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J Physiol (2003), 547.3, pp. 835-847
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.033522
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ABSTRACT |
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One important mechanism cytotoxic T lymphocytes (CTLs) use to kill virus-infected, transplanted or tumour targets is exocytosis of granules that contain cytotoxic agents such as perforin and granzymes. Granule exocytosis-dependent target cell killing is a complex process, involving initial T-cell receptor (TCR)-dependent signalling that includes Ca2+ influx and activation of protein kinase C, shape changes that serve to bind the CTL to the target and, finally, exocytosis of lytic granules at the site of contact with the target cell. Although there is reason to propose that multiple steps in the lytic process could involve the actin cytoskeleton of CTLs, few studies have examined this issue, and those that have do not allow the specific step(s) involved to be determined. We have used the potent membrane-permeant actin cytoskeleton-modifying drugs jasplakinolide and latrunculin A to investigate the actin dependence of defined processes that are expected to be important for granule exocytosis-dependent killing. Our results, obtained using TALL-104 human leukaemic CTLs as a model system, are consistent with the idea that a functional actin cytoskeleton is required for TCR/CD3-dependent signalling, for activation of store-dependent Ca2+ influx and for CTL shape changes. When cells were stimulated with solid-phase anti-CD3 antibodies, treatment with either jasplakinolide or latrunculin A abolished granule exocytosis. However, when cells were stimulated in a manner that bypasses TCR/CD3-dependent signalling, granule exocytosis was not significantly altered, suggesting that the actin cytoskeleton does not function as a barrier to exocytosis.
(Received 30 September 2002; accepted after revision 13 December 2002; first published online 7 February 2003)
Corresponding author A. Zweifach: Department of Physiology and Biophysics, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Denver, CO 80262, USA. Email: adam.zweifach{at}uchsc.edu
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INTRODUCTION |
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Cytotoxic T lymphocytes (CTLs) kill virus-infected cells, tumour cells and cells in transplanted tissues and organs. One important mechanism they use is the target-directed exocytosis of preformed lytic granules that contain cytotoxic agents such as perforin and granzymes (reviewed in Berke, 1994, 1995; Griffiths, 1995). Granule exocytosis-mediated target cell killing is a complex process that involves initial T-cell receptor (TCR)-dependent recognition and signalling events that include activation of protein kinase C (PKC) and influx-dependent increases in intracellular Ca2+ concentration ([Ca2+]i), CTL shape changes that may contribute to the formation of CTL-target conjugates and, finally, fusion of lytic granules with the plasma membrane in the region of the CTL in contact with the target.
There is reason to propose that each of these steps in the lytic interaction could involve the CTL actin cytoskeleton. Initial TCR-dependent signalling events are likely to depend on the actin cytoskeleton, since a functional actin cytoskeleton may be required for efficient TCR/CD3-mediated signalling (Arrieumerlou et al. 2000), and could be required for the formation of a supramolecular activation complex or immunological synapse, a highly organized signalling structure that assembles at sites of contact between T cells and antigen-presenting cells (APCs; Monks et al. 1998; Dustin & Cooper, 2000) or targets (Potter et al. 2001; Stinchcombe et al. 2001). Activation of store-dependent capacitative Ca2+ entry (CCE), which is thought to underlie influx in helper T cells (Lewis, 2001) and CTLs (Zweifach, 2000), has been reported to be actin dependent (Patterson et al. 1999; Rosado & Sage, 2000a, b). The shape changes that the CTL undergoes after contacting a relevant target that are important for conjugate formation are likely to be actin dependent, as the actin cytoskeleton plays important roles in shape changes in many cell types, including T cells (Parsey & Lewis, 1993; Valitutti et al. 1993; Delon et al. 1998; Borroto et al. 2000). Finally, the fusion of lytic granules with the plasma membrane could require the disassembly of cortical actin, as cortical actin has been suggested to function as a barrier to exocytosis in many secretory cell types (Trifaro et al. 1992; Sugawara et al. 1993; Roth & Burgoyne, 1995; Chowdhury et al. 2000; Gil et al. 2000; Yoneda et al. 2000).
The relatively few studies that have used pharmacological agents to examine the role of the actin cytoskeleton in CTL function demonstrate clear effects, but do not allow the specific steps affected to be identified. O'Rourke et al. (1991) reported that cytochalasin D inhibited target cell-stimulated granule exocytosis and target cell killing, but found that exocytosis in response to an immobilized anti-TCR monoclonal antibody (mAb) was not inhibited. Valitutti et al. (1993) showed that cytochalasin D prevented CTL shape changes in response to target cell contact, and suggested that cAMP could modulate CTL function by decreasing filamentous actin. Perez et al. (1985) found that CTL-target conjugate formation was inhibited by cytochalasin B treatment. Lancki et al. (1987) showed that cytochalasin B treatment inhibited the exocytosis stimulated by antigen/major histocompatibility complex (MHC) or mitogenic lectins, but did not affect the exocytosis stimulated by ionomycin and phorbol 12-myristate 13-acetate (PMA).
Our goal in the present work was to test systematically the involvement of the actin cytoskeleton in the various processes described above that could contribute to granule exocytosis-mediated target cell killing. We used potent membrane-permeant pharmacological agents that have defined effects on the actin cytoskeleton: latrunculin A, a marine toxin that sequesters actin monomers to depolymerize actin filaments (Spector et al. 1983; Ayscough, 1998), and jasplakinolide, an agent that inhibits actin filament disassembly to promote the formation of actin filaments (Scott et al. 1988; Bubb et al. 1994). As a model system, we used TALL-104 human leukaemic CTLs (Cesano & Santoli, 1992), which, as we have shown previously, release perforin and granzyme in response to Raji B cells coated with a bispecific antibody (bsAb) that contains an 
-CD3 F(ab') (Lyubchenko et al. 2001). Perforin release in response to bsAb-treated Raji cells is characterized by key features that have been described for other CTL preparations, including: shape changes leading to conjugate formation, granule reorientation, exocytosis that occurs specifically at the point of contact with bsAb-treated target, and Ca2+ dependence (Lyubchenko et al. 2001), indicating that this system duplicates the key features of normal CTL function. Our results suggest that the actin cytoskeleton is in fact involved in multiple processes that are likely to be important for target cell killing.
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METHODS |
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Chemicals and reagents
Salts for physiological solutions and poly-L-lysine were from Sigma-Aldrich (St Louis, MO, USA). Fura-2 AM, calcein AM, latrunculin A, jasplakinolide, Alexa fluor 532-labelled phalloidin, Alexa fluor 488-labelled DNAse 1 and 4',6-diamidino-2-phenylindol (DAPI) were from Molecular Probes (Eugene, OR, USA). Thapsigargin (TG) was purchased from Alexis Biochemicals (San Diego, CA, USA). Fetal calf serum (FCS) and glutamine were purchased from Gemini Bioproducts (Calabassas, CA, USA). -CD3-coated beads (Dynabeads M-450 Pan T) were purchased from Dynal A.S. (Oslo, Norway). The bsAb Hu1D10-Jun x HuM291-Fos (Link et al. 1998) was a gift from Protein Design Laboratories (Mountain View, CA, USA). Fluorescein isothiocyanate (FITC)-labelled OKT3 anti-CD3 mAb was obtained from Caltag Laboratories (Burlingame, CA, USA). UCHT1 anti-CD3 mAb was obtained from ID Labs (London, Ontario, Canada.)
Cells
Raji B lymphoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and were grown in RPMI supplemented with 10 % FCS. TALL-104 cells were also obtained from the American Type Culture Collection (Rockville, MD, USA) and grown in Iscove's medium supplemented with 10 % FCS and 100 i.u. interleukin-2. Both cell types were grown in a humidified incubator at 37 °C in 10 % CO2. Raji cells were loaded with calcein by incubating them with 1 µM calcein AM in cell culture medium for 20 min at room temperature, and were treated with bsAb by incubating them in the presence of 320 µg ml-1 bsAb in Ringer solution for 15 min at room temperature. TALL-104 cells were loaded with fura-2 by incubating them with 1 µM fura-2 AM in cell culture medium for 30 min at room temperature. TALL-104 cells were treated with latrunculin A or jasplakinolide by incubating them with 1 µM drug in cell culture medium for 60 min at room temperature. Control experiments indicate that this room temperature incubation does not affect target cell killing or Ca2+ signalling. Cells were washed twice with fresh medium before use. Except where indicated, in all experiments involving a cytoskeleton inhibitor experimental solutions were supplemented with the inhibitor.
Solutions
Ringer solution contained (mM): 145 NaCl, 4.5 KCl, 1 MgCl2, 2 CaCl2, 5 Hepes and 10 glucose (pH 7.4 with NaOH). Zero Ca2+ Ringer solution was identical, except CaCl2 was replaced with MgCl2 and 1 mM EGTA was added.
Imaging
Experiments were performed at room temperature with imaging systems that have been described previously (Zweifach, 2000), built around Nikon inverted microscopes (Nikon, Melville, NY, USA). On system 1, which was used for analysis of actin-modifying drug effects (Fig. 1), excitation light was provided by a 150 W xenon arc lamp (Optiquip, Highland Mills, NY, USA), and excitation wavelength was selected using appropriate excitation filters in a Sutter Lambda-10 filter wheel (Sutter Instruments, Novato, CA, USA). A fura-BCECF (2',7'-bis(carboxyethyl)-5-carboxyfluorescein) dichroic mirror was used in the filter cube, and emission light was filtered with a second Sutter filter wheel. The filters and the dichroic mirror were obtained from Chroma (Brattleboro, VT, USA). On system 2, which was used for all other experiments, excitation light was provided by a Sutter Lambda light source coupled to the microscope by a liquid light guide, and excitation light was filtered as on system 1. A Chroma UV/FITC/TRITC (tetramethyl rhodamine isothiocyanate) beam splitter and emission filter was used. On both systems, images were acquired with Cooke Sensicam peltier-cooled interline CCD cameras (PCO, Kelhiem, Germany). Hardware was controlled and images were acquired and analysed with SlideBook software from Intelligent Imaging Innovations (Denver, CO, USA) running on Macintosh computers. For analysis of Ca2+ data, background-subtracted images were thresholded on F340, and ratios were computed pixel by pixel. Ratios were converted to [Ca2+]i according to the method of Grynkiewicz et al. (1985) using calibration values obtained in vitro. Data were analysed further using Igor Pro software (Wavemetrics, Lake Oswego, OR, USA).
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Figure 1. Effects of jasplakinolide and latrunculin A on the actin cytoskeleton of TALL-104 cells Formaldehyde-fixed methanol-permeabilized cells were stained with Alexa fluor-532-labelled phalloidin to label f-actin, Alexa fluor 488-labelled DNAse I to label g-actin, and DAPI to label nuclei, and examined on our imaging system. Jasplakinolide increased cellular f-actin content and decreased g-actin levels. Latrunculin A reduced f-actin and decreased the f-actin:g-actin ratio. Images shown were acquired with a | ||
Granzyme release assays
Granyzme released from TALL-104 cells was assayed by measuring hydrolysis of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) essentially as described previously (Takayama et al. 1987).
Analysis of conjugate formation
Control or drug-treated fura-2-loaded TALL-104 cells were mixed with bsAb-treated calcein-loaded Raji cells at room temperature at ~1:1 ratio for 20 min in zero-Ca2+ Ringer solution. They were then adhered to poly-L-lysine-coated coverslips, washed with zero-Ca2+ Ringer solution, and the number of Raji cells with 0, 1, 2, 3 or 4 TALL-104 cells attached was counted.
Flow cytometric analysis of CD3 expression
Control or drug-treated TALL-104 cells were incubated with FITC-labelled OKT3 -CD3 (diluted 1:60 in normal Ringer solution) for 20 min at room temperature, then washed twice with normal Ringer solution and placed on ice. Flow cytometric analysis of surface CD3 expression was performed on a FACSCalibur flow cytometer (Beckton Dickinson Immunocytometry Systems, San Jose, CA, USA).
Statistics
Except where noted, all results are presented as means ± S.E.M. Statistical significance was assessed using free on-line Student's t tests available at http://www.graphpad.com
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RESULTS |
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Effects of jasplakinolide and latrunculin A on the actin cytoskeleton of TALL-104 CTLs
We first assessed the effects of jasplakinolide and latrunculin A on the actin cytoskeleton of TALL-104 cells (Fig. 1 and Table 1). We fixed and then permeabilized control and drug-treated cells, and used Alexa fluor 532-labelled phalloidin to stain filamentous actin (f-actin; Wulf et al. 1979), Alexa fluor 488-labelled DNAse 1 to stain monomeric actin (g-actin; Haugland et al. 1994) and DAPI to stain nuclei. In untreated cells, f-actin was present as a cortical ring, while g-actin was distributed more uniformly throughout the cell (Fig. 1). As expected, jasplakinolide increased actin polymerization by increasing f-actin and concomitantly decreasing g-actin (Fig. 1 and Table 1). Note that as jasplakinolide and phalloidin compete for binding to the same site on actin (Bubb et al. 1994), the data in Fig. 1 and Table 1 are likely to underestimate the true extent of actin polymerization that jasplakinolide promotes. The f-actin:g-actin ratio was significantly enhanced by jasplakinolide (Table 1). Jasplakinolide also caused obvious alterations in cell morphology. As expected based on its ability to sequester actin monomers (g-actin), latrunculin A decreased actin the f-actin:g-actin ratio (Fig. 1 and Table 1). Interestingly, the levels of both f-actin and g-actin were apparently reduced by latrunculin A treatment, suggesting that total cellular actin levels were reduced. The reason for the apparent decrease in g-actin levels in latrunculin A-treated cells is unclear, but may be a result of inhibition of new g-actin synthesis during the course of drug treatment by transiently increased g-actin levels (Lyubimova et al. 1999). Regardless, based on the effects we observed on f-actin staining and on the f-actin:g-actin ratio, we conclude that jasplakinolide and latrunculin A provide tools that allow us to selectively increase or decrease cellular actin polymerization.
Jasplakinolide and latrunculin A inhibit the perforin release stimulated by bsAb-treated Raji cells
As an initial means of testing the idea that the actin cytoskeleton is involved in the lytic interaction between CTLs and targets, we examined the effects of jasplakinolide and latrunculin A on the perforin release triggered by bsAb-treated Raji cells (Fig. 2). We have shown previously that this system duplicates key features of normal CTL- target interactions (Lyubchenko et al. 2001). Granule exocytosis is target directed and is Ca2+-dependent. We monitored perforin release with a method we developed (Zweifach, 2000) based on the CARE-LASS assay, designed to replace the standard 51Cr-release assay of target cell killing (Lichtenfels et al. 1994). Our method detects the insertion of perforin pores in the target cell's membrane in real time by monitoring the rate of dye release from single target cells. In these experiments, bsAb-treated Raji cells that were loaded with calcein were adhered to coverslips. An excess of TALL-104 cells was added to the chamber and allowed to settle into contact with the Raji cells. In control experiments bsAb-treated Raji cells began to exhibit sudden decreases in calcein fluorescence, consistent with dye leakage through perforin pores inserted via granule exocytosis (Lichtenfels et al. 1994; Zweifach, 2000; Lyubchenko et al. 2001) approximately 500 s after TALL-104 addition (Fig. 2A). After ~2000 s, ~70 % of Raji cells had released the dye. In contrast, when Tall-104 cells were pretreated with either of the cytoskeleton modifiers, dye release was essentially eliminated (Fig. 2B and C). The effects of latrunculin A and jasplakinolide on calcein release from bsAb-treated Raji cells are consistent with the idea that actin-dependent processes are involved in at least one critical step in the interaction between CTLs and targets that is required for exocytosis of perforin-containing granules.
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Figure 2. Jasplakinolide and latrunculin A inhibit perforin release stimulated by bsAb-treated Raji cells Top, fluorescence images of calcein-loaded bsAb-treated Raji cells before and 2200 s after the addition of an excess of TALL-104 cells. Bottom, time course of hitting computed from calcein fluorescence traces for 107 cells, 65 cells and 116 cells as in Lyubchenko et al. (2001). Arrows indicate time of addition of TALL-104 cells. Images were acquired with a | ||
Jasplakinolide and latrunculin A inhibit CD3-stimulated CTL [Ca2+]i responses
As the increases in [Ca2+]i and activation of PKC that occur as a result of TCR/CD3 signalling are known to be critical for granule exocytosis (Berebi et al. 1987; Lancki et al. 1987), one mechanism by which latrunculin A and jasplakinolide could inhibit perforin release is by inhibiting signalling via the TCR/CD3 complex. To test whether perturbing the actin cytoskeleton inhibits contact-stimulated [Ca2+]i increases (Fig. 3A), fura-2-loaded TALL-104 cells were adhered to coverslips, and bsAb-treated Raji cells were dropped onto them. In control experiments, single-cell [Ca2+]i signals elicited by contact with bsAb-treated Raji cells were heterogeneous, often consisting of repetitive oscillations from baseline levels of 50-100 nM to values ranging from 300 to 1500 nM (data not shown). Both latrunculin A and jasplakinolide profoundly inhibited [Ca2+]i elevations (Fig. 3A). The average [Ca2+]i in the presence of modifiers showed no increase, although individual cells sometimes displayed small transient responses. Similar effects of latrunculin A and jasplakinolide on [Ca2+]i signals triggered by -CD3-coated polystyrene beads were observed (Fig. 3B), although the effects were not as pronounced.
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Figure 3. Jasplakinolide and latrunculin A inhibit CD3-stimulated [Ca2+]i signals Effects of jasplakinolide and latrunculin A on [Ca2+]i responses induced by bsAb-treated Raji cells (A), | ||
We next tested the effects of jasplakinolide and latrunculin A on [Ca2+]i responses stimulated by soluble -CD3 mAb (Fig. 3C). Soluble antibodies (Abs) cause only minimal cross-linking, and are therefore expected to reveal any deficits in signalling through the TCR/CD3 (Arrieumerlou et al. 2000). Cells were stimulated with 10 µg ml-1 UCHT1 in normal Ringer solution. Both jasplakinolide and latrunculin A profoundly inhibited [Ca2+]i elevations in response to UCHT1. Analysis of single-cell [Ca2+]i traces revealed that in addition to decreasing the amplitude of single-cell [Ca2+]i responses, jasplakinolide and latrunculin A decreased the number of cells that responded to soluble Ab and, for cells that did respond, increased the delay between Ab addition and initiation of [Ca2+]i signalling (data not shown).
Effects of jasplakinolide and latrunculin A on plasma membrane CD3 levels and conjugate formation
A reduction in plasma membrane CD3 levels is one mechanism that could contribute to the effects of jasplakinolide and latrunculin A on -CD3-stimulated [Ca2+]i responses. To test this, control or drug-treated cells were stained with a FITC-conjugated -CD3 mAb, and flow cytometry was used to analyse surface CD3 levels (Fig. 4A). In three experiments we found that jasplakinolide reduced the mean -CD3 fluorescence intensity by 35 ± 0.03 %, while latrunculin A reduced it by 75 ± 0.003 %, consistent with the idea that plasma membrane CD3 levels are lower in drug-treated cells.
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Figure 4. Jasplakinolide and latrunculin A reduce plasma membrane CD3 levels, but do not inhibit conjugate formation A, effects of jasplakinolide and latrunculin A on surface CD3 levels measured using flow cytometry. Cells were stained with a FITC-labelled | ||
Inhibition of conjugate formation by jasplakinolide and latrunculin A could contribute to the inhibition of perforin release and to the reduction in [Ca2+]i signals triggered by the bsAb-treated Raji cells described above. To test this idea, we loaded control or drug-treated TALL-104 cells with fura-2, loaded bsAb-treated Raji cells with calcein, and allowed them to interact for 20 min at room temperature in Ca2+-free Ringer solution in which MgCl2 replaced CaCl2. These conditions were chosen to inhibit granule release and the attendant release of calcein from bsAb-treated Raji cells. Note that conjugate formation does not require the presence of extracellular Ca2+, but does require the presence of Mg2+ (Golstein & Smith, 1976; Perez et al. 1985), and our previous results confirm that this is true for TALL-104 cells (Lyubchenko et al. 2001). We then examined the cell mixtures on our imaging system, using calcein fluorescence to identify Raji cells and the fluorescence of fura-2 excited at 360 nM (the Ca2+-insensitive isosbestic wavelength) to identify TALL-104 cells. We counted the number of bsAb-treated Raji cells that had 0, 1, 2, 3 or 4 TALL-104 cells bound to them (Fig. 4B). Interestingly, we found that latrunculin A did not alter conjugate formation, but jasplakinolide did have inhibitory effects. Further analysis of the data shown in Fig. 4B indicates that Raji cells had 1.17 ± 0.18 untreated TALL-104 cells bound to them, 1.00 ± 0.2 latrunculin A-treated cells bound to them, but only 0.64 ± 0.06 jasplakinolide-treated TALL-104 cells. The effect of latrunculin A was not statistically significant (P = 0.55), but the effect of jasplakinolide was (P = 0.03). Thus, jasplakinolide but not latrunculin A reduces conjugate formation.
Inspection of conjugates revealed a further effect: drug-treated TALL-104 cells did not display the same morphology after binding as control cells (Fig. 4C). Control TALL-104 cells appeared to wrap themselves tightly around the bsAb-treated Raji cells, while drug-treated cells do not. This observation suggests that both jasplakinolide and latrunculin A affect the ability of TALL-104 cells to undergo shape changes that result in the characteristic morphology adopted by control cells.
Latrunculin A inhibits -CD3 stimulated changes in cellular motility
To further explore the idea that the actin cytoskeleton plays a role in the CTL shape changes that are important for normal conjugate formation, we investigated the interaction of cells with -CD3 beads (Fig. 5). We adhered TALL-104 cells to poly-L-lysine-coated coverslips, and dropped beads into contact with them. We reasoned that when cells are immobilized in this manner, processes that would cause shape changes in non-adherent cells would result instead in a relative movement of beads that could be easily measured, since the -CD3 beads we use have FITC-like fluorescence. Despite the fact that we might expect adherence to poly-L-lysine to change the basal state of actin polymerization, we reasoned that these effects would apply to control and drug-treated cells. Furthermore, it was not possible to image cells if they were not adhered.
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Figure 5. The actin cytoskeleton is involved in TCR-stimulated changes in cellular motility A, images showing a representative interaction of an untreated TALL-104 cell (fura-2 fluorescence, blue) with an | ||
Control cells actively extended processes in random directions, although there was a great deal of heterogeneity in the activity of cells. Interactions of TALL-104 cells with -CD3 beads were highly stereotypical. Typically, cells made contact with beads by means of a fine process or pseudopod (Fig. 5A). After contact, the process thickened and extended further before being retracted together with the bead towards the cell body. We analysed these image sequences by computing the centre of the bead's fluorescence and the centre of the cell's fluorescence, and then calculating their relative distance from one another (Fig. 5B). In 15 cases, cells made contact with beads that landed at a centre-to-centre distance of 8.8 ± 0.3 µm. Beads were moved 1.5 ± 0.3 µm away from the centre of the cell before moving back towards the cell body, coming to rest 3.6 ± 0.4 µm from the cell's centre. Beads therefore travelled a total distance of 8.2 ± 0.5 µm. [Ca2+]i signals often began before beads began moving, at the earliest discernible point of contact between the fine pseudopod and the bead (data not shown), suggesting that movement of beads onto the cell body is not required to initiate signalling in response to -CD3 beads, and indicating that relatively small contact areas are required for stimulation. We sometimes observed cells making contact with more than one bead. In such cases, beads were moved in a temporally independent fashion, suggesting that the processes that control motility underlying bead motion are regulated locally, not at the whole-cell level. The movement of beads did not require Ca2+ influx, as it occurred in an apparently indistinguishable manner in the absence of extracellular Ca2+ (data not shown).
To confirm that the motion of -CD3 beads reflects TCR/CD3-stimulated changes in cellular motility and is not simply the result of beads adhering via the Ab that coats them to cells undergoing random motion, we monitored interactions between TALL-104 cells and -CD8 beads in similar experiments (Fig. 5B). These beads do not stimulate [Ca2+]i increases when they are dropped on to TALL-104 cells (data not shown). In these experiments, cells made contact with beads that landed at a centre-to-centre distance of 8.4 ± 0.4 µm, not significantly different from that in the control experiments described above. Beads were not pushed further from the centre of the cell following initial contact, and although -CD8 beads did come to rest closer to the centre of the cell than their initial position (4.8 ± 0.2 µm), the total distance they travelled (3.6 µm) was significantly less (P < 0.05) than the total distance -CD3 beads moved. These results suggest that contact with -CD3 beads triggers changes in cellular motility.
Consistent with the idea that the actin cytoskeleton is involved in cellular motility, latrunculin A-treated cells did not extend processes like untreated control cells, and directed movement of beads was markedly inhibited (Fig. 5B). In eight cases, cells made contact with beads that landed at a centre-to-centre distance of 7.4 ± 0.2 µm. We interpret the significantly decreased distance (P < 0.05) at which contact occurred compared to control experiments as reflecting the fact that latrunculin A-treated cells did not extend processes. Beads were not pushed away from the cell following contact, and moved only 0.8 ± 0.2 µm, coming to rest 6.6 ± 0.3 µm from the centre of the cell, which was significantly different from the value in control cells (P < 0.05). Could this effect reflect decreased signalling in response to beads in latrunculin A-treated cells? We feel that this explanation is unlikely. In some cases, latrunculin A-treated cells gave [Ca2+]i responses that were as large or larger than responses of control cells, despite the fact that on average, [Ca2+]i responses in latrunculin A-treated cells were reduced (Fig. 3). Furthermore, the responses of latrunculin A-treated cells to -CD3 beads were of comparable magnitude to responses of control cells to bsAb-treated Raji cells, and control TALL-104 cells change shape in response to bsAb-treated Raji cells.
TG-stimulated [Ca2+]i influx is reduced by jasplakinolide and latrunculin A
Inhibition of the ability of store depletion to activate Ca2+ influx is another mechanism that could contribute to the reduction in CD3-stimulated [Ca2+]i signals observed here (Fig. 3) and thus to the inhibition of perforin release (Fig. 2), as [Ca2+]i elevation is required for granule exocytosis. To examine whether actin-modifying drugs inhibit CCE, we tested the effects of actin-modifying drugs on the [Ca2+]i signals stimulated by TG, a drug that blocks sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, depleting stores and thereby activating CCE (Thastrup et al. 1989; Fig. 6A). TALL-104 cells were stimulated with TG in the absence of extracellular Ca2+, and then, ~1200 s later, 2 mM extracellular Ca2+ was added to assess the magnitude of influx. In these experiments, the first small transient [Ca2+]i elevation represents Ca2+ release from intracellular stores and the second larger increase once extracellular Ca2+ is added is due to influx. Neither of the actin-modifying drugs appeared to significantly alter TG-stimulated release of Ca2+ from stores (see insets in Fig. 6A). However, the magnitude of [Ca2+]i increases and the rate at which [Ca2+]i increased following addition of extracellular Ca2+ were reduced by each of the drugs. Peak [Ca2+]i following addition of extracellular Ca2+ was reduced by 45 % in latrunculin A-treated cells, and by 56 % in jasplakinolide-treated cells. Applying 2 µM jasplakinolide or latrunculin A to cells after stimulation with TG in the presence of 2 mM extracellular Ca2+ did not affect [Ca2+]i over the course of 675 s (data not shown), indicating that these drugs do not simply act as CCE channel blockers. Thus, these results are consistent with the idea that the ability of store depletion to activate Ca2+ influx is impaired following perturbation of the actin cytoskeleton.
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Figure 6. Jasplakinolide and latrunculin A reduce capacitative calcium entry A, effects of jasplakinolide and latrunculin A on TG-induced [Ca2+]i responses in TALL-104 cells. Each trace is an average of > 200 cells from triplicate experiments. Cells were stimulated with TG in the absence of external Ca2+, and then Ringer solution containing 2 mM external Ca2+ was added to assess influx. The insets (dashed lines) show the release of Ca2+ from stores on an expanded scale. Bars represent 40 nM [Ca2+]i and 200 s. B, histograms of bis-oxonol fluorescence intensity for control cells or cells treated with cytoskeleton drugs. After treatment, cells were adhered to poly-L-lysine-coated dishes, and washed with Ringer solution containing 200 nM bis-oxonol, and fluorescence was measured using our imaging system. Bis-oxonol fluorescence intensity is given in arbitrary units. | ||
CCE channels are not gated by changes in membrane potential (Vm), but the rate of influx is sensitive to Vm, as the driving force for Ca2+ entry is decreased by depolarization (Lewis & Cahalan, 1995). To test whether depolarization of resting Vm could contribute to the reduction of TG-stimulated influx by jasplakinolide and latrunculin A, we used the potential-sensitive dye bis-oxonol (Tatham & Delves, 1984) to assess relative resting Vm in control cells and in cells treated with actin-modifying drugs (Fig. 6B). This anionic dye distributes in a Nernstian fashion, so membrane depolarization increases the amount of dye in the cytoplasm, resulting in higher fluorescence intensities. Cells were treated with jasplakinolide or latrunculin A, or left in medium, plated on poly-L-lysine-treated dishes, and then washed with normal Ringer solution containing 200 nM bis-oxonol and examined 5 min later. Surprisingly, we found that both of the actin-modifying drugs we used decreased average bis-oxonol fluorescence. These results are inconsistent with the idea that jasplakinolide and latrunculin A depolarize Vm, and thus suggest that the effects on TG-stimulated Ca2+ influx reflect decreased activity of CCE channels.
Effects of jasplakinolide and latrunculin A on granule exocytosis
To investigate whether the actin cytoskeleton acts as a barrier to the fusion of lytic granules with the plasma membrane, we used BLT-esterase assays (Takayama et al. 1987) to assess granule exocytosis in populations of TALL-104 cells stimulated with either -CD3 beads or TG and PMA, stimuli that bypass TCR/CD3-mediated signalling (Table 2). We first tested whether latrunculin A and jasplakinolide caused exocytosis in the absence of stimulation. Latrunculin A-treated cells released ~15 % of total BLT-esterase activity during the course of 50 min at room temperature. Jasplakinolide-treated cells did not release granzyme in the absence of stimulation. Although soluble -CD3 antibodies cause a [Ca2+]i increase in TALL-104 cells (Fig. 3C), they do not cause exocytosis in TALL-104 cells (our unpublished results), consistent with results from other CTL systems. In contrast, solid-phase -CD3 beads are a potent stimulus, triggering the release of ~35 % of total cellular BLT esterase activity (Table 2). Latrunculin A reduced bead-stimulated granule exocytosis to 14.6 %, an amount that is not different from the spontaneous release it caused, and jasplakinolide reduced exocytosis to baseline levels (Table 2). Both of the drugs tested thus completely inhibited -CD3-stimulated granule exocytosis.
We next tested the effects of latrunculin A and jasplaknolide on exocytosis in response to TG and PMA, stimuli that bypass signalling through the TCR/CD3 pathway. We first confirmed that granule exocytosis in TALL-104 cells is dependent upon both increases in [Ca2+]i and PKC activity, as has been reported for other CTLs (Berebi et al. 1987; Lancki et al. 1987). Treatment of cells with 50 nM PMA in the presence of 0.25 mM external Ca2+ or with 1 µM TG and 50 nM PMA in Ca2+-free extracellular solution did not trigger exocytosis (data not shown). When cells were stimulated with TG and PMA in the presence of 0.25 mM external Ca2+, they released 35.2 ± 5.1 % of their total granzyme (Table 2). Increasing external Ca2+ to 2 mM increased BLT-esterase release by ~60 % to 52.5 ± 1.9 %, and although there was a significant release of BLT-esterase activity when cells were stimulated with TG alone in the presence of 2 mM external Ca2+ (23.9 ± 4.7 %), this exocytosis was completely blocked when cells are pretreated for 10 min with 10 µM of the PKC inhibitor bisindolylmaleimide I (data not shown). Taken together, these results suggest that, as has been reported in other CTL lines, exocytosis in TALL-104 cells requires concurrent activation of PKC and increases in [Ca2+]i. Treating cells with jasplakinolide or latrunculin A had very different effects on exocytosis stimulated by TG and PMA than on bead-stimulated release (Table 2). In the presence of 0.25 mM or 2 mM external Ca2+, neither jasplakinolide nor latrunculin A had a significant effect on granule exocytosis. These results are inconsistent with the idea that the actin cytoskeleton acts as a barrier to granule exocytosis.
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DISCUSSION |
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We have used powerful drugs with defined effects to investigate the role of the actin cytoskeleton in processes that are expected to be important for granule exocytosis-mediated target cell killing. We hypothesized that the actin cytoskeleton could be involved in multiple steps in the interaction between CTL and the target. Our results are consistent with idea that the actin cytoskeleton is involved in TCR/CD3-mediated signalling, the activation of capacitative Ca2+ entry, and in CTL shape changes, but does not appear to act as a barrier to the exocytosis of lytic granules. Although we have not presented the data here, we obtained identical results with cytochalasin D, which in our hands increased actin polymerization (as assessed by phalloidin staining). Cytochalasin D can increase or decrease actin polymerization depending upon the concentration used and the length of treatment (Mills et al. 2000).
[Ca2+]i signals stimulated by contact with bsAb-treated target cells were essentially eliminated by treatment with latrunculin A or jasplakinolide, and [Ca2+]i signals in response to -CD3 beads and to soluble -CD3 mAb were also substantially reduced (Fig. 3). We report two effects that probably contribute - a reduction in plasma membrane CD3 levels (Fig. 4) and an inhibition in CCE (Fig. 6). Previous studies that have examined the effects of drugs that affect actin polymerization on signalling in T cells in response to soluble -CD3 have yielded inconsistent results. Arrieumerlou et al. (2000) found that latrunculin A treatment reduced [Ca2+]i responses and InsP3 production in response to soluble -CD3 in Jurkat human leukaemic T cells, but cytochalasin D did not. Delon et al. (1998) found that cytochalasin D did not alter -CD3-stimulated [Ca2+]i responses in primary murine T cells. Valitutti et al. (1995) found that responses to soluble -CD3 mAb were enhanced by treatment with cytochalasin D.
In addition to reduced CD3 levels, it is also possible that signalling via the remaining CD3 is impaired. Previous work has shown that a fraction of TCR-
is associated with the actin cytoskeleton (Caplan & Baniyash, 1996), and this fraction increases following -CD3 stimulation (Rozdzial et al. 1995; Caplan & Baniyash, 1996). Stimulation with -CD3 increases actin polymerization (Parsey & Lewis, 1993; Arrieumerlou et al. 2000), and when solid-phase -CD3 is used, actin polymerization occurs at the site of contact with the Ab (Lowin-Kropf et al. 1998; Sedwick et al. 1999). Taken together, these results are consistent with the idea that there is an association between components of the TCR/CD3 signalling apparatus and the actin cytoskeleton that is required for functional signalling, and which would be impaired by actin disruption.
We found that both jasplakinolide and latrunculin A significantly reduced the influx-dependent [Ca2+]i responses stimulated by TG without affecting the release of Ca2+ from stores (Fig. 6). Several recent studies have reported similar findings in other cell types, but this is the first such report in T cells. In DDT1-MF2 smooth muscle cells, jasplakinolide and the phosphatase inhibitor calyculin A increased actin polymerization and inhibited the activation of CCE, while cytochalasin D or latrunculin A decreased actin polymerization and did not affect influx (Patterson et al. 1999). In platelets, latrunculin A, which does not depolymerize actin in resting cells but does prevent TG-stimulated actin polymerization, inhibits CCE (Rosado & Sage, 2000b). Jasplakinolide and calyculin A, which polymerize actin in resting cells (Rosado & Sage, 2000b), also inhibit CCE. In RBL mastocytoma cells, cytochalasin D, calyculin A and jasplakinolide were all without effect on activation of CCE (Bakowski et al. 2001). The reasons for the different effects of actin-modifying drugs on the activation of CCE in different cell types remain unclear, but may arise from different basal actin dynamics in different cell types (Rosado & Sage, 2000a).
The effects of actin-modifying drugs on the activation of CCE have often been interpreted in terms of a secretion-like model, which proposes that drug-driven actin polymerization prevents the translocation of a subset of the endoplasmic reticulum into contact with CCE channels (Patterson et al. 1999; Rosado & Sage, 2000a, b) by forming a physical barrier to vesicle translocation. We feel that the lack of inhibitory effect of jasplakinolide and latrunculin A on granule exocytosis in response to stimulation with TG and PMA (Table 2) argues against a secretion-like interpretation of the effects of actin-modifying drugs on CCE activation. It seems to us unlikely that the actin cytoskeleton would act as a barrier to a vesicular transport process involved in CCE activation, but not to the exocytosis of lytic granules. That latrunculin A, which depolymerized actin in our hands (Fig. 1), also reduced CCE is a further argument against a secretion-like interpretation of the effects of actin-modifying drugs. The inhibition of CCE we observed is unlikely to have been due to an indirect effect of membrane depolarization, as the fluorescence of the potential-sensitive dye bis-oxonol was decreased in cells treated with either latrunculin A or jasplakinolide (Fig. 5), suggesting that cells are in fact relatively hyperpolarized. To our knowledge, effects of changes in resting Vm have never been examined as possible contributors to the effects of actin-modifying drugs on CCE. Assuming that these results do not simply reflect a nonspecific effect of the drugs we used on partitioning of bis-oxonol, how might latrunculin A and jasplakinolide affect resting Vm? In lymphocytes, resting Vm is largely determined by the activity of voltage-gated K+ channels (Lewis & Cahalan, 1995), and has been reported to fluctuate between -40 and -80 mV (Verheugen & Vijverberg, 1995; Verheugen et al. 1995). Some current must therefore depolarize Vm, driving it away from the equilibrium potential for K+ (approximately -80 mV). The identity of this depolarizing current is unknown, but could reflect the activity of electrogenic Na+-coupled transporters or Cl- channels. Hyperpolarization of resting Vm could thus be due either to an increase in the number or activity of voltage-gated K+ channels, to activation of Ca2+-activated K+ channels, or to a reduction in the amplitude of the depolarizing current.
If, as we suggest, secretion-like effects do not underlie the effects of actin-modifying drugs on CCE, what mechanism(s) might? We envisage two possibilities. First, the number of CCE channels at the plasma membrane might be decreased by drug treatment. We found that plasma membrane CD3 levels were reduced in drug-treated cells (Fig. 4). It is possible that there is a generalized decrease in the levels of many membrane proteins in drug-treated cells. Second, the maximum extent to which CCE channels can be activated by store depletion might be reduced by drug treatment, due either to an alteration in channel conformation if channels interact with actin, or to a disruption of the (presently unknown) coupling mechanism between store content and channel activity.
Previous work with primary mouse CTLs showed that cytochalasin B inhibited conjugate formation (Perez et al. 1985). However, the effects of cytochalasin B on the actin cytoskeleton of the CTLs used were not determined. We found that conjugate formation with bsAb-treated Raji cells was inhibited by jasplakinolide but was not affected by latrunculin A. Thus, if cytochalasin B decreased actin polymerization in the previous study (Perez et al. 1985), the results differ from ours. If cytochalasin B increased actin polymerization in the previous study (Perez et al. 1985), then our results are consistent. If there is a discrepancy, it could result from our model system. As the interaction between TALL-104 cells and bsAb-treated Raji cells is mediated via antibody binding to CD3, we would not expect conjugate formation to be affected until surface CD3 levels are reduced to extremely low levels by drug treatment. In the case of antigen-specific CTLs like those used in the previous study, the interactions between the TCR and antigen/MHC are likely to be of much lower affinity, and are more dependent on sustained signalling through the TCR (Valitutti et al. 1995). Consistent with this idea, Valitutti et al. (1995) showed that adding cytochalasin D to preformed conjugates disrupted ongoing Ca2+ signalling. This was not the case in our system: jasplakinolide had no effect when added after bsAb-treated Raji cells had initiated Ca2+ signals in TALL-104 cells (data not shown).
That jasplakinolide and latrunculin A blocked TALL-104 shape changes after contact with Raji cells and latrunculin inhibited shape changes in response to contact with -CD3 beads is consistent with a great deal of evidence that supports the idea that the actin cytoskeleton plays a critical role in TCR-stimulated motility in T cells. Valitutti et al. (1993) reported that CTL shape changes in response to contact target cells was blocked by treatment of cells with cytochalasin D. Delon et al. (1998) showed that CD4+ T-cell recognition of and shape changes in response to APCs was inhibited by cytochalasin D. Signalling as assessed by [Ca2+]i signals was also inhibited. Bunnel et al. (2001) showed that spreading of Jurkat human leukaemic T cells on -CD3-coated coverslips, a process that is accompanied by dynamic actin remodelling that requires the linker of activated T cells, was blocked by cytochalasin D. Interestingly, we found that beads were moved in a similar manner in the absence of extracellular Ca2+, suggesting that Ca2+ influx is not required for bead movement. Assuming that our assay reflects the same actin-dependent events as the spreading assay, these results contradict those of Bunnel et al. (2001), who found that spreading on -CD3-coated coverslips is reduced by chelating extracellular Ca2+ with EGTA and intracellular Ca2+ with BAPTA. One possible explanation for these divergent results is that release of Ca2+ from stores is required for TCR-stimulated actin polymerization, but influx is not. An additional difference is that our experiments were conducted at room temperature, while Bunnel et al. (2001) worked at 37 °C, although it is hard to see how temperature could account for this discrepancy.
A number of studies have examined the role of the actin cytoskeleton in exocytosis in various secretory cell types (Trifaro et al. 1992; Roth & Burgoyne, 1995; Chowdhury et al. 2000; Gil et al. 2000; Yoneda et al. 2000). This work has given rise to the idea that the actin cytoskeleton can act as a barrier to exocytosis, a barrier that must be dismantled before exocytic vesicles can gain access to the plasma membrane. The results presented in Table 1 argue against the idea that the actin cytoskeleton forms a barrier to the exocytosis of CTL lytic granules. When cells were stimulated with -CD3 beads, latrunculin A and jasplakinolide reduced granule exocytosis, a result that may be explained by the fact that they reduce signalling, as assessed by [Ca2+]i signals, and thus are also likely to inhibit activation of PKC, long known to be a second signal required for exocytosis (Berebi et al. 1987; Lancki et al. 1987). These results contradict those of O'Rourke et al. (1991), who found that granule exocytosis in response to solid-phase -CD3 was not affected by cytochalasin D treatment. One possible explanation for these results is that since they were monitoring release stimulated by Ab adsorbed onto plates onto which the cells settled, they were delivering a stronger stimulus. The reduction in signalling may not have been sufficient to inhibit exocytosis. Also, our experiments were conducted at room temperature, whereas theirs were conducted at 37 °C; increased temperature would likely increase signalling, which could contribute to stronger responses. Importantly, we found that when cells were stimulated with TG and PMA, jasplakinolide and latrunculin A did not inhibit granule exocytosis. Similar results have been reported by Lancki et al. (1987) for cytochalasin-D-treated CTLs stimulated with ionomycin and PMA. That exocytosis is not altered when signalling via TCR/CD3 is bypassed does not support the idea that the actin cytoskeleton functions as a barrier to exocytosis of CTL lytic granules.
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REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Arrieumerlou C, Randriamampita C, Bismuth G & Trautmann A (2000). Rac is involved in early TCR signaling. J Immunol 165, 3182-3189 | [Abstract/Full Text] |
| Ayscough K, (1998). Use of latrunculin-A, an actin monomer-binding drug. Methods Enzymol 298, 18-25 | [Medline] |
| Bakowski D, Glitsch MD & Parekh AB (2001). An examination of the secretion-like coupling model for the activation of the Ca2+ release-activated Ca2+ current I(CRAC) in RBL-1 cells. J Physiol 532, 55-71 | [Abstract/Full Text] |
| Berebi G, Takayama H & Sitkovsky MV (1987). Antigen-receptor interaction requirement for conjugate formation and lethal-hit triggering by cytotoxic T lymphocytes can be bypassed by protein kinase C activators and calcium ionophores. Proc Natl Acad Sci U S A 84, 1364-1368 | [Medline] |
| Berke G, (1994). The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Ann Rev Immunol 12, 736-753 | |
| Berke G, (1995). The CTL's kiss of death. Cell 81, 9-12 | [Medline] |
| Borroto A, Gil D, Delgado P, Vicente-Manzanares M, Alcover A, Sanchez-Madrid F & Alarcon B (2000). Rho regulates T cell receptor ITAM-induced lymphocyte spreading in an integrin-independent manner. Eur J Immunol 30, 3403-3410 | [Medline] |
| Bubb MR, Senderowicz AM, Sausville EA, Duncan KL & Korn ED (1994). Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin. J Biol Chem 269, 14869-14871 | [Abstract] |
| Bunnell SC, Kapoor V, Trible RP, Zhang W & Samelson LE (2001). Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14, 315-329 | [Medline] |
| Caplan S , & Baniyash M (1996). Normal T cells express two T cell antigen receptor populations, one of which is linked to the cytoskeleton via zeta chain and displays a unique activation-dependent phosphorylation pattern. J Biol Chem 271, 20705-20712 | [Abstract/Full Text] |
| Cesano A , & Santoli D (1992). Two unique human leukemic T-cell lines endowed with a stable cytotoxic function and a different spectrum of target reactivity analysis and modulation of their lytic mechanisms. In Vitro Cell Dev Biol 28A, 648-656 | |
| Chowdhury HH, Popoff MR & Zorec R (2000). Actin cytoskeleton and exocytosis in rat melanotrophs. Pflugers Arch 439, R148-149 | [Medline] |
| Delon J, Bercovici N, Liblau R & Trautmann A (1998). Imaging antigen recognition by naive CD4+ T cells: compulsory cytoskeletal alterations for the triggering of an intracellular calcium response. Eur J Immunol 28, 716-729 | [Medline] |
| Dustin ML , & Cooper JA (2000). The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling. Nat Immunol 1, 23-29 | [Medline] |
| Gil A, Rueda J, Viniegra S & Gutierrez LM (2000). The F-actin cytoskeleton modulates slow secretory components rather than readily releasable vesicle pools in bovine chromaffin cells. Neuroscience 98, 605-614 | [Medline] |
| Golstein P , & Smith ET (1976). The lethal hit stage of mouse T and non-T cell-mediated cytolysis: differences in cation requirements and characterization of an analytical 'cation pulse' method. Eur J Immunol 6, 31-37 | [Medline] |
| Griffiths GM, (1995). The cell biology of CTL killing. Curr Opin Immunol 7, 343-348 | [Medline] |
| Grynkiewicz G, Poenie M & Tsien RY (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260, 3440-3450 | [Abstract] |
| Haugland RP, You W, Paragas VB, Wells KS & Dubose DA (1994). Simultaneous visualization of G- and F-actin in endothelial cells. J Histochem Cytochem 42, 345-350 | [Abstract] |
| Lancki DW, Weiss A & Fitch FW (1987). Requirements for triggering of lysis by cytolytic T lymphocyte clones. J Immunol 138, 3646-3653 | [Abstract] |
| Lewis RS, (2001). Calcium signaling mechanisms in T lymphocytes. Annu Rev Immunol 19, 497-521 | [Abstract/Full Text] |
| Lewis RS , & Cahalan MD (1995). Potassium and calcium channels in lymphocytes. Annu Rev Immunol 13, 623-653 | [Abstract] |
| Lichtenfels R, Biddison WE, Schulz H, Vogt AB & Martin R (1994). CARE-LASS (calcein-release-assay), an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity. J Immunol Methods 172, 227-239 | [Medline] |
| Link B, Kostelny S, Cole MS, Fusselman W, Tso J & Weiner G (1998). Anti-CD3-based bispecific antibody designed for therapy of human B-cell malignancy can induce T-cell activation by antigen-dependent and antigen independent mechanisms. Int J Cancer 77, 251-256 | [Medline] |
| Lowin-Kropf B, Shapiro VS & Weiss A (1998). Cytoskeletal polarization of T cells is regulated by an immunoreceptor tyrosine-based activation motif-dependent mechanism. J Cell Biol 140, 861-871 | [Abstract/Full Text] |
| Lyubchenko TA, Wurth GA & Zweifach A (2001). Role of calcium influx in cytotoxic T lymphocyte lytic granule exocytosis during target cell killing. Immunity 15, 847-859 | [Medline] |
| Lyubimova A, Bershadsky AD & Ben-Ze'ev A (1999). Autoregulation of actin synthesis requires the 3'-UTR of actin mRNA and protects cells from actin overproduction. J Cell Biochem 76, 1-12 | [Medline] |
| Mills JW, Falsig Pedersen S, Walmod PS & Hoffmann EK (2000). Effect of cytochalasins on F-actin and morphology of Ehrlich ascites tumor cells. Exp Cell Res 261, 209-219 | [Medline] |
| Monks CR, Freiberg BA, Kupfer H, Sciaky N & Kupfer A (1998). Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82-86 | [Medline] |
| O'Rourke AM, Apgar JR, Kane KP, Martz E & Mescher MF (1991). Cytoskeletal function in CD8- and T cell receptor-mediated interaction of cytotoxic T lymphocytes with class I protein. J Exp Med 173, 241-249 | [Abstract] |
| Parsey MV , & Lewis GK (1993). Actin polymerization and pseudopod reorganization accompany anti-CD3-induced growth arrest in Jurkat T cells. J Immunol 151, 1881-1893 | [Abstract] |
| Patterson RL, van Rossum DB & Gill DL (1999). Store-operated Ca2+ entry: evidence for a secretion-like coupling model. Cell 98, 487-499 | [Medline] |
| Perez P, Bluestone JA, Stephany DA & Segal DM (1985). Quantitative measurements of the specificity and kinetics of conjugate formation between cloned cytotoxic T lymphocytes and splenic target cells by dual parameter flow cytometry. J Immunol 134, 478-485 | [Abstract] |
| Potter TA, Grebe K, Freiberg B & Kupfer A (2001). Formation of supramolecular activation clusters on fresh ex vivo CD8+ T cells after engagement of the T cell antigen receptor and CD8 by antigen-presenting cells. Proc Natl Acad Sci U S A 98, 12624-12629 | [Abstract/Full Text] |
| Rosado JA , & Sage SO (2000a). The actin cytoskeleton in store-mediated calcium entry. J Physiol 526, 221-229 | [Abstract/Full Text] |
| Rosado JA , & Sage SO (2000b). A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Trends Cardiovasc Med 10, 327-332 | [Medline] |
| Roth D , & Burgoyne RD (1995). Stimulation of catecholamine secretion from adrenal chromaffin cells by 14-3-3 proteins is due to reorganisation of the cortical actin network. FEBS Lett 374, 77-81 | [Medline] |
| Rozdzial MM, Malissen B & Finkel TH (1995). Tyrosine-phosphorylated T cell receptor zeta chain associates with the actin cytoskeleton upon activation of mature T lymphocytes. Immunity 3, 623-633 | [Medline] |
| Scott VR, Boehme R & Matthews TR (1988). New class of antifungal agents: jasplakinolide, a cyclodepsipeptide from the marine sponge, Jaspis species. Antimicrob Agents Chemother 32, 1154-1157 | [Medline] |
| Sedwick CE, Morgan MM, Jusino L, Cannon JL, Miller J & Burkhardt JK (1999). TCR, LFA-1, and CD28 play unique and complementary roles in signaling T cell cytoskeletal reorganization. J Immunol 162, 1367-1375 | [Abstract/Full Text] |
| Spector I, Shochet NR, Kashman Y & Groweiss A (1983). Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 219, 493-495 | |
| Stinchcombe JC, Bossi G, Booth S & Griffiths GM (2001). The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15, 751-761 | [Medline] |
| Sugawara S, Kaslow HR & Dennert G (1993). CTX-B inhibits CTL cytotoxicity and cytoskeletal movements. Immunopharmacology 26, 93-104 | [Medline] |
| Takayama H, Trenn G & Sitkovsky MV (1987). A novel cytotoxic T lymphocyte activation assay: optimized conditions for antigen receptor triggered granule enzyme secretion. J Immunol Methods 104, 183-190 | [Medline] |
| Tatham PER , & Delves PJ (1984). Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A. Biochem J 221, 137-146 | [Medline] |
| Thastrup O, Dawson AP, Scharff O, Foder B, Cullen PJ, Drobak BK, Bjerrum PJ, Christensen SB & Hanley MR (1989). Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents Actions 27, 17-23 | [Medline] |
| Trifaro JM, Rodriguez del Castillo A & Vitale ML (1992). Dynamic changes in chromaffin cell cytoskeleton as prelude to exocytosis. Mol Neurobiol 6, 339-358 | [Medline] |
| Valitutti S, Dessing M, Aktories K, Gallati H & Lanzavecchia A (1995). Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton. J Exp Med 181, 577-584 | [Abstract] |
| Valitutti S, Dessing M & Lanzavecchia A (1993). Role of cAMP in regulating cytotoxic T lymphocyte adhesion and motility. Eur J Immunol 23, 790-795 | [Medline] |
| Verheugen JA , & Vijverberg HP (1995). Intracellular Ca2+ oscillations and membrane potential fluctuations in intact human T lymphocytes: role of K+ channels in Ca2+ signaling. Cell Calcium 17, 287-300 | [Medline] |
| Verheugen JA, Vijverberg HP, Oortgiesen M & Cahalan MD (1995). Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium. J Gen Physiol 105, 765-794 | |
| Wulf E, Deboben A, Bautz FA, Faulstich H & Wieland T (1979). Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A 76, 4498-4502 | [Medline] |
| Yoneda M, Nishizaki T, Tasaka K, Kurachi H, Miyake A & Murata Y (2000). Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly. J Endocrinol 166, 677-687 | [Medline] |
| Zweifach A, (2000). Target-cell contact activates a highly selective capacitative calcium entry pathway in cytotoxic T lymphocytes. J Cell Biol 148, 603-614 | [Abstract/Full Text] |
Acknowledgements
We thank Drs Joseph K. Angleson and Yiannis Koutalos for helpful discussions. We also thank Protein Design Laboratories for the gift of the bispecific antibody. This work was supported by NIH grant AI42964 to A.Z.
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100 objective, and images for a given stain were acquired at identical exposure times and are scaled identically. Scale bar is 7.5 µm.