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Lipid raft domains and protein networks in T-cell receptor signal transduction
Lipid raft domains and protein networks in T-cell receptor signal transduction Thomas Harder Activation of the T-cell antigen receptor (TCR) is a key event in triggering the physiological responses of T lymphocytes to antigen. The earliest TCR-evoked signalling steps, such as tyrosine phosphorylations, ras activation and induction of Ca2þ fluxes, are initiated in the T-cell plasma membrane. It has been implicated that cholesterol- and sphingolipid-rich membrane domains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals at the plasma membrane; however, recent experiments have now differentiated distinct roles for lipid-raft-mediated and protein-mediated interactions in the formation of TCR signalling membrane domains. Addresses Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK e-mail: [email protected]
Current Opinion in Immunology 2004, 16:353–359 This review comes from a themed issue on Lymphocyte activation Edited by John C Cambier and Arthur Weiss 0952-7915/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2004.03.013
Abbreviations BCR B-cell antigen receptor Csk carboxy-terminal Src kinase DRM detergent-resistant membrane GFP green fluorescent protein GPI glycosylphosphatidylinositol HA influenza haemagglutinin LAT linker for activation of T cells Ld liquid-disordered Lo liquid-ordered PLC phospholipase C SH2 Src homology 2 TCR T-cell antigen receptor TLA TCR–LAT signalling assembly
Introduction The lateral segregation of proteins and lipids in the plane of the plasma membrane is a key feature of T-lymphocyte activation. T-cell plasma membrane domains range from small and highly dynamic lipid rafts, through multimolecular assemblies of signalling proteins that form in the vicinity of activated T-cell antigen receptor (TCR), to large membrane domains that form the immunological synapse. The clarification of the distinct roles of these membrane domains requires an understanding of their www.sciencedirect.com
physical, biochemical and cell biological properties. This review will focus on the relationship between lipid raft domains and TCR signalling foci in the plasma membrane and how they may interact in early TCR signal transduction.
Lipid raft domains: physics and chemistry Membrane rafts are lipid-based membrane domains with distinctive physical properties and chemical composition. From a biophysical perspective, rafts are modelled by phase separation in lipid membranes. Coexisting liquidordered (Lo) and liquid-disordered (Ld) phases have been extensively described in many artificial membrane systems [1]. ‘Simple’ artificial model membranes composed of cholesterol, sphingolipids and glycerophospholipid were initially used to model the phase behaviour of cell membranes [2]. Accordingly, rafts resemble Lo membrane phases — tightly packed with fluid domains of cholesterol and saturated acyl chain lipids — surrounded by an Ld phase of non-raft regions [1]. More recently, raft domains have been proposed to correspond to the socalled ‘condensed complexes’ of cholesterol and sphingomyelin found in artificial membrane models [3]. These condensed complexes may form a shell of 80 or so lipid molecules around the membrane anchor of raft proteins [4]. Membrane lipids and proteins that preferentially form ordered membrane phases convey a specific chemical composition to raft membrane domains [5]. Accordingly, sphingolipids (such as sphingomyelin and ganglioside GM1 [GM1]) and cholesterol form raft domains in cell membranes. Glycerophospholipids preferentially partition into Ld phases because they generally carry a polyunsaturated fatty acid, which is difficult to accommodate into ordered membrane phases, and hence are believed to form non-raft regions. Most raft membrane proteins harbour hydrophobic lipid-membrane anchors that provide saturated acyl/alkyl chains. These include proteins with a glycosylphosphatidylinositol (GPI)-lipid anchor, which inserts into the outer leaflet of the plasma membrane [5]. Most inner-leaflet-anchored raft proteins carry, via cysteine thioester bond, a C16-saturated palmitoyl fatty acid, which inserts into the lipid bilayer. These proteins generally contain an additional membrane anchor, for example an amino-terminal myristoyl C14 fatty acylation or multiple S-palmitoylations [6]. The Src-related tyrosine kinases Lck and Fyn, which phosphorylate TCR components following triggering by a ligand, are prominent examples of this class of inner-leaflet raft-anchored Current Opinion in Immunology 2004, 16:353–359
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proteins. Most raft-associated transmembrane proteins, such as the linker for activation of T cells (LAT; [7,8]) and influenza haemagglutinin (HA; [9,10]) are critically modified by an S-palmitoyl inner-leaflet membrane anchor.
Small and dynamic lipid rafts in cell membranes Not surprisingly, the phase behaviour of the cellular plasma membrane is far more complex then that of the artificial membrane models. GPI-anchored or acylated raft proteins readily partition into Lo domains in artificial membranes, but at the resolution of fluorescence light microscopy appear evenly distributed in the plasma membrane of living cells [11–13]. However, several studies using fluorescence resonance energy transfer (FRET) measurements have provided strong support for the notion that raft markers are clustered in cholesterol-dependent lipid-based domains. This has been shown for outer-leaflet GPI-anchored proteins as well as for inner-leaflet raftassociated fluorescent reporter proteins anchored by fatty acylations (see also Update; [14,15,16]. Electron-paramagnetic-resonance (EPR) spectroscopy was used to dissect coexisting ordered and disordered phases in influenza virus membranes with an extremely high temporal resolution. This study revealed the separation of highly ordered raft-like and non-raft-like phases, and a very fast exchange rate of lipid probes between the ordered phase and their environment (104 sec1; [17,18]). These results highlight the highly dynamic and/or transient nature of raft membrane domains. A further approach to elucidate the dynamics of membrane constituents in the plasma membrane is to track the trajectories of single molecule diffusion. These studies show a confined diffusion of probes in zones of 110 nm diameter. At time intervals around 25 ms the probes move from one confinement zone to another in a process called hopdiffusion [19] The hop-diffusion rates of a non-raft glycerophospholipid and a GPI-anchored raft probe were indistinguishable in these experiments. These observations led to the conclusion that the lifetimes of rafts (or the probe’s residence time in the raft) must be much shorter than the residence time in a confinement zone, which is 25 ms [18]. However, recent studies using fluorescence recovery after photobleaching (FRAP) technology showed that the global diffusion rate of plasma membrane raftassociated HA was lower than that of non-raft-associated HA variants. This retarded diffusion of the raft-associated HA is cholesterol dependent, suggesting that it is due to transient interactions of HA with raft domains [20]. The structure of raft domains in the plasma membrane remains a subject of intense debate [18,21]. It is a current view that, in the untouched plasma membrane, raft domains contain few raft molecules and exchange components with their environment in the sub-millisecond scale. It was proposed that the cells maintain a Current Opinion in Immunology 2004, 16:353–359
homeostatic equilibrium of small and dynamic rafts in the plasma membrane [18]. Importantly, the crosslinking of raft components (for example, by antibodies to raft markers or using the GM1-binding pentavalent choleratoxin B subunit) leads to the coalescence of rafts and their clear segregation from crosslinked non-raft proteins [22,23]. These stabilized raft domains function as signalling centres and resemble signalling domains generated by TCR crosslinking [24–26].
Structure of T-cell receptor signalling domains beyond detergent-resistant membranes Assaying the composition of detergent resistant membranes (DRMs) has proved to be an extremely powerful tool for identifying raft-associated TCR signalling machinery [27–29]. As discussed in [21] there are several limitations of DRM analysis, but the key drawback of this method is the disruption of the membrane’s spatial organisation by detergent extraction. Therefore, it not possible to conclude that proteins, when co-isolated in DRM vesicles, are physically coupled in the native membrane. Understanding raft-controlled dynamic interactions between proteins and lipids will be a key challenge for future studies. These experiments will require the biophysical measurement of membrane order in the vicinity of membrane proteins, and measurements of transient protein encounters. Several lines of evidence suggest that rafts regulate dynamic interactions between T-cell signalling proteins, as discussed below. Furthermore, several recent studies revealed the lateral sequestration of early TCR signalling proteins in T-cell plasma membrane domains. Formation of this TCR signalling machinery in membrane domains is largely driven by protein– protein interactions.
Raft-controlled interactions between T-cell membrane proteins Because of their dynamic nature, lipid-raft-mediated interactions facilitate transient encounters between two raft-resident membrane proteins, whereas the interactions of raft proteins with non-raft proteins are limited by their segregation in different membrane phases. This concept was supported by elucidating the role of raft targeting in the function of CD8 as an MHC class I coreceptor (Figure 1). The a subunit of the CD8ab heterodimer provides an extracellular binding site for the MHC class I ligand and contains a cytoplasmic protein motif, which binds the raft-associated tyrosine kinase Lck. The CD8b subunit is palmitoylated and thus responsible for targeting CD8 to DRMs [30]. CD8b palmitoylation strongly enhances the binding of Lck to the CD8a subunit and increases the coreceptor function of CD8ab [31]. Moreover, the CD8b subunit mediates the association with the TCR complex, increasing the fraction of TCR in DRMs and the avidity of CD8/TCR www.sciencedirect.com
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Figure 1
(a) Lck
Lck CD8αβ
CD8αβ
Phases miscible (b) Lck
Lck CD8αα
CD8αα
Phases non-miscible Current Opinion in Immunology
Model for the regulation of CD8–Lck interaction by differential phase miscibility. (a) CD8ab is targeted to DRMs via palmitoylation of the CD8b subunit [30]. This facilitates dynamic interactions with DRM-associated Lck, as both membrane proteins are targeted to a common raft environment. (b) Lck–CD8a binding is mediated by specific cytoplasmic protein–protein interactions. CD8aa resides in non-raft domains (as defined by DRM association). As a consequence of phase immiscibility dynamic encounters of Lck with CD8aa are less favoured, leading to a decreased binding of Lck to CD8aa form.
for MHC class I [31]. Consistently, the non-raft CD8aa variant, which is expressed on certain NK cells, intestinal T cells and gd T cells, binds less Lck and is less efficient as an MHC I coreceptor [31]. Hence, CD8 raft targeting by the CD8b subunit facilitates its interaction with Lck, and the binding of Lck to the CD8a subunit is mediated by cytoplasmic protein–protein interactions. These results highlight the protein and lipid raft contributions to Lck–CD8 complex formation. Similar raft-controlled interactions are thought to play a role in the regulation of Lck activity by tyrosine phosphatase CD45 and the carboxy-terminal Src kinase (Csk) [32]. CD45 and Csk both act on Lck negative regulatory tyrosine, Tyr505. Phosphorylation of Tyr505 by Csk inactivates Lck by inducing an SH2-mediated intramolecular interaction that masks the catalytic domain of Lck. Dephosphorylation of Tyr505 by CD45 in turn relieves this inhibition and thus allows Lck activation. Although CD45 was initially believed to be excluded from raft domains, several reports have now demonstrated that a small fraction of CD45 resides in DRMs. By replacing the extracellular domains of CD45 with those of Thy-1, CD2 and CD43 it was found that CD45 extracellular variants that fail to partition into DRMs were incapable of activating Lck [33]. Encounters between CD45 and Lck, leading to Lck Tyr505 dephosphorylation appear to occur via a small fraction of CD45 in raft membranes, and it will be exciting to understand the factors that regulate this raft localisation [33]. The Lck-inactivating kinase Csk is recruited to the plasma membrane by binding to the www.sciencedirect.com
tyrosine phosphorylated transmembrane adaptors PAG/ cbp (phosphoprotein associated with glycosphingolipidenriched microdomains; [32]) and LIME (Lck-interacting molecule), which additionally binds Lck [34,35]. The recruitment of Csk to DRMs is regulated by tyrosine phosphorylation of these adaptors. PAG/cbp and LIME belong to an emerging class of tyrosine-phosphorylated Spalmitoylated transmembrane adaptor proteins, including LAT and LAB (linker for activation of B cells)/NTAL (non-T-cell activation linker) [36,37]. Modulating lipid order in the environment of a membrane protein may provide an important mechanism for controlling the encounters of immunoreceptors with raft-associated signalling machinery. Engagement/crosslinking of immunoreceptors, including the TCR, increases their association with DRMs, suggesting higher lipid order in their membrane environment ([38,39], but see also [29] for an alternative view). The lipid environment of the B-cell antigen receptor (BCR) was addressed using the fluorescent DiIC16 lipid probe for ordered membrane phases. Indeed, it appears that BCR engagement increases the energy transfer efficiency between BCR (tagged by a fluorescently labelled anti-BCR Fab fragment) and the DiIC16 probe, suggesting an increase in ordered membrane phase upon BCR engagement (S Pierce, unpublished). This may facilitate encounters between the BCR and raft-associated Src-related kinases, leading to BCR phosphorylation and activation, potentially providing a general mechanism for immunoreceptor triggering [38].
TCR–LAT signalling assemblies are more than rafts TCR activation triggers tyrosine phosphorylation cascades, involving sequential activation of the protein tyrosine kinases Lck and zeta-chain-associated protein 70 (ZAP-70). Activated ZAP-70 phosphorylates the transmembrane adaptor LAT, which, via phosphotyrosine-based docking motifs, recruits SH2-domain-containing signalling proteins [40]. Most prominently, these include phospholipase Cg (PLCg) and cytoplasmic adaptor proteins, such as grb-2, crk-L and gads. Grb-2 sequestration to the plasma membrane leads to ras activation via recruitment of the GDP/ GTP exchange factor Sos. PLCg catalyses the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) to generate the second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3), which mediate the induction of Ca2þ fluxes and the activation of ras and protein kinase C (PKC) [40]. These early TCR signalling events trigger cascades of biochemical reactions that eventually lead to changes in gene transcription and the physiological response of the activated T cell. The structure and dynamics of TCR signalling domains in the plasma membrane were addressed by biochemical and light microscopical approaches. Native (i.e. not detergent extracted) plasma membrane fragments containing Current Opinion in Immunology 2004, 16:353–359
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TCRs and associated signalling machinery were immunoisolated using anti-TCR antibody-coated magnetic beads. Biochemical analysis of these TCR membrane domains revealed that LAT accumulated in the vicinity of activated TCR, forming TCR–LAT signalling assemblies (TLAs; [41]). Active tyrosine phosphorylation and LAT tyrosine-based docking motifs are required for LAT, PLCg and grb-2 accumulation in TLAs [41,42]. In elegant microscopical studies the assembly and disassembly of green fluorescent protein (GFP)-tagged LAT and cytoplasmic signalling proteins in TLAs was visualized by video microscopy [43]. Moreover, the dynamic exchange of their constituents was analysed by photobleaching experiments [43,44]. These studies showed that LAT was stably anchored in TLAs within seconds of TCR activation, followed by dissociation of LAT and associated signalling proteins after several minutes of TCR engagement [43]. In both biochemical and microscopical experiments the recruitment of general raft markers into TLAs was investigated: GM-1 and cholesterol enrichment was not detected in immunoisolated TLAs, nor did the microscopical analysis show an incorporation of a generic raft-marker GPI-anchored GFP into TLAs [41,43]. Importantly, this does not mean that TLAs are not raft domains — considering the clustering of LAT they probably do represent ordered membrane phases. However, these results make a clear distinction of transient raft-
facilitated interactions, insufficient to detectably enrich general raft markers in TLAs, from the protein-mediated scaffolds, which selectively anchor raft-associated LAT and associated signalling machinery in the vicinity of TCR.
A network of interactions in TLAs provides specificity and determines thresholds LAT contains numerous phosphotyrosine-based proteindocking sites that bind grb2, gads and PLCg. Analysis of their individual contributions to TLA formation showed that these docking sites cooperated in recruiting LAT, grb2 and PLCg into TLAs [42]. Moreover, analysis of LAT tyrosine mutants showed that multiple grb-2 docking sites on LAT were required to recruit grb-2, even though single grb-2 docking sites were efficiently phosphorylated [45]. The functional significance of synergy between LAT tyrosine-based docking sites was highlighted by the finding that gads/grb-2 and PLCg docking sites had to reside on the same LAT molecule in order to transduce TCR signalling [46]. Cooperativity of TLA signalling complex formation is likely to be caused by networks of interactions between proteins recruited into TLAs. A tetrameric complex of LAT, PLCg, SLP-76 and Gads is a prototypic example for such a potentially cooperative network [47]. In addition to protein–protein interactions, LAT requires palmitoylations to transduce TCR signals and form TLAs [8,41]. Therefore, the cooperative TLA model includes the
Figure 2
Signalling complex formation
PM
Stable signalling assembly Unstable interactions
PM x x
Rate of tyrosine phosphorylation Current Opinion in Immunology
Model for cooperative TLA formation. In this ‘reduced’ version, a transmembrane raft-associated LAT-like adaptor molecule harbours two tyrosine-based docking sites for a cytosolic adaptor that has a weak potential to dimerise. If a fraction of tyrosine-based docking sites are not phosphorylated (x) the adaptor bound to phosphorylated sites () remains weakly bound and readily dissociates. Upon phosphorylation of all docking sites the adaptors bridge two of the LAT-like molecules and the ensemble is stabilized by a network of protein-based and lipid membrane domain-based interactions. PM, plasma membrane. Current Opinion in Immunology 2004, 16:353–359
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Lipid raft domains and protein networks Harder 357
plasma membrane and raft domains in the network of interactions required for signalling complex assembly. Consequently, TLAs may represent lateral LAT assemblies in which adaptor proteins and enzymes form bridges between individual LAT molecules (Figure 2).
anchored fluorescent reporters were actually monomeric. This low fraction of clustered GPI-anchored proteins is likely to cause the difficulties encountered by other FRET approaches to detect GPI-anchored protein clustering [16,55].
Multiple cooperative interactions will define the specific position of a component within the TLA network, even if some individual interactions in the network are weak and/ or relatively unselective. Moreover, the cooperative network defines a tyrosine phosphorylation threshold below which protein–protein and protein–lipid interactions are relatively unstable, and above which formation of a specific complex is achieved. Fully formed TLAs may be further stabilized by limiting the access of phosphatases to the phosphotyrosyl binding sites occupied by SH2 domains of signalling proteins. Moreover, it is conceivable that specific tyrosine phosphorylation patterns on LAT drive the assembly of different types of TLAs. A hypothetical differential phosphorylation pattern on LAT has been put forward as a mechanism of negatively discriminating and positively selecting TCR signals in thymocyte development [48]. Moreover, it was observed that mice homozygously expressing single tyrosine mutants of LAT exhibit reduced thymic T-cell development and developed a strongly Th2-biased cytokine expression [49–51]. Thus, different TCR signalling regimen may evoke TLAs of different structure or dynamics, leading to differential flavours of TCR signalling.
A recent study takes a novel approach to monitoring the phase behaviour of membranes; that is, taking a biophysical perspective of raft domains [56]. Using the lipid dye Laurdan, which allows spectrometric measurement of the degree of lipid ordering in a membrane bilayer, the separation of Lo and Ld phases in the plasma membrane of living macrophages was shown. These domains covered a significant proportion of the cell surface under physiological conditions. They were frequently associated with actin-rich membrane protrusions and may form via protein scaffolds that stabilise these membrane domains.
Acknowledgements The work in Thomas Harder’s laboratory is supported by Medical Research Council and EPA Trust.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Conclusions and future outlook In addition to central TCR-evoked signalling, costimulatory and negative signals regulate the activation of primary T cells upon interaction with an antigenpresenting cell (APC). These signals converge in the immunological synapse, which forms the contact zone between the T cell and the APC [52,53]. Raft accumulation, formation of signalling protein assemblies and active cell biological transport of signalling proteins to the immunological synapse are closely intertwined. It will be a huge challenge to unravel the relationship between these different elements of membrane domain formation.
Update Several reports have addressed the properties of raft membrane domains in the plasma membrane of cells. By using FRET measurements a recent study elucidated the important features of cholesterol-dependent clusters formed by GPI-anchored proteins [54]. This study provides evidence for small (4 nm) and dense clusters of maximally four GPI-anchored reporter protein molecules in the plasma membrane. These clusters accommodated only 20–40% of the GPI-anchored reporter proteins and were composed of several types of GPIanchored proteins. Hence, a large fraction of the GPIwww.sciencedirect.com
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32. Lindquist JA, Simeoni L, Schraven B: Transmembrane adapters: attractants for cytoplasmic effectors. Immunol Rev 2003, 191:165-182. 33. Irles C, Symons A, Michel F, Bakker TR, van der Merwe PA, Acuto O: CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling. Nat Immunol 2003, 4:189-197. CD45 variants were generated by replacing the CD45 extracellular domain with that of Thy-1, CD2 and CD43. This paper describes a close correlation between DRM targeting of these CD45 variants and their capability to restore Lck activation and TCR signalling in CD45-deficient cell lines. 34. Brdickova N, Brdicka T, Angelisova P, Horvath O, Spicka J, Hilgert I, Paces J, Simeoni L, Kliche S, Merten C et al.: LIME: A new membrane raft-associated adaptor protein involved in CD4 and CD8 coreceptor signaling. J Exp Med 2003, 198:1453-1462. See annotation to [37]. 35. Hur EM, Son M, Lee OH, Choi YB, Park C, Lee H, Yun Y: LIME, a novel transmembrane adaptor protein, associates with p56lck and mediates T cell activation. J Exp Med 2003, 198:1463-1473. See annotation to [37]. 36. Janssen E, Zhu M, Zhang W, Koonpaew S: LAB: a new membrane-associated adaptor molecule in B cell activation. Nat Immunol 2003, 4:117-123. See annotation to [37]. 37. Brdicka T, Imrich M, Angelisova P, Brdickova N, Horvath O, Spicka J, Hilgert I, Luskova P, Draber P, Novak P et al.: Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling. J Exp Med 2002, 196:1617-1626. A series of papers [34–37] describes further members of a class of palmitoylated transmembrane adaptors that recruit signalling proteins into plasma-membrane-associated complexes. The formation of multimolecular assemblies as described for the adaptor LAT may be a common theme in the activity of these molecules. 38. Dykstra M, Cherukuri A, Sohn HW, Tzeng SJ, Pierce SK: Location is everything: lipid rafts and immune cell signaling. Annu Rev Immunol 2003, 21:457-481. 39. Giurisato E, McIntosh DP, Tassi M, Gamberucci A, Benedetti A: T cell receptor can be recruited to a subset of plasma membrane rafts, independently of cell signaling and attendantly to raft clustering. J Biol Chem 2003, 278:6771-6778. 40. Zhang W, Samelson LE: The role of membrane-associated adaptors in T cell receptor signalling. Semin Immunol 2000, 12:35-41. 41. Harder T, Kuhn M: Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies. J Cell Biol 2000, 151:199-208. 42. Hartgroves LC, Lin J, Langen H, Zech T, Weiss A, Harder T: Synergistic assembly of linker for activation of T cells signaling protein complexes in T cell plasma membrane domains. J Biol Chem 2003, 278:20389-20394. www.sciencedirect.com
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The authors analysed immunoisolated TCR signalling domains from Jurkat T-cell lines expressing LAT tyrosine mutants. They found that different tyrosine-based docking sites on LAT cooperated in the formation of TLAs, suggesting a cooperative assembly of LAT, SH2 and SH3 adaptors, and phospholipase C at the site of TCR triggering. 43. Bunnell SC, Hong DI, Kardon JR, Yamazaki T, McGlade CJ, Barr VA, Samelson LE: T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J Cell Biol 2002, 158:1263-1275. Video microscopy was used to follow the dynamic formation of TLAs. In line with [41], the researchers describe LAT accumulation in the vicinity of activated TCRs. The TLAs dissociated within several minutes after TCR triggering. Interestingly, the authors describe that different elements of the signalling assemblies appear to use different ways to depart from the TLAs. The generic raft marker GPI–GFP was not incorporated into TLAs. Together with [41] and [44], this work underlines the role of protein– protein interactions in TLA formation. 44. Tanimura N, Nagafuku M, Minaki Y, Umeda Y, Hayashi F, Sakakura J, Kato A, Liddicoat DR, Ogata M, Hamaoka T et al.: Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation. J Cell Biol 2003, 160:125-135. Measuring fluorescence recovery after photobleaching showed that LAT is stably anchored at the site of TCR activation. This anchoring depends on LAT tyrosine-based docking sites for signalling proteins. 45. Zhu M, Janssen E, Zhang W: Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development. J Immunol 2003, 170:325-333. This is a comprehensive analysis of different LAT tyrosine mutants in reconstituting thymic development and recruiting signalling proteins to LAT. It provides evidence that grb-2 docking to LAT depends on the presence of multiple grb-2-docking sites, suggesting cooperativity in grb-2 association to LAT. 46. Lin J, Weiss A: Identification of the minimal tyrosine residues required for linker for activation of T cell function. J Biol Chem 2001, 276:29588-29595. 47. Yablonski D, Kadlecek T, Weiss A: Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domainbinding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT. Mol Cell Biol 2001, 21:4208-4218. 48. Werlen G, Hausmann B, Naeher D, Palmer E: Signaling life and death in the thymus: timing is everything. Science 2003, 299:1859-1863. This review proposes that differential phosphorylation of LAT is key to the distinct downstream signalling elicited by positively and negatively selecting TCR ligands in thymocyte development. 49. Nunez-Cruz S, Aguado E, Richelme S, Chetaille B, Mura AM, Richelme M, Pouyet L, Jouvin-Marche E, Xerri L, Malissen B et al.: LAT regulates gammadelta T cell homeostasis and differentiation. Nat Immunol 2003, 4:999-1008. See annotation to [51].
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50. Aguado E, Richelme S, Nunez-Cruz S, Miazek A, Mura AM, Richelme M, Guo XJ, Sainty D, He HT, Malissen B et al.: Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science 2002, 296:2036-2040. See annotation to [51]. 51. Sommers CL, Park CS, Lee J, Feng C, Fuller CL, Grinberg A, Hildebrand JA, Lacana E, Menon RK, Shores EW et al.: A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 2002, 296:2040-2043. Mice homozygously expressing a tyrosine point mutation disrupting a PLCg docking site [49,50] or three distal grb-2 docking sites [51] exhibit inhibition of thymocyte development. T lymphocytes reaching the periphery are strongly skewed towards a Th2 phenotype, leading to general lymphocyte proliferation. 52. Huppa JB, Davis MM: T-cell-antigen recognition and the immunological synapse. Nat Rev Immunol 2003, 3:973-983. 53. van der Merwe PA: Formation and function of the immunological synapse. Curr Opin Immunol 2002, 14:293-298. 54. Sharma P, Varma R, Sarasij RC, Ira, Gousset K, Krishnamoorthy G, Rao M, Mayor S: Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 2004, 116:577-589. Using a homotypic FRET set-up (i.e. using the same donor and acceptor fluorophore) cholesterol-dependent clustering of GPI-anchored proteins (see [14]) was confirmed and studied in great detail. Based on dilution and bleaching experiments it is estimated that GPI-anchored protein clusters are very small, containing up to four receptor molecules, whereas many GPI-anchored reporter molecules may actually be monomeric. The study also shows clustering between diverse GPI-anchored protein molecules. Furthermore, antibody-mediated clustering of a specific GPI-anchored protein reorganizes the nanoscale organization of native GPI-anchored protein clusters. 55. Glebov OO, Nichols BJ: Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nat Cell Biol 2004 (published online 8 February 2004) DOI:10.1038/ ncb1103 AOP. This paper reports a heterotypic FRET analysis (i.e. using different donor and acceptor fluorophores) that did not reveal clustering of raft markers. In addition, this study indicates that the accumulation of raft markers at the site of T-cell activation, as frequently observed by fluorescence microscopy, can be at least partially attributable to enhanced membrane convolution caused by localised TCR activation. 56. Gaus K, Gratton E, Kable EP, Jones AS, Gelissen I, Kritharides L, Jessup W: Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc Natl Acad Sci USA 2003, 100:15554-15559. This study makes use of the lipid dye Laurdan, which displays a spectral shift depending on the degree of order of its lipid environment. This spectral shift was used to monitor the phase behaviour of macrophage plasma membranes. These experiments revealed distinct ordered and disordered regions providing the first direct evidence for segregated physical phases in cell plasma membranes as predicted by a raft–nonraft equilibrium.
Current Opinion in Immunology 2004, 16:353–359
Current Opinion in Immunology 2004, 16:353–359 This review comes from a themed issue on Lymphocyte activation Edited by John C Cambier and Arthur Weiss 0952-7915/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2004.03.013
Abbreviations BCR B-cell antigen receptor Csk carboxy-terminal Src kinase DRM detergent-resistant membrane GFP green fluorescent protein GPI glycosylphosphatidylinositol HA influenza haemagglutinin LAT linker for activation of T cells Ld liquid-disordered Lo liquid-ordered PLC phospholipase C SH2 Src homology 2 TCR T-cell antigen receptor TLA TCR–LAT signalling assembly
Introduction The lateral segregation of proteins and lipids in the plane of the plasma membrane is a key feature of T-lymphocyte activation. T-cell plasma membrane domains range from small and highly dynamic lipid rafts, through multimolecular assemblies of signalling proteins that form in the vicinity of activated T-cell antigen receptor (TCR), to large membrane domains that form the immunological synapse. The clarification of the distinct roles of these membrane domains requires an understanding of their www.sciencedirect.com
physical, biochemical and cell biological properties. This review will focus on the relationship between lipid raft domains and TCR signalling foci in the plasma membrane and how they may interact in early TCR signal transduction.
Lipid raft domains: physics and chemistry Membrane rafts are lipid-based membrane domains with distinctive physical properties and chemical composition. From a biophysical perspective, rafts are modelled by phase separation in lipid membranes. Coexisting liquidordered (Lo) and liquid-disordered (Ld) phases have been extensively described in many artificial membrane systems [1]. ‘Simple’ artificial model membranes composed of cholesterol, sphingolipids and glycerophospholipid were initially used to model the phase behaviour of cell membranes [2]. Accordingly, rafts resemble Lo membrane phases — tightly packed with fluid domains of cholesterol and saturated acyl chain lipids — surrounded by an Ld phase of non-raft regions [1]. More recently, raft domains have been proposed to correspond to the socalled ‘condensed complexes’ of cholesterol and sphingomyelin found in artificial membrane models [3]. These condensed complexes may form a shell of 80 or so lipid molecules around the membrane anchor of raft proteins [4]. Membrane lipids and proteins that preferentially form ordered membrane phases convey a specific chemical composition to raft membrane domains [5]. Accordingly, sphingolipids (such as sphingomyelin and ganglioside GM1 [GM1]) and cholesterol form raft domains in cell membranes. Glycerophospholipids preferentially partition into Ld phases because they generally carry a polyunsaturated fatty acid, which is difficult to accommodate into ordered membrane phases, and hence are believed to form non-raft regions. Most raft membrane proteins harbour hydrophobic lipid-membrane anchors that provide saturated acyl/alkyl chains. These include proteins with a glycosylphosphatidylinositol (GPI)-lipid anchor, which inserts into the outer leaflet of the plasma membrane [5]. Most inner-leaflet-anchored raft proteins carry, via cysteine thioester bond, a C16-saturated palmitoyl fatty acid, which inserts into the lipid bilayer. These proteins generally contain an additional membrane anchor, for example an amino-terminal myristoyl C14 fatty acylation or multiple S-palmitoylations [6]. The Src-related tyrosine kinases Lck and Fyn, which phosphorylate TCR components following triggering by a ligand, are prominent examples of this class of inner-leaflet raft-anchored Current Opinion in Immunology 2004, 16:353–359
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proteins. Most raft-associated transmembrane proteins, such as the linker for activation of T cells (LAT; [7,8]) and influenza haemagglutinin (HA; [9,10]) are critically modified by an S-palmitoyl inner-leaflet membrane anchor.
Small and dynamic lipid rafts in cell membranes Not surprisingly, the phase behaviour of the cellular plasma membrane is far more complex then that of the artificial membrane models. GPI-anchored or acylated raft proteins readily partition into Lo domains in artificial membranes, but at the resolution of fluorescence light microscopy appear evenly distributed in the plasma membrane of living cells [11–13]. However, several studies using fluorescence resonance energy transfer (FRET) measurements have provided strong support for the notion that raft markers are clustered in cholesterol-dependent lipid-based domains. This has been shown for outer-leaflet GPI-anchored proteins as well as for inner-leaflet raftassociated fluorescent reporter proteins anchored by fatty acylations (see also Update; [14,15,16]. Electron-paramagnetic-resonance (EPR) spectroscopy was used to dissect coexisting ordered and disordered phases in influenza virus membranes with an extremely high temporal resolution. This study revealed the separation of highly ordered raft-like and non-raft-like phases, and a very fast exchange rate of lipid probes between the ordered phase and their environment (104 sec1; [17,18]). These results highlight the highly dynamic and/or transient nature of raft membrane domains. A further approach to elucidate the dynamics of membrane constituents in the plasma membrane is to track the trajectories of single molecule diffusion. These studies show a confined diffusion of probes in zones of 110 nm diameter. At time intervals around 25 ms the probes move from one confinement zone to another in a process called hopdiffusion [19] The hop-diffusion rates of a non-raft glycerophospholipid and a GPI-anchored raft probe were indistinguishable in these experiments. These observations led to the conclusion that the lifetimes of rafts (or the probe’s residence time in the raft) must be much shorter than the residence time in a confinement zone, which is 25 ms [18]. However, recent studies using fluorescence recovery after photobleaching (FRAP) technology showed that the global diffusion rate of plasma membrane raftassociated HA was lower than that of non-raft-associated HA variants. This retarded diffusion of the raft-associated HA is cholesterol dependent, suggesting that it is due to transient interactions of HA with raft domains [20]. The structure of raft domains in the plasma membrane remains a subject of intense debate [18,21]. It is a current view that, in the untouched plasma membrane, raft domains contain few raft molecules and exchange components with their environment in the sub-millisecond scale. It was proposed that the cells maintain a Current Opinion in Immunology 2004, 16:353–359
homeostatic equilibrium of small and dynamic rafts in the plasma membrane [18]. Importantly, the crosslinking of raft components (for example, by antibodies to raft markers or using the GM1-binding pentavalent choleratoxin B subunit) leads to the coalescence of rafts and their clear segregation from crosslinked non-raft proteins [22,23]. These stabilized raft domains function as signalling centres and resemble signalling domains generated by TCR crosslinking [24–26].
Structure of T-cell receptor signalling domains beyond detergent-resistant membranes Assaying the composition of detergent resistant membranes (DRMs) has proved to be an extremely powerful tool for identifying raft-associated TCR signalling machinery [27–29]. As discussed in [21] there are several limitations of DRM analysis, but the key drawback of this method is the disruption of the membrane’s spatial organisation by detergent extraction. Therefore, it not possible to conclude that proteins, when co-isolated in DRM vesicles, are physically coupled in the native membrane. Understanding raft-controlled dynamic interactions between proteins and lipids will be a key challenge for future studies. These experiments will require the biophysical measurement of membrane order in the vicinity of membrane proteins, and measurements of transient protein encounters. Several lines of evidence suggest that rafts regulate dynamic interactions between T-cell signalling proteins, as discussed below. Furthermore, several recent studies revealed the lateral sequestration of early TCR signalling proteins in T-cell plasma membrane domains. Formation of this TCR signalling machinery in membrane domains is largely driven by protein– protein interactions.
Raft-controlled interactions between T-cell membrane proteins Because of their dynamic nature, lipid-raft-mediated interactions facilitate transient encounters between two raft-resident membrane proteins, whereas the interactions of raft proteins with non-raft proteins are limited by their segregation in different membrane phases. This concept was supported by elucidating the role of raft targeting in the function of CD8 as an MHC class I coreceptor (Figure 1). The a subunit of the CD8ab heterodimer provides an extracellular binding site for the MHC class I ligand and contains a cytoplasmic protein motif, which binds the raft-associated tyrosine kinase Lck. The CD8b subunit is palmitoylated and thus responsible for targeting CD8 to DRMs [30]. CD8b palmitoylation strongly enhances the binding of Lck to the CD8a subunit and increases the coreceptor function of CD8ab [31]. Moreover, the CD8b subunit mediates the association with the TCR complex, increasing the fraction of TCR in DRMs and the avidity of CD8/TCR www.sciencedirect.com
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Figure 1
(a) Lck
Lck CD8αβ
CD8αβ
Phases miscible (b) Lck
Lck CD8αα
CD8αα
Phases non-miscible Current Opinion in Immunology
Model for the regulation of CD8–Lck interaction by differential phase miscibility. (a) CD8ab is targeted to DRMs via palmitoylation of the CD8b subunit [30]. This facilitates dynamic interactions with DRM-associated Lck, as both membrane proteins are targeted to a common raft environment. (b) Lck–CD8a binding is mediated by specific cytoplasmic protein–protein interactions. CD8aa resides in non-raft domains (as defined by DRM association). As a consequence of phase immiscibility dynamic encounters of Lck with CD8aa are less favoured, leading to a decreased binding of Lck to CD8aa form.
for MHC class I [31]. Consistently, the non-raft CD8aa variant, which is expressed on certain NK cells, intestinal T cells and gd T cells, binds less Lck and is less efficient as an MHC I coreceptor [31]. Hence, CD8 raft targeting by the CD8b subunit facilitates its interaction with Lck, and the binding of Lck to the CD8a subunit is mediated by cytoplasmic protein–protein interactions. These results highlight the protein and lipid raft contributions to Lck–CD8 complex formation. Similar raft-controlled interactions are thought to play a role in the regulation of Lck activity by tyrosine phosphatase CD45 and the carboxy-terminal Src kinase (Csk) [32]. CD45 and Csk both act on Lck negative regulatory tyrosine, Tyr505. Phosphorylation of Tyr505 by Csk inactivates Lck by inducing an SH2-mediated intramolecular interaction that masks the catalytic domain of Lck. Dephosphorylation of Tyr505 by CD45 in turn relieves this inhibition and thus allows Lck activation. Although CD45 was initially believed to be excluded from raft domains, several reports have now demonstrated that a small fraction of CD45 resides in DRMs. By replacing the extracellular domains of CD45 with those of Thy-1, CD2 and CD43 it was found that CD45 extracellular variants that fail to partition into DRMs were incapable of activating Lck [33]. Encounters between CD45 and Lck, leading to Lck Tyr505 dephosphorylation appear to occur via a small fraction of CD45 in raft membranes, and it will be exciting to understand the factors that regulate this raft localisation [33]. The Lck-inactivating kinase Csk is recruited to the plasma membrane by binding to the www.sciencedirect.com
tyrosine phosphorylated transmembrane adaptors PAG/ cbp (phosphoprotein associated with glycosphingolipidenriched microdomains; [32]) and LIME (Lck-interacting molecule), which additionally binds Lck [34,35]. The recruitment of Csk to DRMs is regulated by tyrosine phosphorylation of these adaptors. PAG/cbp and LIME belong to an emerging class of tyrosine-phosphorylated Spalmitoylated transmembrane adaptor proteins, including LAT and LAB (linker for activation of B cells)/NTAL (non-T-cell activation linker) [36,37]. Modulating lipid order in the environment of a membrane protein may provide an important mechanism for controlling the encounters of immunoreceptors with raft-associated signalling machinery. Engagement/crosslinking of immunoreceptors, including the TCR, increases their association with DRMs, suggesting higher lipid order in their membrane environment ([38,39], but see also [29] for an alternative view). The lipid environment of the B-cell antigen receptor (BCR) was addressed using the fluorescent DiIC16 lipid probe for ordered membrane phases. Indeed, it appears that BCR engagement increases the energy transfer efficiency between BCR (tagged by a fluorescently labelled anti-BCR Fab fragment) and the DiIC16 probe, suggesting an increase in ordered membrane phase upon BCR engagement (S Pierce, unpublished). This may facilitate encounters between the BCR and raft-associated Src-related kinases, leading to BCR phosphorylation and activation, potentially providing a general mechanism for immunoreceptor triggering [38].
TCR–LAT signalling assemblies are more than rafts TCR activation triggers tyrosine phosphorylation cascades, involving sequential activation of the protein tyrosine kinases Lck and zeta-chain-associated protein 70 (ZAP-70). Activated ZAP-70 phosphorylates the transmembrane adaptor LAT, which, via phosphotyrosine-based docking motifs, recruits SH2-domain-containing signalling proteins [40]. Most prominently, these include phospholipase Cg (PLCg) and cytoplasmic adaptor proteins, such as grb-2, crk-L and gads. Grb-2 sequestration to the plasma membrane leads to ras activation via recruitment of the GDP/ GTP exchange factor Sos. PLCg catalyses the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) to generate the second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3), which mediate the induction of Ca2þ fluxes and the activation of ras and protein kinase C (PKC) [40]. These early TCR signalling events trigger cascades of biochemical reactions that eventually lead to changes in gene transcription and the physiological response of the activated T cell. The structure and dynamics of TCR signalling domains in the plasma membrane were addressed by biochemical and light microscopical approaches. Native (i.e. not detergent extracted) plasma membrane fragments containing Current Opinion in Immunology 2004, 16:353–359
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TCRs and associated signalling machinery were immunoisolated using anti-TCR antibody-coated magnetic beads. Biochemical analysis of these TCR membrane domains revealed that LAT accumulated in the vicinity of activated TCR, forming TCR–LAT signalling assemblies (TLAs; [41]). Active tyrosine phosphorylation and LAT tyrosine-based docking motifs are required for LAT, PLCg and grb-2 accumulation in TLAs [41,42]. In elegant microscopical studies the assembly and disassembly of green fluorescent protein (GFP)-tagged LAT and cytoplasmic signalling proteins in TLAs was visualized by video microscopy [43]. Moreover, the dynamic exchange of their constituents was analysed by photobleaching experiments [43,44]. These studies showed that LAT was stably anchored in TLAs within seconds of TCR activation, followed by dissociation of LAT and associated signalling proteins after several minutes of TCR engagement [43]. In both biochemical and microscopical experiments the recruitment of general raft markers into TLAs was investigated: GM-1 and cholesterol enrichment was not detected in immunoisolated TLAs, nor did the microscopical analysis show an incorporation of a generic raft-marker GPI-anchored GFP into TLAs [41,43]. Importantly, this does not mean that TLAs are not raft domains — considering the clustering of LAT they probably do represent ordered membrane phases. However, these results make a clear distinction of transient raft-
facilitated interactions, insufficient to detectably enrich general raft markers in TLAs, from the protein-mediated scaffolds, which selectively anchor raft-associated LAT and associated signalling machinery in the vicinity of TCR.
A network of interactions in TLAs provides specificity and determines thresholds LAT contains numerous phosphotyrosine-based proteindocking sites that bind grb2, gads and PLCg. Analysis of their individual contributions to TLA formation showed that these docking sites cooperated in recruiting LAT, grb2 and PLCg into TLAs [42]. Moreover, analysis of LAT tyrosine mutants showed that multiple grb-2 docking sites on LAT were required to recruit grb-2, even though single grb-2 docking sites were efficiently phosphorylated [45]. The functional significance of synergy between LAT tyrosine-based docking sites was highlighted by the finding that gads/grb-2 and PLCg docking sites had to reside on the same LAT molecule in order to transduce TCR signalling [46]. Cooperativity of TLA signalling complex formation is likely to be caused by networks of interactions between proteins recruited into TLAs. A tetrameric complex of LAT, PLCg, SLP-76 and Gads is a prototypic example for such a potentially cooperative network [47]. In addition to protein–protein interactions, LAT requires palmitoylations to transduce TCR signals and form TLAs [8,41]. Therefore, the cooperative TLA model includes the
Figure 2
Signalling complex formation
PM
Stable signalling assembly Unstable interactions
PM x x
Rate of tyrosine phosphorylation Current Opinion in Immunology
Model for cooperative TLA formation. In this ‘reduced’ version, a transmembrane raft-associated LAT-like adaptor molecule harbours two tyrosine-based docking sites for a cytosolic adaptor that has a weak potential to dimerise. If a fraction of tyrosine-based docking sites are not phosphorylated (x) the adaptor bound to phosphorylated sites () remains weakly bound and readily dissociates. Upon phosphorylation of all docking sites the adaptors bridge two of the LAT-like molecules and the ensemble is stabilized by a network of protein-based and lipid membrane domain-based interactions. PM, plasma membrane. Current Opinion in Immunology 2004, 16:353–359
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Lipid raft domains and protein networks Harder 357
plasma membrane and raft domains in the network of interactions required for signalling complex assembly. Consequently, TLAs may represent lateral LAT assemblies in which adaptor proteins and enzymes form bridges between individual LAT molecules (Figure 2).
anchored fluorescent reporters were actually monomeric. This low fraction of clustered GPI-anchored proteins is likely to cause the difficulties encountered by other FRET approaches to detect GPI-anchored protein clustering [16,55].
Multiple cooperative interactions will define the specific position of a component within the TLA network, even if some individual interactions in the network are weak and/ or relatively unselective. Moreover, the cooperative network defines a tyrosine phosphorylation threshold below which protein–protein and protein–lipid interactions are relatively unstable, and above which formation of a specific complex is achieved. Fully formed TLAs may be further stabilized by limiting the access of phosphatases to the phosphotyrosyl binding sites occupied by SH2 domains of signalling proteins. Moreover, it is conceivable that specific tyrosine phosphorylation patterns on LAT drive the assembly of different types of TLAs. A hypothetical differential phosphorylation pattern on LAT has been put forward as a mechanism of negatively discriminating and positively selecting TCR signals in thymocyte development [48]. Moreover, it was observed that mice homozygously expressing single tyrosine mutants of LAT exhibit reduced thymic T-cell development and developed a strongly Th2-biased cytokine expression [49–51]. Thus, different TCR signalling regimen may evoke TLAs of different structure or dynamics, leading to differential flavours of TCR signalling.
A recent study takes a novel approach to monitoring the phase behaviour of membranes; that is, taking a biophysical perspective of raft domains [56]. Using the lipid dye Laurdan, which allows spectrometric measurement of the degree of lipid ordering in a membrane bilayer, the separation of Lo and Ld phases in the plasma membrane of living macrophages was shown. These domains covered a significant proportion of the cell surface under physiological conditions. They were frequently associated with actin-rich membrane protrusions and may form via protein scaffolds that stabilise these membrane domains.
Acknowledgements The work in Thomas Harder’s laboratory is supported by Medical Research Council and EPA Trust.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Conclusions and future outlook In addition to central TCR-evoked signalling, costimulatory and negative signals regulate the activation of primary T cells upon interaction with an antigenpresenting cell (APC). These signals converge in the immunological synapse, which forms the contact zone between the T cell and the APC [52,53]. Raft accumulation, formation of signalling protein assemblies and active cell biological transport of signalling proteins to the immunological synapse are closely intertwined. It will be a huge challenge to unravel the relationship between these different elements of membrane domain formation.
Update Several reports have addressed the properties of raft membrane domains in the plasma membrane of cells. By using FRET measurements a recent study elucidated the important features of cholesterol-dependent clusters formed by GPI-anchored proteins [54]. This study provides evidence for small (4 nm) and dense clusters of maximally four GPI-anchored reporter protein molecules in the plasma membrane. These clusters accommodated only 20–40% of the GPI-anchored reporter proteins and were composed of several types of GPIanchored proteins. Hence, a large fraction of the GPIwww.sciencedirect.com
10. Melkonian KA, Ostermeyer AG, Chen JZ, Roth MG, Brown DA: Role of lipid modifications in targeting proteins to detergent- resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J Biol Chem 1999, 274:3910-3917. Current Opinion in Immunology 2004, 16:353–359
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13. Khan TK, Yang B, Thompson NL, Maekawa S, Epand RM, Jacobson K: Binding of NAP-22, a calmodulin-binding neuronal protein, to raft-like domains in model membranes. Biochemistry 2003, 42:4780-4786.
29. Drevot P, Langlet C, Guo XJ, Bernard AM, Colard O, Chauvin JP, Lasserre R, He HT: TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts. EMBO J 2002, 21:1899-1908.
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30. Arcaro A, Gregoire C, Boucheron N, Stotz S, Palmer E, Malissen B, Luescher IF: Essential role of CD8 palmitoylation in CD8 coreceptor function. J Immunol 2000, 165:2068-2076.
15. Zacharias DA, Violin JD, Newton AC, Tsien RY: Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 2002, 296:913-916. Energy transfer between enhanced green fluorescent protein (EGFP) and enhanced yellow fluorescent protein (EYFP) was employed to measure the proximity between proteins anchored in the cytoplasmic leaflet of the plasma membrane. It was found that raft-anchored reporters (myristoyl palmitoyl anchors) exhibited an energy transfer pattern that was consistent with cholesterol-dependent clustering in small domains. This provided the first direct support for rafts in the inner leaflet of the plasma membrane. Interestingly, geranylgeranyl-anchored reporters induced reporter-protein clustering that was insensitive to cholesterol depletion.
31. Arcaro A, Gregoire C, Bakker TR, Baldi L, Jordan M, Goffin L, Boucheron N, Wurm F, van der Merwe PA, Malissen B et al.: CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes. J Exp Med 2001, 194:1485-1495.
16. Kenworthy AK, Petranova N, Edidin M: High-resolution FRET microscopy of cholera toxin B-subunit and GPI- anchored proteins in cell plasma membranes. Mol Biol Cell 2000, 11:1645-1655. 17. Kawasaki K, Yin JJ, Subczynski WK, Hyde JS, Kusumi A: Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: methodology development and its application to studies of influenza viral membrane. Biophys J 2001, 80:738-748. 18. Subczynski WK, Kusumi A: Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy. Biochim Biophys Acta 2003, 1610:231-243. Two leading biophysicists discuss the evidence for liquid phase separation in biological membranes. On the basis of a large body of data they present a view of highly dynamic raft membrane domains. Some of the details in this review may be hard to digest for non-biophysicists, but the message is conveyed in a very clear fashion. 19. Fujiwara T, Ritchie K, Murakoshi H, Jacobson K, Kusumi A: Phospholipids undergo hop diffusion in compartmentalized cell membrane. J Cell Biol 2002, 157:1071-1081. 20. Shvartsman DE, Kotler M, Tall RD, Roth MG, Henis YI: Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts. J Cell Biol 2003, 163:879-888. 21. Munro S: Lipid rafts. Elusive or illusive? Cell 2003, 115:377-388. This review critically analyses evidence for the existence of raft domains in native cell membranes. It points the finger at the dangers associated with some frequently used tools such as cholesterol depletion, DRM analysis, choleratoxin B staining and others. 22. Brown D: The tyrosine kinase connection: how GPI-anchored proteins activate T cells. Curr Opin Immunol 1993, 5:349-354. 23. Harder T, Scheiffele P, Verkade P, Simons K: Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 1998, 141:929-942. 24. Gouy H, Deterre P, Debre P, Bismuth G: Cell calcium signaling via GM1 cell surface gangliosides in the human Jurkat T cell line. J Immunol 1994, 152:3271-3281. 25. Janes PW, Ley SC, Magee AI: Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J Cell Biol 1999, 147:447-461. 26. Harder T, Simons K: Clusters of glycolipid and glycosylphosphatidylinositol-anchored proteins in lymphoid Current Opinion in Immunology 2004, 16:353–359
32. Lindquist JA, Simeoni L, Schraven B: Transmembrane adapters: attractants for cytoplasmic effectors. Immunol Rev 2003, 191:165-182. 33. Irles C, Symons A, Michel F, Bakker TR, van der Merwe PA, Acuto O: CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling. Nat Immunol 2003, 4:189-197. CD45 variants were generated by replacing the CD45 extracellular domain with that of Thy-1, CD2 and CD43. This paper describes a close correlation between DRM targeting of these CD45 variants and their capability to restore Lck activation and TCR signalling in CD45-deficient cell lines. 34. Brdickova N, Brdicka T, Angelisova P, Horvath O, Spicka J, Hilgert I, Paces J, Simeoni L, Kliche S, Merten C et al.: LIME: A new membrane raft-associated adaptor protein involved in CD4 and CD8 coreceptor signaling. J Exp Med 2003, 198:1453-1462. See annotation to [37]. 35. Hur EM, Son M, Lee OH, Choi YB, Park C, Lee H, Yun Y: LIME, a novel transmembrane adaptor protein, associates with p56lck and mediates T cell activation. J Exp Med 2003, 198:1463-1473. See annotation to [37]. 36. Janssen E, Zhu M, Zhang W, Koonpaew S: LAB: a new membrane-associated adaptor molecule in B cell activation. Nat Immunol 2003, 4:117-123. See annotation to [37]. 37. Brdicka T, Imrich M, Angelisova P, Brdickova N, Horvath O, Spicka J, Hilgert I, Luskova P, Draber P, Novak P et al.: Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling. J Exp Med 2002, 196:1617-1626. A series of papers [34–37] describes further members of a class of palmitoylated transmembrane adaptors that recruit signalling proteins into plasma-membrane-associated complexes. The formation of multimolecular assemblies as described for the adaptor LAT may be a common theme in the activity of these molecules. 38. Dykstra M, Cherukuri A, Sohn HW, Tzeng SJ, Pierce SK: Location is everything: lipid rafts and immune cell signaling. Annu Rev Immunol 2003, 21:457-481. 39. Giurisato E, McIntosh DP, Tassi M, Gamberucci A, Benedetti A: T cell receptor can be recruited to a subset of plasma membrane rafts, independently of cell signaling and attendantly to raft clustering. J Biol Chem 2003, 278:6771-6778. 40. Zhang W, Samelson LE: The role of membrane-associated adaptors in T cell receptor signalling. Semin Immunol 2000, 12:35-41. 41. Harder T, Kuhn M: Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies. J Cell Biol 2000, 151:199-208. 42. Hartgroves LC, Lin J, Langen H, Zech T, Weiss A, Harder T: Synergistic assembly of linker for activation of T cells signaling protein complexes in T cell plasma membrane domains. J Biol Chem 2003, 278:20389-20394. www.sciencedirect.com
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The authors analysed immunoisolated TCR signalling domains from Jurkat T-cell lines expressing LAT tyrosine mutants. They found that different tyrosine-based docking sites on LAT cooperated in the formation of TLAs, suggesting a cooperative assembly of LAT, SH2 and SH3 adaptors, and phospholipase C at the site of TCR triggering. 43. Bunnell SC, Hong DI, Kardon JR, Yamazaki T, McGlade CJ, Barr VA, Samelson LE: T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J Cell Biol 2002, 158:1263-1275. Video microscopy was used to follow the dynamic formation of TLAs. In line with [41], the researchers describe LAT accumulation in the vicinity of activated TCRs. The TLAs dissociated within several minutes after TCR triggering. Interestingly, the authors describe that different elements of the signalling assemblies appear to use different ways to depart from the TLAs. The generic raft marker GPI–GFP was not incorporated into TLAs. Together with [41] and [44], this work underlines the role of protein– protein interactions in TLA formation. 44. Tanimura N, Nagafuku M, Minaki Y, Umeda Y, Hayashi F, Sakakura J, Kato A, Liddicoat DR, Ogata M, Hamaoka T et al.: Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation. J Cell Biol 2003, 160:125-135. Measuring fluorescence recovery after photobleaching showed that LAT is stably anchored at the site of TCR activation. This anchoring depends on LAT tyrosine-based docking sites for signalling proteins. 45. Zhu M, Janssen E, Zhang W: Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development. J Immunol 2003, 170:325-333. This is a comprehensive analysis of different LAT tyrosine mutants in reconstituting thymic development and recruiting signalling proteins to LAT. It provides evidence that grb-2 docking to LAT depends on the presence of multiple grb-2-docking sites, suggesting cooperativity in grb-2 association to LAT. 46. Lin J, Weiss A: Identification of the minimal tyrosine residues required for linker for activation of T cell function. J Biol Chem 2001, 276:29588-29595. 47. Yablonski D, Kadlecek T, Weiss A: Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domainbinding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT. Mol Cell Biol 2001, 21:4208-4218. 48. Werlen G, Hausmann B, Naeher D, Palmer E: Signaling life and death in the thymus: timing is everything. Science 2003, 299:1859-1863. This review proposes that differential phosphorylation of LAT is key to the distinct downstream signalling elicited by positively and negatively selecting TCR ligands in thymocyte development. 49. Nunez-Cruz S, Aguado E, Richelme S, Chetaille B, Mura AM, Richelme M, Pouyet L, Jouvin-Marche E, Xerri L, Malissen B et al.: LAT regulates gammadelta T cell homeostasis and differentiation. Nat Immunol 2003, 4:999-1008. See annotation to [51].
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50. Aguado E, Richelme S, Nunez-Cruz S, Miazek A, Mura AM, Richelme M, Guo XJ, Sainty D, He HT, Malissen B et al.: Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science 2002, 296:2036-2040. See annotation to [51]. 51. Sommers CL, Park CS, Lee J, Feng C, Fuller CL, Grinberg A, Hildebrand JA, Lacana E, Menon RK, Shores EW et al.: A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 2002, 296:2040-2043. Mice homozygously expressing a tyrosine point mutation disrupting a PLCg docking site [49,50] or three distal grb-2 docking sites [51] exhibit inhibition of thymocyte development. T lymphocytes reaching the periphery are strongly skewed towards a Th2 phenotype, leading to general lymphocyte proliferation. 52. Huppa JB, Davis MM: T-cell-antigen recognition and the immunological synapse. Nat Rev Immunol 2003, 3:973-983. 53. van der Merwe PA: Formation and function of the immunological synapse. Curr Opin Immunol 2002, 14:293-298. 54. Sharma P, Varma R, Sarasij RC, Ira, Gousset K, Krishnamoorthy G, Rao M, Mayor S: Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 2004, 116:577-589. Using a homotypic FRET set-up (i.e. using the same donor and acceptor fluorophore) cholesterol-dependent clustering of GPI-anchored proteins (see [14]) was confirmed and studied in great detail. Based on dilution and bleaching experiments it is estimated that GPI-anchored protein clusters are very small, containing up to four receptor molecules, whereas many GPI-anchored reporter molecules may actually be monomeric. The study also shows clustering between diverse GPI-anchored protein molecules. Furthermore, antibody-mediated clustering of a specific GPI-anchored protein reorganizes the nanoscale organization of native GPI-anchored protein clusters. 55. Glebov OO, Nichols BJ: Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nat Cell Biol 2004 (published online 8 February 2004) DOI:10.1038/ ncb1103 AOP. This paper reports a heterotypic FRET analysis (i.e. using different donor and acceptor fluorophores) that did not reveal clustering of raft markers. In addition, this study indicates that the accumulation of raft markers at the site of T-cell activation, as frequently observed by fluorescence microscopy, can be at least partially attributable to enhanced membrane convolution caused by localised TCR activation. 56. Gaus K, Gratton E, Kable EP, Jones AS, Gelissen I, Kritharides L, Jessup W: Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc Natl Acad Sci USA 2003, 100:15554-15559. This study makes use of the lipid dye Laurdan, which displays a spectral shift depending on the degree of order of its lipid environment. This spectral shift was used to monitor the phase behaviour of macrophage plasma membranes. These experiments revealed distinct ordered and disordered regions providing the first direct evidence for segregated physical phases in cell plasma membranes as predicted by a raft–nonraft equilibrium.
Current Opinion in Immunology 2004, 16:353–359