Precision, Seattle, WA), images from stacks of >30 z-sections of 0.2 µm were deconvolved with 10 cycles and then projected along the z-plane as described previously (Ouasti et al., 2007). For time-lapse videomicroscopy, cells previously labeled with various probes were seeded at 5 x 106/ml in RB onto round coverslips coated with poly-lysine (MatTeK, Ashland, MA). Raw data were acquired after 8 min of FasL addition within a thermostated chamber (37°C), by using a Leica DMIRE2 microscope, a high-sensitivity camera (Photometrics cascade II) and 63x or 100x objectives. Images were processed with ASMDW Y1.21 software (Leica).
Evaluation of Cell Death
To assess the levels of incipient cell death that occurred under the same conditions as those used for endocytosis studies we used visual inspection of irreversible membrane blebbing (termed "terminal") as described previously (Stinchcombe et al., 2001). This early hallmark of Fas-induced death (Weis et al., 1995) was evaluated by integrating image analysis of fixed cells with a wealth of live cell images of prolonged experiments in which Fas stimulation led to loss of mitochondrial and cell integrity (cf. Matarrese et al., 2008). The cumulative scoring of terminal blebbing by independent observers correlated well with positive staining for caspase-3 activation.
Other Assays
Activation of caspase-8 was evaluated by using either flow cytometry (Ouasti et al., 2007) or cytofluorescence after loading cells with 10–20 µM Rho-IETD-bis (Packard et al., 2001). The same substrate was used for measurements with a plate reader (Fluoroskan Ascent; Thermo, Basingstoke, United Kingdom) of caspase activation in cell subfractions (Ouasti et al., 2007). Phagocytosis was evaluated using Congo-red stained yeast (Lugini et al., 2003), whereas macropinocytosis was measured using fluid-phase tracers. The uptake of fixable Ruby-dextran (10 kDa; Sigma Chemie) was undertaken as reported previously (Nichols et al., 2001) and evaluated as described previously (Sabharanjak et al., 2002), whereas steady-state traffic of transferrin (Tf) was followed with Alexa594-conjugated transferrin (Blanchard et al., 2002; Naslavsky et al., 2004). HPA and wheat germ agglutinin (WGA) conjugates were used as broad markers of membrane traffic, as well as for surface cell decoration (Degli Esposti, 2008).
Image Analysis
Images were processed with the program ImageJ (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/) or occasionally with the software routine of the DeltaVision RT program (with colocalization threshold set at 50). Quantitative analysis of colocalization was routinely undertaken using the plugin "colocalization threshold" of ImageJ, which uses the threshold algorithm of Costes et al. (2004) (http://www.uhnresearch.ca/facilities/wcif/imagej/appendix_ii). This yielded two complementary but independent parameters, the single-channel specific Mander's coefficient adjusted for threshold, tM1 or tM2, and Pearson's correlation index of global colocalization (Rtot). Mander's tM1 and tM2 coefficients are normalized to the total pixel intensity (appropriately subtracted for background) of each channel and are thus independent of the absolute intensity of channel fluorescence (Costes et al., 2004). Conversely, Pearson's index represents the r of all nonzero-zero pixels that overlay in the images of the channels. It is independent on background but sensitive to individual channel intensity and has values systematically lower than Mander's coefficients, in part because it ranges from a theoretical minimum of –1 to a maximum of 1 (Ménager et al., 2007), whereas Mander's coefficients vary from a unbiased threshold equivalent to a Pearson's value of 0 to a maximum value of 1. RGB.tiff files of deconvolved z-projections were split in 8-bit images with 256 channels of grayscale intensity for each color and then cleared of background using a region of interest (ROI) outside cells and the "subtract background from ROI" routine in ImageJ. Complete elimination of bleed-through and nonspecific background was attained with a scaling factor of 10, as verified using costaining of red and green HPA, or red and green secondary antibodies after differential staining of endogenous FasL. The standard settings of threshold colocalization are listed in the legend of Figure 2.
Evaluation of CD59 and CD81 Spreading
We have evaluated the internalization and intracellular distribution of fluorescent conjugates of monoclonal antibodies specific for diverse surface proteins CD59 and CD81. The constitutive traffic of these proteins follows different portals of endocytosis (Fritzsching et al., 2002; Naslavsky et al., 2005; Meertens et al., 2006). Three independent observers undertook morphological analysis of high-resolution images from at least four separate experiments and scored cells to have "spreading" when they exhibited antibodies staining that was strongly altered from the normal surface pattern. The levels of this spreading were highly correlated with colocalization values of the antibodies staining and subunit B of cholera toxin (CtxB).
Statistical Analysis
Results were presented as either histograms containing the mean ± SE or in interval plots defining the 95% confidence in data variation. Statistical significance of the difference between samples was undertaken with the nonparametric Mann–Whitney test and, whenever normal distribution was evident, also with parametric tests such as analysis of variance (ANOVA), by using the software package MiniTab15 (www.4ulr.com/products/statisticalanalysis). Statistical significance was considered strong when at least two independent tests yielded levels of significance or p values <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and HPA (Degli Esposti, 2008). HPA has been used here as the reference membrane marker because it can access multiple pathways of membrane traffic, similarly to other lectins such as WGA (Vetterlein et al., 2002; Cresawn et al., 2007; Degli Esposti, 2008).
Fas Activation Enhances More Pinocytosis than Clathrin-dependent Endocytosis
Given that previous studies have reported that Fas signaling increases either clathrin-dependent endocytosis (Austin et al., 2006; Lee et al., 2006; Kohlhaas et al., 2007) or pinocytosis (Kenis et al., 2004; Matarrese et al., 2008), we undertook a direct comparison of these different portals in Jurkat cells. Red fluorescent markers were incubated under conditions of steady-state equilibrium with external blue HPA, allowing rigorous colocalization analysis because of the large separation in the fluorescence of the probes. To label an established portal of clathrin-independent endocytosis, we initially used Ruby-dextran, which had been characterized previously as a fluid-phase marker of endocytic elements internalized via clathrin- and dynamin-independent endocytosis (Sabharanjak et al., 2002; Kirkham et al., 2005; Cheng et al., 2006). Confirming flow cytometry studies (Supplemental Figure S1 and Matarrese et al., 2008), images of Jurkat cells showed a clear increase in the uptake of Ruby-dextran after FasL treatment (Figure 1, A and B). A few vesicles loaded with Ruby-dextran were also positive for endocytosed HPA (Figure 1A, arrow). Although quantitative analysis (Costes et al., 2004) indicated modest levels of overall colocalization between Ruby-dextran and blue HPA in FasL-treated cells, these levels were highly significant because untreated cells exhibited a low uptake of the dextran, with consequent negligible colocalization (Supplemental Figure S2A). Of note, Ruby-dextran is extensively regurgitated early after internalization (Chadda et al., 2007).
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; Austin et al., 2006). Tf staining displayed a combination of cortical and juxtanuclear distribution, but only the latter increased after 30 min of FasL treatment (Figure 1C). Colocalization of internalized HPA with Tf-labeled elements was limited and concentrated around the Golgi region, in which recycling endosomes cluster (an average Pearson's index of 0.14 was determined in the experiment of Figure 1C, n = 14 cells). Consequently, Fas-mediated changes in Tf distribution looked similar to the polarized traffic of Tf receptors that has been observed after T cell activation (Blanchard et al., 2002). By analogy, Fas-induced polarization of Tf distribution (Figure 1C) could reflect enhanced entry into recycling endosomes, in which different portals of endocytosis empty their cargos (Sabharanjak et al., 2002; van Ijzendoorn, 2006). Hence, early Fas signaling increased the uptake of a pinocytic marker that follows clathrin-independent endocytosis (Mayor and Pagano, 2007) more than that of Tf, the classical marker of clathrin-dependent endocytosis. However, Fas also polarized membrane traffic toward the region containing recycling endosomes, thereafter labeled "peri-Golgi" (Supplemental Figure S2B).
Fas Stimulation Induces Peri-Golgi Migration of HPA
To efficiently visualize membrane traffic around the peri-Golgi region of T cells, we subsequently used markers that permanently bind to membrane components like fluorescent derivatives of CtxB, which is rapidly internalized following clathrin-independent routes converging toward recycling endosomes around the Golgi (Sabharanjak et al., 2002; Kirkham et al., 2005; Cheng et al., 2006; Chadda et al., 2007). These routes did not include caveolin-dependent endocytosis, which normally contributes to CtxB traffic (Kirkham et al., 2005; Chadda et al., 2007), because T cells do not express caveolin (Deckert et al., 1996; Orlandi and Fishman, 1998). In Jurkat and primary T cells, CtxB traffic reached equilibrium within 30 min of incubation (Figure 2). At longer times of Fas stimulation, CtxB staining became progressively scattered toward the cell periphery, following the dispersal of Golgi-related membranes that depends upon caspase activation (Ouasti et al., 2007; Degli Esposti, 2008). Hence, the characteristic peri-Golgi distribution of CtxB (verified by counterstaining with the Golgi-specific marker GM130; Supplemental Figure S2B) provided a valuable internal reference for observing membrane traffic changes before caspases became activated.
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), the mean value for threshold-adjusted Mander's coefficient (tM1) of red HPA over green CtxB increased more than twofold after FasL treatment (Figure 2B, significant at 0.04, Mann–Whitney test). The converse tM2 values for colocalization of green CtxB over red HPA also increased, albeit with weaker statistical significance than tM1 values (Figure 2B). However, complementary quantitative analysis using Pearson's index of overall colocalization (Rtot), which is distinct and has a wider spread of potential values than Mander's coefficients, showed a highly significant increase in the overall colocalization of HPA with CtxB after Fas stimulation (Figure 2C).
In permeabilized cells, HPA effectively labeled the same peri-Golgi region in which CtxB accumulated (Perez-Vilar et al., 1991; Ouasti et al., 2007). Triple labeling of permeabilized cells indicated that Fas stimulation strongly increased the colocalization of endocytosed red HPA with both green CtxB and internal blue HPA (Figure 3, A and B). This occurred without altering the strong colocalization of CtxB with internal HPA, because their average threshold-adjusted Mander's coefficient varied from 0.684–0.657 after FasL treatment. The increased peri-Golgi distribution of internalized HPA was additionally confirmed in dual HPA experiments (cf. Ouasti et al., 2007). The same experiments conducted in caspase-8–deficient Jurkat cells showed equivalent results of strong colocalization between internalized HPA and internal HPA (Figure 3C, right histograms), confirming that Fas-enhanced colocalization of endocytic and Golgi-related membranes was independent of apical caspases.
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Fas Stimulates the Traffic of CD59
We next followed the cellular distribution of fluorescently labeled antibodies specific for CD59, an abundant protein of T cells (Deckert et al., 1996) that is anchored to the exterior of the plasma membrane via glycosyl-phosphatidyl-inositol (GPI). As for other GPI-anchored proteins, the constitutive traffic of CD59 is specifically driven by clathrin- and dynamin-independent portals of endocytosis (Nichols et al., 2001; Sabharanjak et al., 2002; Naslavsky et al., 2004; Mayor and Pagano, 2007). Moreover, CD59 distribution is normally restricted to clusters lying at the surface of Jurkat cells (Deckert et al., 1996). Hence, we surmised that CD59 could be an ideal reporter for evaluating Fas-induced changes in constitutive endocytosis.
The surface-confined distribution of fluorescent anti-CD59 antibodies was lost soon after FasL treatment due to rapid internalization, which was followed by extensive redistribution in the cell interior (Figure 4, A and B). Within 30 min of Fas stimulation, several cells presented with CD59-positive membranes converging onto endocytosed HPA, increasing their colocalization around the peri-Golgi region (Figure 4, B and C). As a pertinent control, we followed the internalization of fluorescent antibodies specific for CD81, a surface protein trafficking predominantly via clathrin-dependent endocytosis (Fritzsching et al., 2002; Meertens et al., 2006). Contrary to CD59, CD81 remained confined in cortical and surface elements after Fas-stimulation, showing very little internal colocalization with HPA (Figure 4D). The specific accumulation of CD59 in the peri-Golgi region was subsequently confirmed, also quantitatively, in costaining experiments with CtxB (Figure 5).
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; Pilzer et al., 2005) and is reported to be present in Fas-stimulated Jurkat cells too (Elward et al., 2005). However, the release of membrane particles enriched in CD59 was strongly dependent upon the activation of apical caspases (Elward et al., 2005). Blocking caspases by the general inhibitor z-VAD not only reduced Fas-induced ejection of membrane particles but also enhanced the internalization of anti-CD59 antibodies (Figure 6A and Supplemental Figure S3A). Importantly, Fas stimulation enhanced CD59 internalization also in primary activated T lymphocytes (Supplemental Figure S3B). This indicated that rapid alteration of CD59 traffic was a general response of Fas-stimulated T cells.
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; Kirkham et al., 2005; Cheng et al., 2006), which are cumulatively blocked by toxin B of C. difficile (Mayor and Pagano, 2007). Pretreatment with C. difficile toxin B at concentrations that blocked Rho GTPases without impairing viability (Audoly et al., 2005) strongly inhibited Fas-induced internalization of CD59 and its colocalization with CtxB and HPA (Figures 6 and 7). Complementary results were obtained by quantitative analysis of colocalization between CD59 and CtxB (Figure 6A) and by the scoring of cellular spreading of CD59-labeled membranes that, contrary to that of CD81, strongly increased in Fas-stimulated cells (Figure 6, B and C). Indeed, CD59 staining hardly changed before and after Fas stimulation when the toxin was present, similarly to the unaltered pattern of CD81 staining (Figure 7A and Supplemental Figure S4A). We verified that C. difficile toxin predominantly blocked CDC42 by using secramine A, a compound that specifically inhibits CDC42 (Pelish et al., 2006). Results with secramine A were essentially superimposable to those obtained with C. difficile toxin B, following the colocalization between CD59 and CtxB (Figure 6A) and the internal distribution of CD59 (Figures 6B and 7B).
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; Eramo et al., 2004; Söderström et al., 2005; Lee et al., 2006). In type I cells, ligated death receptors and their associated DISC follow rapid clathrin-dependent internalization, ending up into late endosomes/lysosomes. This was marginally present in Jurkat cells (Lee et al., 2006), whereas other type II cells showed limited or delayed internalization of death receptors (Eramo et al., 2004; Austin et al., 2006; Matarrese et al., 2008).
To follow the path of ligated CD95/Fas in T cells, we first analyzed the internalization of FasL added at concentrations saturating its cognate receptor (Figure 8A, cf. Eramo et al., 2004). After 40–60 min, a proportion of FasL bound to Fas was found in intracellular elements that colocalized with either CtxB (Figure 8A) or endocytosed HPA (Figure 8, A and B; also see Figure 9A and Supplemental Figure S4B). Because DISC assembly and recruitment of procaspase-8 occur also during receptor internalization, the intracellular location of ligated Fas coincides with the initial intracellular activation of apical caspases (Lee et al., 2006). We then studied the subcellular distribution of active caspases using a fluorogenic substrate nominally specific for caspase-8, Rhodamine110-IETD-bisamide (Rho-IETD-bis), enabling direct measurements of caspase activation within cells (Packard et al., 2001). When, occasionally, a control cell displayed bright green staining after loading with Rho-IETD-bis, it also exhibited signs of advanced death with accumulated vacuoles, in part because the substrate reacted with cathepsin D under acidic conditions (S. Ivanova, unpublished data). Besides this background, punctuate staining of Rho-IETD-bis occurred between 30 and 60 min of Fas stimulation, progressively accruing around the peri-Golgi region. Consequently, active caspases became extensively colocalized with endocytosed HPA, as quantitatively verified with the usual colocalization analysis (Figure 8, C and D).
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). By using the latter approach, we followed the intracellular distribution of activated caspase-3, the major substrate of caspase-8 and dominant executioner caspase (Figure 9B). Along the progression of Fas signaling, caspase-3–positive elements moved from the periphery to the interior of cells, accumulating in the peri-Golgi region where they colocalized with endocytosed HPA (Figure 9B). Subsequent to caspase-dependent fragmentation of the Golgi (Ouasti et al., 2007), caspase-3- and HPA-positive membranes continued to be closely associated throughout the cell (Figure 9B, bottom).
Intriguingly, Rho GTPase inhibition marginally reduced the overall intensity of caspase activation within Fas-stimulated cells (Supplemental Figure S4C), indicating that Rho GTPase-dependent routes of membrane traffic were unlikely to contribute to DISC assembly. Did then Rho GTPases influence downstream steps of death propagation? To answer this crucial question, we resorted to analytically determine the levels of terminal blebbing, an early hallmark of Fas-induced death (Weis et al., 1995) that could be evaluated under the same conditions of our endocytosis studies (see Materials and Methods and Supplemental Figure S4D). Concomitantly with the initial increase in caspase activation, the basal level of terminal blebbing increased over 10-fold (Figure 9C). As expected, blocking caspases with z-VAD reduced this indicator of incipient death (Figure 9C). However, also secramine and C. difficile toxin B significantly reduced Fas-enhanced terminal blebbing (Figure 9C). Our results thus suggested that Rho GTPases activated early after DISC assembly may contribute to death propagation in T cells, consistent with previous evidence for a link between receptor-stimulated endocytosis and apoptosis signaling (Lee et al., 2006; Matarrese et al., 2008).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kenis et al., 2004; Lee et al., 2006; Ouasti et al., 2007; Matarrese et al., 2008), the mechanism and significance of Fas-enhanced endocytosis have remained obscure (Siegel et al., 2004; Austin et al., 2006; Chaigne-Delalande et al., 2008; Reinehr and Häussinger, 2008). Here, we clarify that Fas-stimulated T cells open Rho GTPase-dependent portals that drive the traffic of CD59 (Figures 4
–7). Fas signaling also induces a global polarization of endocytic membranes toward the Golgi apparatus, a novel finding with the important implications discussed below.
We have extensively used HPA as a general membrane marker with access to different portals of endocytosis as well as secretory organelles. This marker has been essential for visualizing the peculiar alteration in membrane traffic that Fas stimulation induces in T cells, namely, the accumulation of early and recycling endosomes in the peri-Golgi region. Previously, we reported that FasL-treated cells showed an increased merging of internalized HPA with GM130 and ERGIC-53, membrane markers of the early secretory system (Ouasti et al., 2007). Concomitantly, HPA-labeled membranes also colocalized with mitochondria (Ouasti et al., 2007), which in turn became associated with early endosomes (Kawasaki et al., 2000; Matarrese et al., 2008). Other studies have shown an intracellular colocalization of Fas with CtxB (Siegel et al., 2004; Legembre et al., 2005), which typically concentrates in the peri-Golgi region (Figure 2 and Supplemental Figure S2B; cf. Sabharanjak et al., 2002). We can now rationalize all this evidence as an expression of Fas-enhanced endocytic traffic concentrating membrane elements in the peri-Golgi region, in which Golgi membranes and mitochondria cluster together (for review, see Degli Esposti, 2008). Consequently, the intermixing of mitochondria and other organelles observed previously may represent a reporter for the Fas-induced polarization of membrane traffic.
These considerations could be extended to other type II cells but not to type I cells. Abundant evidence obtained after surface down-modulation of CD95/Fas has indicated that type I cells respond to Fas stimulation by enhancing clathrin-dependent endocytosis, along which ligated receptors are rapidly internalized and form a mobile signaling platform (Algeciras-Schimnich et al., 2002; Lee et al., 2006). This pathway of endocytosis ultimately sorts Fas and its associated DISC for endolysosomal degradation, thereby leading to signal attenuation.
Type II cells do not show an equivalent sorting for rapid degradation (Lee et al., 2006; Ouasti et al., 2007), suggesting a diversion of membrane traffic from late endosomes/lysosomes. Confirming this possibility, we demonstrate here that Fas stimulation concentrates and polarizes membrane traffic into the peri-Golgi region of T cells, in which recycling endosomes connect the endocytic pathway to the exocytic pathway (van Ijzendoorn, 2006; Ménager et al., 2007). The traffic diversion into recycling endosomes could provide spatial segregation of Fas signaling into the cell, creating two interconnections: 1) between endocytic elements and exocytosis, which also drives a Fas-induced delivery of new death receptor molecules to the cell surface (Rheiner and Häussinger, 2008); and 2) between endocytic elements and mitochondria, the intermixing of which may facilitate the priming of mitochondrial membranes to the proapoptotic action of Bcl-2 proteins (Matarrese et al., 2008).
Both interconnections underline characteristic features of type II cells, such as the delayed down-modulation of ligated Fas receptors (Eramo et al., 2004; Chaigne-Delalande et al., 2008) and the crucial engagement of mitochondria for caspase amplification (Peter and Krammer, 2003). Moreover, enhanced traffic into recycling endosomes could be instrumental for routing active caspases to selected cellular compartments, in particular secretory organelles where their action promotes outward movement of endomembranes (Elward et al., 2005; Ouasti et al., 2007; Rheiner and Häussinger, 2008). These endomembranes contain newly synthesized Fas that, once delivered to the cell surface, could produce signal persistence by binding to additional FasL molecules. This process would partially compensate for ongoing internalization of ligated receptors and perhaps potentiate intracellular signaling until mitochondria are fully engaged.
In the new view of traffic diversion to recycling endosomes, how would Fas signaling produce a different sorting of endocytic membranes in different cell types? Our simplest explanation is that the assembled DISC promotes activation of Rho GTPases in T and other type II cells but not in type I cells. Besides our data of Rho-GTPase inhibition (Figures 6 and 7), the studies of Subauste et al. (2000) and Söderström et al. (2005) provide supportive evidence for early activation of CDC42 and related GTPases, which is not present in type I cells. Moreover, Rho GTPases also reside in recycling endosomes, in which they may further stimulate membrane traffic.
The small Rho GTPase CDC42 predominantly drives Fas-stimulated membrane traffic in T cells, for the following reasons: 1) it promotes the selective traffic of GPI-anchored proteins such as CD59 (Sabharanjak et al., 2002; Mayor and Pagano, 2007), which we found to be rapidly altered after Fas activation (Figures 4–7); 2) it is activated early after Fas stimulation (Subauste et al., 2000), as confirmed in our experiments (unpublished data); 3) it is selectively inhibited by the semisynthetic compound secramine A (Pelish et al., 2006), which abolishes the alteration of CD59 traffic (Figures 6 and 7); 4) it is upstream of Rac in promoting filopodia and other surface protrusions, which are blocked by C. difficile toxin B (Malorni et al., 2003), together with the reduction in CD59 traffic alterations (Figure 6); 5) it is the central regulator of cell polarity (Etienne-Manneville, 2004), and we found that Fas stimulation increases the polarization of membrane traffic toward the Golgi region (Figures 2–7); and 6) when expressed in its constitutively active form, it promotes cell death in Jurkat cells (Chuang et al., 1997), and we found that its inhibition with secramine reduces incipient cell death (Figure 9C).
In clarifying the link between endocytosis and the intracellular signaling of Fas, our work opens new perspectives to appreciate the biological role of membrane traffic in the death program of T cells. Of note, polarized recycling of membrane traffic is a key property of activated T cells, which enables intercellular communication with other cells of the immune system (Krummel and Macara, 2006). We are currently investigating the connections between intracellular membrane traffic and intercellular forms of communication during Fas-induced cell death.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Mauro Degli Esposti (mauro.esposti{at}manchester.ac.uk)
Abbreviations used: APC, allophycocyanin; HPA, Helix pomatia agglutinin; Rho-IETD-bis, rhodamine110carbonyl-Ile-Glu-Thr-Asp-bisamide; PE, phychoerythrin; RB, modified Ringer buffer; z-VAD, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; WGA, wheat germ agglutinin.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Audoly, G., Popoff, M. R., and Gluschankof, P. (2005). Involvement of a small GTP binding protein in HIV-1 release. Retrovirology 2, 48.[CrossRef][Medline]
Austin, C. D. et al. (2006). Death-receptor activation halts clathrin-dependent endocytosis. Proc. Natl. Acad. Sci. USA 103, 10283–10288.
Blanchard, N., Di Bartolo, V., and Hivroz, C. (2002). In the immune synapse, ZAP-70 controls T cell polarization and recruitment of signaling proteins but not formation of the synaptic pattern. Immunity 17, 389–399.[CrossRef][Medline]
Chadda, R., Howes, M. T., Plowman, S. J., Hancock, J. F., Parton, R. G., and Mayor, S. (2007). Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway. Traffic 8, 702–717.[CrossRef][Medline]
Chaigne-Delalande, B., Moreau, J. F., and Legembre, P. (2008). Rewinding the DISC. Arch. Immunol. Ther. Exp 56, 9–14.[CrossRef]
Cheng, Z. J., Singh, R. D., Sharma, D. K., Holicky, E. L., Hanada, K., Marks, D. L., and Pagano, R. E. (2006). Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements. Mol. Biol. Cell 17, 3197–3210.
Chuang, T. H., Hahn, K. M., Lee, J. D., Danley, D. E., and Bokoch, G. M. (1997). The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes. Mol. Biol. Cell 8, 1687–1698.[Abstract]
Conner, S. D., and Schmid, S. L. (2003). Regulated portals of entry into the cell. Nature 422, 37–44.[CrossRef][Medline]
Costes, S. V., Daelemans, D., Cho, E. H., Dobbin, Z., Pavlakis, G., and Lockett, S. (2004). Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys. J 86, 3993–4003.[CrossRef][Medline]
Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., Apodaca, G., and Weisz, O. A. (2007). Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins. EMBO J 26, 3737–3748.[CrossRef][Medline]
Davis, D. M., and Sowinski, S. (2008). Membrane nanotubes: dynamic long-distance connections between animal cells. Nat. Rev. Mol. Cell Biol 9, 43–436.
Degli Esposti, M. (2008). Organelle intermixing and membrane scrambling in cell death. Methods Enzymol 422, 421–438.
Deckert, M., Ticchioni, M., and Bernard, A. (1996). Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases. J. Cell Biol 133, 791–799.
Di Fiore, P. P., and De Camilli, P. (2001). Endocytosis and signaling. An inseparable partnership. Cell 106, 1–4.
Distler, J. H., Huber, L. C., Hueber, A. J., Reich, C. F., 3rd, Gay, S., Distler, O., and Pisetsky, D. S. (2005). The release of microparticles by apoptotic cells and their effects on macrophages. Apoptosis 10, 731–741.[CrossRef][Medline]
Eramo, A. et al. (2004). CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. Eur. J. Immunol 34, 1930–1940.[CrossRef][Medline]
Etienne-Manneville, S. (2004). Cdc42–the centre of polarity. J. Cell Sci 117, 1291–1300.
Elward, K., Griffiths, M., Mizuno, M., Harris, C. L., Neal, J. W., Morgan, B. P., and Gasque, P. (2005). CD46 plays a key role in tailoring innate immune recognition of apoptotic and necrotic cells. J. Biol. Chem 280, 36342–36454.
Fritzsching, B., Schwer, B., Kartenbeck, J., Pedal, A., Horejsi, V., and Ott, M. (2002). Release and intercellular transfer of cell surface CD81 via microparticles. J. Immunol 169, 5531–5537.
Green, D. R., Droin, N., and Pinkoski, M. (2003). Activation-induced cell death in T cells. Immunol. Rev 193, 70–81.[CrossRef][Medline]
Kawasaki, Y., Saito, T., Shirota-Someya, Y., Ikegami, Y., Komano, H., Lee, M. H., Froelich, C. J., Shinohara, N., and Takayama, H. (2000). Cell death-associated translocation of plasma membrane components induced by CTL. J. Immunol 164, 4641–4648.
Kenis, H., van Genderen, H., Bennaghmouch, A., Rinia, H. A., Frederik, P., Narula, J., Hofstra, L., and Reutelingsperger, C. P. (2004). Cell surface-expressed phosphatidylserine and annexin A5 open a novel portal of cell entry. J. Biol. Chem 279, 52623–52629.
Kohlhaas, S. L., Craxton, A., Sun, X. M., Pinkoski, M. J., and Cohen, G. M. (2007). Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. J. Biol. Chem 282, 12831–12841.
Kirkham, M., Fujita, A., Chadda, R., Nixon, S. J., Kurzchalia, T. V., Sharma, D. K., Pagano, R. E., Hancock, J. F., Mayor, S., and Parton, R. G. (2005). Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol 168, 465–476.
Krummel, M. F., and Macara, I. (2006). Maintenance and modulation of T cell polarity. Nat. Immunol 7, 1143–1149.[CrossRef][Medline]
Lee, K. H., Feig. C., Tchikov, V., Schickel, R., Hallas, C., Schutze, S., Peter, M. E., and Chan, A. C. (2006). The role of receptor internalization in CD95 signaling. EMBO J 25, 1009–1023.[CrossRef][Medline]
Legembre, P., Daburon, S., Moreau, P., Ichas, F., de Giorgi, F., Moreau, J. F., and Taupin, J. L. (2005). Amplification of Fas-mediated apoptosis in type II cells via microdomain recruitment. Mol. Cell. Biol 25, 6811–6820.
Lugini, L. et al. (2003). Potent phagocytic activity discriminates metastatic and primary human malignant melanomas: a key role of ezrin. Lab. Invest 83, 1555–1567.[CrossRef][Medline]
Malorni, W., Quaranta, M. G., Straface, E., Falzano, L., Fabbri, A., Viora, M., and Fiorentini, C. (2003). The Rac-activating toxin cytotoxic necrotizing factor 1 oversees NK cell-mediated activity by regulating the actin/microtubule interplay. J. Immunol 171, 4195–41202.
Matarrese, P., Manganelli, V., Garofalo, T., Tinari, A., Gambardella, L., Ndebele, K., Khosravi-Far, R., Sorice, M., Degli Esposti, M., and Malorni, W. (2008). Endosomal compartment contributes to the propagation of CD95/Fas-mediated signals in type II cells. Biochem. J 413, 467–478.[CrossRef][Medline]
Mayor, S., and Pagano, R. E. (2007). Pathways of clathrin-independent endocytosis. Nat. Rev. Mol. Cell Biol 8, 603–612.[CrossRef][Medline]
Meertens, L., Bertaux, C., and Dragic, T. (2006). Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles. J. Virol 80, 11571–11578.
Ménager, M. M., Ménasché, G., Romao, M., Knapnougel, P., Ho, C. H., Garfa, M., Raposo, G., Feldmann, J., Fischer, A., and de Saint Basile, G. (2007). Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4. Nat. Immunol 8, 257–267.[CrossRef][Medline]
Naslavsky, N., Weigert, R., and Donaldson, J. G. (2004). Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements. Mol. Biol. Cell 15, 3542–3552.
Nichols, B. J., Kenworthy, A. K., Polishchuk, R. S., Lodge, R., Roberts, T. H., Hirschberg, K., Phair, R. D., and Lippincott-Schwartz, J. (2001). Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol 153, 529–541.
Orlandi, P. A., and Fishman, P. H. (1998). Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J. Cell Biol 141, 905–915.
Ouasti, S., Matarrese, P., Paddon, Khosravi-Far, R. R., Sorice, M., Tinari, A., Malorni, W., and Degli Esposti, M. (2007). Death receptor ligation triggers membrane scrambling between Golgi and mitochondria. Cell Death Differ 14, 453–461.[CrossRef][Medline]
Packard, B. Z., Komoriya, A., Brotz, T. M., and Henkart, P. A. (2001). Caspase 8 activity in membrane blebs after anti-Fas ligation. J. Immunol 167, 5061–5066.
Pelish, H. E., Peterson, J. R., Salvarezza, S. B., Rodriguez-Boulan, E., Chen, J. L., Stamnes, M., Macia, E., Feng, Y., Shair, M. D., and Kirchhausen, T. (2006). Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro. Nat. Chem. Biol 2, 39–46.[CrossRef][Medline]
Perez-Vilar, J., Hidalgo, J., and Velasco, A. (1991). Presence of terminal N-acetylgalactosamine residues in subregions of the endoplasmic reticulum is influenced by cell differentiation in culture. J. Biol. Chem 266, 23967–23976.
Peter, M. E., and Krammer, P. H. (2003). The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ 10, 26–35.[CrossRef][Medline]
Pilzer, D., Gasser, O., Moskovich, O., Schifferli, J. A., and Fishelson, Z. (2005). Emission of membrane vesicles: roles in complement resistance, immunity and cancer. Springer Semin. Immunopathol 27, 375–387.[CrossRef][Medline]
Reinehr, R., and Häussinger, D. (2008). CD95 ligation and intercellular membrane flow. Biochem. J 413, e11–e12.[CrossRef][Medline]
Sabharanjak, S., Sharma Parton, P.R.G., and Mayor, S. (2002). GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423.[CrossRef][Medline]
Siegel, R. M., Muppidi, J. R., Sarker, M., Lobito, A., Jen, M., Martin, D., Straus, S. E., and Lenardo, M. J. (2004). SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane. J. Cell Biol 167, 735–744.
Söderström, T. S., Nyberg, S. D., and Eriksson, J. E. (2005). CD95 capping is ROCK-dependent and dispensable for apoptosis. J. Cell Sci 118, 2211–2223.
Stinchcombe, J. C., Bossi, G., Booth, S., and Griffiths, G. M. (2001). The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15, 751–761.[CrossRef][Medline]
Subauste, M. C., Von Herrath, M., Benard, V., Chamberlain, C. E., Chuang, T. H., Chu, K., Bokoch, G. M., and Hahn, K. M. (2000). Rho family proteins modulate rapid apoptosis induced by cytotoxic T lymphocytes and Fas. J. Biol. Chem 275, 9725–9733.
van Ijzendoorn, S. C. (2006). Recycling endosomes. J. Cell Sci 119, 1679–1681.
Vetterlein, M., Ellinger, A., Neumuller, J., and Pavelka, M. (2002). Golgi apparatus and TGN during endocytosis. Histochem. Cell Biol 117, 143–150.[CrossRef][Medline]
Weis, M., Schlegel, J., Kass, G. E., Holmstrom, T. H., Peters, I., Eriksson, J., Orrenius, S., and Chow, S. C. (1995). Cellular events in Fas/APO-1-mediated apoptosis in JURKAT T lymphocytes. Exp. Cell Res 219, 699–708.[CrossRef][Medline]
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