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Address correspondence to Lawrence Samelson, Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 37, Rm. 1E24, Bethesda, MD 20892. Tel.: (301) 496-9683. Fax: (301) 496-8479. E-mail: samelson{at}helix.nih.gov
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Abstract |
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Key Words: T cell receptor; adapters; signaling complexes; tyrosine phosphorylation; confocal microscopy
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Introduction |
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Various imaging studies have established that T cells that engage antigen-presenting cells (APCs) bearing stimulatory MHC-peptide complexes undergo macromolecular rearrangements that result in the formation of an immune synapse. This structure develops over 530 min as a result of active cytoskeletal processes and subdivides the T cellAPC interface into two concentric zones: the central and peripheral supramolecular activation clusters (cSMAC and pSMAC) (for review see Delon and Germain, 2000). The cSMAC is enriched in the TCR, CD2, and CD28, whereas the pSMAC contains integrins and cytoskeletal proteins. Larger glycoproteins, such as CD43 and CD45, are initially excluded from the entire synapse (Sperling et al., 1998; Johnson et al., 2000). Despite the dramatic nature of these rearrangements, the function of the immune synapse remains elusive.
Initial reports describing the immune synapse emphasized the correlation between SMAC formation and productive T cell activation (Monks et al., 1998; Grakoui et al., 1999). However, neither thymocytes nor T helper 2 CD4-positive T cells (Th2) cells develop immune synapses during their activation (Balamuth et al., 2001; Richie et al., 2002). Additionally, increases in cellular phosphotyrosine, intracellular calcium elevations, the dephosphorylation of moesin, and cytoskeletal rearrangements are all initiated within seconds of receptor engagement and peak within 2 to 3 min of TCR engagement (Negulescu et al., 1996; Wulfing and Davis, 1998; Bunnell et al., 2001; Delon et al., 2001). Therefore, the signals driving these events must be derived from TCRs ligated before the formation of a cSMAC (Monks et al., 1998; Grakoui et al., 1999; Dustin and Cooper, 2000; Lee et al., 2002). Several reports have indicated that TCRs are initially engaged at the periphery of the T cellAPC contact or in small clusters within the maturing synapse (Grakoui et al., 1999; Dustin and Cooper, 2000; Krummel and Davis, 2002). More recently, peak levels of activated Lck and ZAP-70 were found with the TCR in a peripheral zone before the coalescence of the engaged TCRs into a cSMAC (Lee et al., 2002), suggesting that signal transduction is initiated in these small, TCR-rich clusters, before the formation of a mature SMAC.
Dynamic studies of high temporal resolution are needed to relate the biochemical events described above to the complex morphological changes associated with T cell activation. To this end, we adapted a method developed in order to simultaneously visualize T cell contacts and the rearrangement of enhanced GFP (EGFP)-tagged actin in live, activated T cells (Bunnell et al., 2001). This approach constrains responding T cells to the plane of a stimulatory coverslip, allowing the rapid collection of high-resolution images of chimeric proteins participating in signaling complexes within developing contacts. Our results establish that fully functional signaling complexes form rapidly within membrane domains tightly associated with the stimulatory surface, that components of these complexes are continuously dissociating and reassociating, and that the components of these complexes exit by distinct mechanisms. These results reveal both the speed of TCR-induced signal initiation and the dynamically changing composition of the resulting signaling complexes.
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Results |
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Discussion |
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The assembly of TCR-containing complexes
The most critical event in the initiation of antigen receptor-dependent signaling in T cells is the aggregation of the TCR (Boniface et al., 1998). In our system, the TCR is recruited into clusters even though the TCR-specific stimulatory antibodies we use are uniformly distributed across our coverslips (unpublished data). The clustering of the TCR is PP2-insensitive, and is therefore independent of downstream PTK-driven signaling pathways. Strikingly, TCR-rich clusters preferentially form in membrane domains tightly apposed to the stimulatory coverslip. These observations are consistent with models of immune synapse formation in which the TCR is preferentially engaged within membrane domains separated by a narrow gap compatible with TCR-ligand engagement (Shaw and Dustin, 1997; Anton van der Merwe et al., 2000). Because we rarely observe TCR-rich clusters that are not tyrosine phosphorylated, PTK-dependent phosphorylation must be rapidly triggered after the clustering of the TCR. ZAP-70, which binds directly to the signal-transducing subunits of the TCR upon their phosphorylation, is recruited into clusters within 615 s of contact initiation, supporting this conclusion. As expected, the clustering of ZAP-70 is sensitive to PP2. Thus, the formation of functional signaling complexes requires phosphotyrosine-dependent interactions, whereas the initial clustering of the TCR does not.
The assembly of LAT-nucleated complexes
LAT nucleates the formation of signaling complexes critical to T cell activation. Our live-cell studies have shown that LAT is specifically recruited into clusters within 15 s of TCR ligation. Several components of the LAT-nucleated complex, including Grb2, Gads, and SLP-76, are recruited into clusters within a similar time frame. Furthermore, our fixed cell studies have shown these LAT-containing complexes are coincident with the ZAP-70containing clusters described above. This observation confirms that ZAP-70 is poised to phosphorylate two of its most prominent substrates, LAT and SLP-76, in intact T cells. Previously, these interactions had only been observed in purified membranes (Harder and Kuhn, 2000; Wilson et al., 2001).
Lipid rafts and complex assembly
Normal T cell activation requires the association of LAT with lipid rafts (Zhang et al., 1998b). Because lipid raft components have been reported to accumulate with crosslinked antigen receptors, TCR ligation may govern the clustering of LAT by directing the local accumulation of lipid rafts (Xavier et al., 1998; Janes et al., 1999; Holowka et al., 2000). However, the extensive accumulation of lipid rafts at sites of receptor ligation does not appear to be an essential prerequisite of T cell activation. For example, Th2 cells responding to antigen-loaded APCs do not significantly accumulate lipid raft components in their contacts (Balamuth et al., 2001). Additionally, the direct crosslinking of the TCR at 37°C does not necessarily result in the enrichment of either cholesterol or GM1 in receptor-associated membranes (Harder and Kuhn, 2000). Here, we were unable to observe the recruitment of a lipid raft marker, EYFP-GPI, into signaling clusters. Therefore, interactions between LAT and receptor-associated raft components are unlikely to explain the clustering of LAT with the TCR. In contrast, the recruitment of LAT into clusters is PP2-sensitive, indicating that PTK-dependent scaffolds play a dominant role in the clustering of LAT. Nevertheless, lipid rafts could be required for the initiation, rather than the maintenance, of interactions between LAT and TCR-associated signaling proteins. Given the basal association of LAT with lipid rafts, these observations suggest that LAT is either abstracted from lipid rafts during cluster formation, or present in a subset of lipid rafts distinct from that containing EYFP-GPI. The induced segregation of LAT from other lipid raft components could contribute to T cell activation by limiting the recruitment of lipid raft-associated negative regulators of activation, such as Cbp/PAG, to the TCR (Brdicka et al., 2000; Kawabuchi et al., 2000).
Signal initiation by minimal complexes
Calcium elevations typically begin within 12 s of contact initiation, when the T cell coverslip contact consists of a few small points. This lag between the initiation of contact and the elevation of calcium is similar to the time required for complex formation. Because the point contacts present at this time are the size of a single TCR-rich cluster the minimal functional unit required for calcium elevations may be a single cluster. The early onset of these calcium elevations is consistent with the permissive role of intracellular calcium in T cell spreading (Bunnell et al., 2001). Strikingly, this behavior parallels that of T cells engaging APCs, which also elevate intracellular calcium before initiating more active contact with their targets, but do so after a variable lag which may correspond to the time required to form a single functional signaling complex (Negulescu et al., 1996; Delon et al., 1998).
The dynamic composition of signaling complexes
Signaling complexes quickly disassemble, reassemble, and change in composition as specific proteins dissociate. ZAP-70 achieves a steady state where it remains associated with laterally immobile clusters for 30 min or more. In contrast, LAT, Grb2, Gads, and SLP-76 are transiently recruited to signaling complexes, and disappear from these structures within 13 min. Our FRAP studies have shown that early in the spreading process ZAP-70, which appears to be immobile, disassociates from and reassociates with signaling complexes quickly enough to half maximally repopulate bleached complexes within 7.510 s. This rate of recovery is consistent with that observed in a previous FRAP study that examined the reassociation of ZAP-70 with the activated TCR in a nonT cell model (Sloan-Lancaster et al., 1998). Cells bleached later in the spreading process displayed similar rates of fluorescence recovery, but recovered to a lesser extent. Because the rate of recovery remains constant, the dissociation and reassociation rates are probably unchanged at these later timepoints. The reduced extent of fluorescence recovery could be explained by three mechanisms: TCRs are rendered incapable of rebinding ZAP-70, free ZAP-70 becomes incapable of rebinding the TCR, or existing receptor-kinase complexes are prohibited from dissociating. Because the kinetics with which fluorescence recovery declines are similar to the kinetics with which receptor phosphorylation is lost, the time-dependent reduction in fluorescence recovery may be caused by the dephosphorylation of the TCR.
Signaling complexes as protosynapses
Multiple signals are triggered by TCR engagement prior to the formation of a central TCR-rich cluster, or cSMAC (Negulescu et al., 1996; Delon et al., 1998; Grakoui et al., 1999; Lee et al., 2002). These signals may be initiated by TCR-rich clusters, or protosynapses, that develop in advance of the mature cSMAC (Grakoui et al., 1999; Dustin and Cooper, 2000; Krummel et al., 2000). The TCR-rich clusters observed here resemble these protosynapses in several respects, including their size, kinetics of formation, and ability to exclude CD43 and CD45 (Sperling et al., 1998; Johnson et al., 2000). Although interactions between engaged TCRs and coverslip-bound stimulatory antibodies preclude the translocation of TCRs into a central cluster, we observe most of the biochemical outputs induced by antigen-loaded APCs. Our results suggest that protosynapses are capable of directing T cell activation in the absence of cSMAC formation and could drive the activation of thymocytes and Th2 cells, which do not form well-defined SMACs (Balamuth et al., 2001; Richie et al., 2002). We also observed the continuous dissociation of components from these signaling complexes, suggesting that the generation of new complexes throughout contact formation is required to maintain the levels of tyrosine phosphorylation observed when T cell contacts and synapses reach their greatest extent (Lee et al., 2002). In addition, the formation of these new clusters at the leading edge may facilitate the coupling of the TCR to the active remodeling of the actin cytoskeleton through downstream adaptors, such as SLP-76 (Krause et al., 2000).
Molecular fates postclustering
The molecular clusters observed here function as both signaling and sorting structures. These complexes are assembled from proteins residing in multiple cellular compartments. ZAP-70 remains associated with signaling complexes, LAT departs the complexes by undefined mechanisms, and both Grb2 and Gads dissociate into the cytosol. In contrast, SLP-76 departs TCR-rich signaling complexes in large structures specifically induced by TCR ligation. These structures are transported along microtubules as discrete units and accumulate in an undefined perinuclear structure not labeled with markers specific for early endosomes, recycling endosomes, the Golgi apparatus, or the TGN. The association of SLP-76 with these structures persists to timepoints at which neither clusters of Grb2 nor Gads can be detected. Although amounts of LAT and Gads beneath the threshold for visualization may direct the movement and localization of SLP-76, we believe that SLP-76 dissociates from LAT-nucleated complexes and enters these novel structures by a distinct and as of yet undefined mechanism.
Potential consequences of late trafficking events
The termination of signal transduction through the targeting of signaling molecules into degradative compartments has been well described (Strous and Govers, 1999). In nonT cells, internalization has also been shown to direct signaling molecules into intracellular compartments from which they can initiate novel signals (Daaka et al., 1998). The consequences of the protein trafficking described here are not yet clear. The sustained recruitment of SLP-76 to a perinuclear vesicular compartment might in some way contribute to T cell activation. Many questions remain regarding the functional consequences of these molecular sorting events and the identities of the structures that contain sorted signaling molecules. In future studies we will image the redistribution of multiple chimeric proteins, lipid markers, and labeled proteins during T cell activation, helping to resolve these issues.
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Materials and methods |
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Constructs and the generation of stable cell lines
The ZAP-70EGFP, Grb2-EYFP, and EYFPGL-GPI chimeras have been described (Sloan-Lancaster et al., 1998; Keller et al., 2001; Yamazaki et al., 2002). The TAC-EGFP chimera was provided by Dr. J. Donaldson (National Institutes of Health, Bethesda, MD). The remaining chimeric proteins were derived by standard methods. All chimeras were fully functional, as assessed by biochemical complex formation, and, where possible, by the reconstitution of deficient cell lines. Jurkat E6.1 cells stably expressing EGFP-actin have been described. Jurkat E6.1 cells expressing either TAC-EGFP, ZAP-70EGFP, LAT-EGFP, Grb2-EYFP, or EYFP-Gads, and J14 cells expressing SLP-76EYFP were generated as described (Bunnell et al., 2001). The stable lines used in these studies express the chimeric signaling proteins at approximately physiological levels. EYFPGL-GPI was transiently transfected into Jurkat E6.1 cells using the AMAXA electroporation system.
Spreading assays
Spreading assays were performed as described, with the following modifications for either immunofluorescent staining or for biochemical analyses (Bunnell et al., 2001). For inhibitor studies, cells were preincubated with the corresponding drugs for 1 h, and then plated and imaged in the presence of the drug. For staining procedures, four-chambered coverglasses (Lab-Tek II; Nunc/Nalgene) were coated with either UCHT1 or HIT3a, as required, warmed, and preloaded with 300 µl ml of normal culture media supplemented with 25 mM Hepes. Cells (2 x 105) were injected into the bottoms of the chambers in 100 µl of normal media and fixed at the indicated times by the addition of 600 µl 4% paraformaldehyde in PBS. After 30 min of fixation at 37°C, the chambers were rinsed three times in PFN buffer (PBS, 10% FBS, and 0.02% sodium azide), permeabilized for 5 min with 0.1% Triton X-100 in PBS, rinsed three times in PFN, and blocked for 1 h in PFN-G (PFN supplemented with 2% normal goat serum). Blocked chambers were incubated for 1 h with the indicated primary antibodies diluted in PFN-G, rinsed three times in PFN, and stained for 1 h with isotype-specific Alexa-conjugated goat antisera diluted in PFN-G. Stained cells were rinsed three times in PBS and imaged as below. For the immunofluorescent visualization of TCR subunits cells were fixed in a final paraformaldehyde concentration of 0.25% and 500 µg/ml digitonin was used for permeabilization.
Cellular imaging
All IRM and corresponding fluorescent images were collected on a Zeiss LSM 410 confocal microscope, as described (Bunnell et al., 2001). Ratiometric calcium measurements correlated with IRM images were collected by alternately visualizing Fluo-4 and Fura-Red in the cell body with a wide pinhole, and taking images of the contact interface under normal conditions. Jurkat E6.1 cells were loaded with calcium indicators, as described, and maintained in the presence of 0.5 mM probenecid (Liu et al., 1998; Jung et al., 2001). The dynamic rearrangements of the actin cytoskeleton and of chimeric signaling molecules during spreading responses were monitored using a Perkin-Elmer Ultraview spinning wheel confocal system equipped with an Orca-ERII CCD camera (Hamamatsu), and filters suitable for the visualization of both EGFP and red dyes. For these live studies, samples were illuminated with either the 488- or 568-nm laser lines from a krypton/argon laser. Live images were collected as vertical Z-stacks which were projected in three dimensions or subsampled for the plane of the coverslip. All photobleaching studies were performed using a Zeiss LSM 510. Cells were imaged for 20 s, photobleached within a specific region of interest (ROI) using the combined 458- and 488-nm laser lines, and then imaged over time. All images were collected using a 63x Plan-Apochromat objective (Carl Zeiss). For live studies, the temperature of the sample was maintained at 37°C using a hot air blower (Nevtek) and an objective heater (Bioptechs).
Image processing and quantitation
IP Lab 3.5 was used for most image processing. Adobe Photoshop (v. 5.0) was used to prepare composite images and for all annotations. Calculations for the photobleaching studies were performed in Excel. Mean fluorescence recovery traces were calculated by monitoring fluorescence intensity over time in the bleached region and in a control region containing similar total fluorescence, normalizing fluorescence in these regions by the total cell fluorescence; and setting the normalized fluorescence during the prebleach interval to 100% before averaging. The percent recovery of fluorescence in bleached regions is calculated by taking the differential between the normalized mean fluorescence intensities within the control and bleached regions, and scaling so that the maximum differential corresponds to a 100% loss of fluorescence.
Online supplemental materials
Supplemental materials are available online at http://www.jcb.org/cgi/content/full/jcb.200203043/DC1. These materials include movies of IRM-correlated calcium elevations, the localization of EGFP-actin, ZAP-70EGFP, LAT-EGFP, Grb2-EGFP, EYFP-Gads, and SLP-76EYFP during TCR-induced spreading, as well as FRAP and inhibitor studies.
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Footnotes |
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Tetsuo Yamazaki's present address is Dept. of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan.
* Abbreviations used in this paper: APC, antigen-presenting cell; cSMAC, central supramolecular activation cluster; EGFP, enhanced GFP; EYFP, enhanced yellow fluorescent protein; IRM, interference reflection microscopy; pSMAC, peripheral SMAC; PTK, protein tyrosine kinase; TCR, T cell receptor; Th2 cells, T helper 2 CD4-positive T cells.
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Acknowledgments |
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S.C. Bunnell is a Fellow of the Cancer Research Institute. D.I. Hong was a Howard Hughes Medical InstituteNational Institutes of Health Research Scholar.
Submitted: 8 March 2002
Revised: 23 August 2002
Accepted: 27 August 2002
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