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Organisation and dynamics of antigen receptors: implications for lymphocyte signalling
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Organisation and dynamics of antigen receptors: implications for lymphocyte signalling Bebhinn Treanor and Facundo D Batista In contrast to the fluid mosaic model proposed by Singer and Nicolson over thirty years ago, the emergent paradigm is one of plasma membrane compartmentalisation mediated by protein– protein interactions, differences in lipid composition, and interactions with the underlying actin cytoskeleton. Extensive research has focused on examining the distribution and organisation of cell surface receptors upon ligand binding; however, relatively little is known about the steady-state organisation and dynamics of cell surface proteins. Here, we discuss recent findings on the regulation of steady-state organisation and diffusion dynamics of immunoreceptors, and how this might impact on our understanding of basal and ligand-dependent signalling. Address Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK Corresponding author: Treanor, Bebhinn ([email protected]) and Batista, Facundo D ([email protected])
Current Opinion in Immunology 2010, 22:299–307 This review comes from a themed issue on Lymphocyte activation and effector functions Edited by Gabrielle Belz and John Wherry Available online 29th April 2010 0952-7915/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2010.03.009
Introduction Lymphocyte activation is triggered by immunoreceptor recognition of antigen and is associated with dramatic morphological changes and the spatial reorganisation of antigen receptors and signalling molecules. However, it appears that some, and perhaps all, immunoreceptors transmit a low level constitutive (tonic) signal independently of ligand [1]. This tonic signal is below the threshold of signalling to induce full-blown activation, but is essential for lymphocyte development and survival [2,3] (Figure 1A). Currently, the mechanism for this tonic signal is not clear, but given that ligand-induced activation is critically linked to alterations in the distribution and dynamics of antigen receptors, it is likely that the steady-state characteristics of antigen receptors has important implications for both tonic and ligand-induced signalling. Despite this, relatively little is known about www.sciencedirect.com
the steady-state organisation and dynamics of antigen receptors. Here, we consider potential mechanisms regulating steady-state diffusion of antigen receptors and discuss how this might impact on our understanding of tonic and ligand-dependent signalling.
Plasma membrane compartmentalisation The Singer–Nicolson fluid mosaic model of the cell membrane in the early 1970s proposed that the cell membrane was organised such that transmembrane proteins are interspersed in the phospholipid bilayer [4]. The model predicted a random distribution of molecular components in the membrane and the unrestricted lateral freedom of proteins and lipids. While this model no doubt had significant impact, several lines of experimental evidence were to emerge which could not be explained by this model. The first studies to question this model revealed that the diffusion of both proteins and lipids within the plasma membrane was several orders of magnitude slower than the diffusion of proteins within reconstituted membranes or liposomes [5–9]. These early studies employed fluorescence recovery after photobleaching (FRAP), a technique that enables the measurement of diffusion of fluorescently labelled membrane components from a non-bleached area into bleached areas. Subsequent FRAP and single particle tracking (SPT) studies clearly identified lateral restrictions in the mobility of both proteins and lipids within the plasma membrane [10– 13]. These measurements were made on the micrometer scale over milliseconds, providing what is referred to as macroscopic diffusion coefficients. More recently, SPT at a frame rate of 40,000 frames per second has revealed that proteins and lipids undergo ‘hop’ diffusion within the plasma membrane, that is, protein and lipid molecules appeared to undergo short-term confined diffusion within a small region (50–300 nm compartment) and long-term hop movement between compartments [14] (Figure 1B). The improved temporal resolution allowed researchers to see that diffusion within a compartment was consistent with diffusion within liposomes and reconstituted membranes, but the temporary confinement within these compartments and long-range hop diffusion resulted in macroscopic diffusion coefficients which were consistent with previous video-rate SPT and FRAP measurements. As a result of these studies it was proposed that the entire plasma membrane is partitioned into nanoscale-sized compartments with regard to translational diffusion of membrane proteins. But what defines these boundaries Current Opinion in Immunology 2010, 22:299–307
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Figure 1
that restrict the lateral diffusion of proteins and lipids within the membrane?
The regulation of protein diffusion on the plasma membrane Actin corrals
The earliest studies to suggest a role for the actin cytoskeleton in the lateral restriction of membrane proteins used experimental models in which the interaction between the plasma membrane and the underlying actin cortex was weakened; for example, by altering the spectrin-actin based membrane skeleton in erythrocytes [8,11,15]. Subsequent studies in which diffusion of membrane proteins with truncated cytoplasmic domains was markedly increased further suggested that the underlying actin cytoskeleton might modulate membrane protein diffusion [10,16]. Indeed, the diffusion of both proteins and lipids has been shown to increase upon treatment of cells with pharmacological agents that disrupt the actin cytoskeleton [13,14,17–19]. These and other studies led to the proposal of the membrane-skeleton fence, or membrane skeleton corralling model [20]. According to this model, transmembrane proteins protrude into the cytoplasm and collide with the membrane-skeleton causing temporary confinement or ‘corralling’ of the proteins within the membrane-skeleton mesh. Molecules transition between actin defined ‘corrals’ by ‘hop’ diffusion. This model has been supported by reconstruction of the underlying actin cytoskeleton by electron tomography, which revealed actin-defined compartments consistent in size (50–200 nm) with that of the compartments determined from high-speed SPT [21].
General concepts. (A) Thresholds of signalling. Schematic diagram to indicate that signalling is not an all or none event but rather there is a continuum of signalling, and the outcome of that signalling is determined by the magnitude or intensity of signalling. Lymphocyte survival is dependent on a low level constitutive (tonic) signal, which may be independent of ligand. Ligand-dependent immunoreceptor signalling exceeds this threshold and leads to cellular activation. (B) Hop diffusion. Schematic diagram to depict hop diffusion within the plasma membrane. Protein and lipid molecules undergo short-term confined diffusion within a small region and long-term hop movement between compartments. Red arrows indicate ‘hop’ transition between compartments. Scale bar represents approximately 50 nm. Current Opinion in Immunology 2010, 22:299–307
However, to directly visualise actin-defined corrals while simultaneously tracking single molecules necessitated the development of high-speed Dual-View total internal reflection fluorescence microscopy (TIRF) (Figure 2A). In contrast to conventional two-colour TIRF microscopy, which sequentially illuminates at different wavelengths and uses a motorized filter wheel to switch between emission filters, Dual-View TIRF simultaneously illuminates at two wavelengths and uses a splitter containing a dichroic and appropriate emission filters to simultaneously collect fluorescent emission. This means that the two fluorophore-labelled proteins of interest are actually visualised at the exact same moment, rather than milliseconds apart while the instrument switches laser illumination and emission filters. This is of critical importance for visualising dynamic processes. Using this technique, Lidke and colleagues provided the first direct demonstration of actin-defined corrals restricting steady-state diffusion of FceRI [22]. While it is not possible to visualise nanometer-sized actin structures because of limitations in optical resolution, this study revealed micron-scale actin features that limit receptor diffusion. Diffusion of single molecules of FceRI was www.sciencedirect.com
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Figure 2
An ezrin-defined and actin-defined membrane-skeleton regulates BCR diffusion. (A) Set up of Dual-View TIRF microscopy in which two fluorophores are simultaneously illuminated at the appropriate wavelengths and a splitter containing a dichroic and appropriate emission filters is used to simultaneously collect fluorescent emission from the sample. (B) Single frame of Dual-View TIRF microscopy showing single particles of BCR (red) and actin (green) (top row) and overlayed imaged (bottom row). Scale bar 2 mm. (C) Schematic model to show that an ezrin (blue) and actin (green) network regulates BCR (red) diffusion dynamics by creating barriers, which confine (red trajectories) BCR diffusion in actin-rich and ezrin-rich areas and define the boundaries where BCR can more freely diffuse (yellow trajectories).
limited to actin-poor regions and the average size of these actin-defined regions was approximately 1 mm, in contrast to the nanoscale confinement zones described by Kusumi and colleagues. Very recently, we have simultaneously visualised the actin cytoskeleton and single molecules of the B cell receptor (BCR) [23]. We observed single molecules of BCR with limited mobility trapped within actin-rich regions, which may consist of membrane compartments on the nanometer scale as suggested by the membrane-skeleton fence model [20]. In contrast, BCR diffusion was increased within actin-poor areas; however, the boundaries for diffusion tracks within these regions are still defined by the actin network, which appears to act as a diffusion barrier (Figure 2B,C). Moreover, we observed that mobile single particles of BCR become confined once they enter an actin-rich region. Taken together, these studies firmly identify the importance of the actin cytoskeleton in regulating the steady-state diffusion of a wide variety of membrane proteins. However, according to the membrane-skeleton fence model, long-range diffusion of transmembrane proteins occurs by proteins ‘hopping’ between adjacent compartments. What allows such a ‘hop’ to occur? Membrane-cytoskeleton linker proteins
Presumably, this ‘hop’ occurs when a space is formed between the plasma membrane and the underlying actin cytoskeleton that allows the passage of the cytoplasmic domain of the transmembrane protein. This is likely to occur when actin filaments temporarily dissociate from www.sciencedirect.com
the plasma membrane and could be regulated through proteins such as the ezrin-radixin-moesin (ERM) family of proteins. These proteins are a family of highly conserved and widely distributed membrane-associated proteins that provide a regulated linkage between plasma membrane proteins and the actin cytoskeleton. Phosphorylation of ERM proteins on a threonine within the C-terminal domain induces a conformational opening of the protein to expose a FERM domain in the N-terminus, which binds to integral membrane proteins, and an actinbinding domain in the C-terminus [24]. We recently employed high-speed Dual-View TIRF to simultaneously visualise GFP-tagged ezrin and single particles of BCR [23]. We observed that the BCR was largely slow moving, or confined, within ezrin-rich regions and more mobile in regions of reduced ezrin intensity (Figure 2C). Strikingly, ezrin-GFP was rapidly reorganized and appeared to create ‘gates’ [15] allowing the BCR to transition between compartments. Consistent with these observations, diffusion of cystic fibrosis transmembrane conductance regulator (CFTR) [25] and some G-protein coupled receptors [26] is increased in cells expressing chimeric molecules of ERM-binding protein 50 (EBP50) which lack the ERM-binding motif. Thus, ERM proteins may influence the diffusion of a wide variety of proteins. Indeed, at the frame rate used for single particle tracking, the ezrin network was highly dynamic and as such, we propose that ERM proteins provide a mechanism to very quickly modify membrane Current Opinion in Immunology 2010, 22:299–307
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protein diffusion by altering the association of the plasma membrane and the underlying actin cytoskeleton. Protein islands
In addition to the actin cytoskeleton and associated membrane linker proteins, emerging evidence suggests that the plasma membrane is compartmentalised into ‘protein islands’. The first evidence to suggest such an organisation utilised a combination of fluorescence resonance energy transfer (FRET) (measures molecular proximity) and electron and scanning force microscopy [27]. This study provided evidence of a higher hierarchical level of receptor clustering in lymphoid cells in which nanometer-scale islets of MHC class I were organised into micrometer-sized ‘island groups’ of 400–800 nm in size. Further support for such hierarchical protein clustering was observed more recently using transmission electron microscopy (TEM) of ‘plasma membrane sheets’, where the cytoplasmic surface of the plasma membrane is exposed by adhering cells to coated EM grids and then ‘ripping’ the adherent membrane away from the rest of the cell [28,29,30]. Using this method, Lillemeier and colleagues very elegantly showed that membrane-associated proteins were found clustered in cholesterolenriched domains that were segregated by ‘protein free’ and cholesterol-low membrane domains [28]. Actin staining showed that these structures are connected to the cytoskeleton suggesting that it may be important for their formation and/or maintenance. How might such an organisation affect protein diffusion within the membrane? Protein islands might induce an ‘oligomerisation-induced trapping’ [20] effect leading to reduced diffusion and enhanced confinement. It should be noted that given the low level of protein labelling in most SPT studies, it is not clear whether tracked particles are in fact monomers or contained within such ‘protein islands’. Moreover, it has yet to be established if molecules within these ‘protein islands’ diffuse independently or whether the ‘island’ diffuses as a whole. However, using a mixture of IgE coupled to two different fluorophores, Andrews and colleagues found that despite co-confinement of IgE molecules, the trajectories of individual molecules did not appear to be correlated [22]. This might suggest that individual molecules within an island can diffuse independently, however correlated trajectories might exist on longer time scales if diffusion of the ‘protein island’ is much slower. Lipid microdomains
Perhaps the most widely discussed revision to the Singer– Nicolson fluid mosaic model is the postulation that the cell membrane is composed of specialised lipid microdomains with distinct composition and function [31]. These cholesterol-rich microdomains, or ‘membrane rafts’, which selectively associate with proteins through non-covalent interactions, were proposed to function as Current Opinion in Immunology 2010, 22:299–307
rafts for the transport of selected membrane proteins or as relay stations in intracellular signalling. According to this idea, proteins in the membrane are localised to either lipid rafts, or the intervening fluid regions of glycerolipids. The idea of these lipid domains was based on the observation of membrane fragment insolubility in nonionic detergents (referred to as detergent-resistant membranes; DRMs). In lymphocytes, several studies have suggested the constitutive localisation of Src family kinases, such as Lck and Lyn [32,33] and the transient localisation of immunoreceptors upon activation to lipid microdomains [reviewed in 34]. But how would such lipid microdomains affect the lateral mobility of cell surface proteins? If the diffusion characteristics in the raft domain are similar to those in the liquid-ordered domain in artificial membranes, then it would be expected that diffusion would be reduced within these domains [35]. Moreover, electrostatic interaction between the cytoplasmic domain of proteins and phospholipids within the membrane may alter the conformation of membrane proteins and thus the lateral mobility [36]. However, several GPI-anchored and putative ‘raft’-associated proteins display high lateral mobility within the plasma membrane [37–39], suggesting that lipid domains may have a limited influence on diffusion within the plasma membrane. Moreover, disruption of lipid rafts using cholesterol-depleting agents results in a loss of membrane fluidity and decreased diffusion of membrane proteins [40,41], further complicating the examination of the effect of membrane rafts on the lateral mobility of membrane proteins. Still, it may be that the small size of lipid rafts precludes accurate measurement of diffusion within these domains. Indeed, Eggeling and colleagues have shown that sphingolipids and glycosylphosphatidylinositiol (GPI)-anchored proteins are transiently confined within cholesterol-dependent domains on the scale of <20 nm [42]. Observation of such an effect was made using stimulated emission depletion (STED), a microscopy technique that provides enhanced lateral resolution, of the order predicted for lipid raft size. Whether this temporary confinement is purely because of raft characteristics, or is indicative of raft association with the actin cytoskeleton, as suggested by Kusumi and colleagues [20], remains to be determined. Galectin lattice
While much attention has focused on the influence of membrane and cytoskeletal features in defining physical barriers to membrane proteins, the potential influence of extracellular aspects has been largely overlooked. However, glycan-based domains generated by galectin binding to cell surface glycoproteins have been proposed [43]. Galectins are a family of b-galactoside-binding proteins that can cross-link glycoproteins to form an extracellular molecular lattice. This galectin lattice may influence glycoprotein localisation and lateral mobility at the cell www.sciencedirect.com
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surface [reviewed in 44,45]. Indeed, recent evidence suggests a role for the galectin lattice in the compartmentalisation of the T cell receptor and CD45 [46]. Moreover, a possible role for the galectin lattice in the regulation of membrane protein diffusion has recently emerged [47]. This study showed that the galectin lattice reduced the lateral mobility of EGF receptor. However, the organisation and composition as well as the dynamics of galectin lattices in different cell types have yet to be determined. How this additional layer of membrane complexity integrates together with protein scaffolds and the actin cytoskeleton represents an exciting new area. Indeed, the emergence of several lines of evidence implicating the galectin lattice in the regulation of basal and activationinduced signalling and autoimmunity [reviewed in 44] suggests that it may play an important role in the regulation of immunoreceptor signalling.
How might this observation provide insight into tonic BCR signalling that is absolutely necessary for B cell development and survival [2,3]? In contrast to ligandinduced signalling, where the activation of cells is simultaneously induced by triggering many receptors, tonic signalling is much more difficult to measure. How do you synchronize a constitutive signal that is emitted by only a few receptors at any one moment? Given this difficulty, the mechanism for tonic signalling is not clear, but appears to be ligand independent [1]. We propose that the steady-state dynamism of the actin cytoskeleton may play an important role in regulating tonic BCR signalling by regulating the diffusion dynamics of the BCR. Our observations suggest that the signalling induced by alteration of the actin cytoskeleton is strongly correlated with changes in BCR diffusion, and in particular, with a reduction in the proportion of very slow (or immobile) BCR.
Implications for signalling It is clear that numerous cell surface proteins including immunoreceptors exhibit restricted steady-state diffusion. This raises the question of what is the functional significance of restricted steady-state diffusion? Surprisingly, we recently found that simple alteration of the actin cytoskeleton was sufficient to induce robust intracellular signalling in B cells in the absence of BCR stimulation [23]. This signalling was comparable to that observed upon BCR cross-linking and resulted in not only early signalling events, such as robust calcium flux, but also activation of downstream signalling pathways including ERK and Akt, and even transcriptional activation. We found that the signal induced by alteration of the actin cytoskeleton was abrogated in B cells lacking key BCR signalling molecules such as PLCg2 and Vav, strongly suggesting that this signal is mediated by the BCR.
How might changes in BCR diffusion facilitate signalling? One possible explanation might be that the membraneskeleton restricts BCR mobility and thus limits the interaction between the BCR and activated kinases or coreceptors. Disruption of the diffusion barrier defined by the actin cytoskeleton increases BCR diffusion and thus may increase the probability that the BCR will encounter an activated kinase or coreceptor. It may also be that the actin cytoskeleton defines the distribution of proteins at the cell surface, which may limit BCR signalling, for example, by immobilising BCR and phosphatases together (Figure 3). Moreover, since ligand-induced BCR signalling leads to alterations in the actin cytoskeleton [48], it is conceivable that tonic BCR signalling may to some degree regulate steady-state actin organisation or dynamics. In line with this we observed that B cells deficient in key BCR signalling molecules such as Lyn
Figure 3
A role for actin-restricted BCR diffusion in tonic signalling? We suggest that the steady-state dynamism of the actin cytoskeleton may play a role in regulating tonic BCR signalling by regulating the diffusion dynamics of the BCR. For example, the membrane-skeleton restricts BCR mobility and thus may limit the interaction between the BCR and activated kinases or co-receptors, or immobilise BCR and phosphatases together. Stochastic alterations in the distribution or density of the actin cytoskeleton (indicated in light brown) may release BCRs and lead to increased BCR diffusion and thus increase the probability that the BCR will encounter an activated kinase or coreceptor, which produces a low level or transient signal (left panel). This transient signal is rapidly switched off but continual actin dynamism permits previously resting BCRs to signal (right panel). www.sciencedirect.com
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and Syk have dramatically altered organisation of the actin cytoskeleton [23,49]. Moreover, we found that diffusion of the BCR was reduced in B cells lacking PLCg2 and Vav, suggesting that tonic signalling through the BCR may indeed influence steady-state actin dynamics or organisation. Such interplay between tonic BCR signalling and actin dynamics may provide a mechanism for regulating this low level constitutive signalling.
Figure 4
Of course, several additional models for tonic signalling have been proposed (Figure 4), which are not necessarily antithetical to our model. For example, homotypic BCR aggregation [50,51] proposes that the BCR exists as an oligomeric complex on the surface of B cells and generates a tonic signal. Alternatively, it has been proposed that the selective association of the preBCR with lipid raft domains results in constitutive signalling [52], or that tonic signals are generated by the homeostatic equilibrium established by the stochastic activation of kinases and their rapid termination by tyrosine phosphatases [53]. What is implicit in each of these models is that the organisation and mobility of the BCR is likely to be an important aspect controlling tonic signalling. How might the organisation and mobility of the immunoreceptors impact on ligand-induced signalling? Interestingly, Hao and colleagues recently reported that BCR stimulation is accompanied by the rapid depolymerisation of the actin cytoskeleton. Importantly, they found that pre-treatment of B cells with actin depolymerising agents enhanced ligand-induced BCR signalling. These observations suggest that disruption of the actin cytoskeleton is a critical aspect of ligand-induced signalling. We suggest that this is likely important because it alters the organisation and mobility of the BCR. As previously discussed, this may permit the association of the BCR with coreceptors or activated kinases. Interestingly, in the context of ‘protein islands’, Lillemeier and colleagues recently reported that TCR and Lat were preclustered into separate domains in unstimulated T cells and these domains transiently concatenate after antigen recognition [54]. Notably, treatment with actin depolymerising agents led to the formation of large clusters suggesting that the actin cytoskeleton prevents the aggregation of protein islands. Since functional association between these interacting components is necessary for cellular activation, restricted diffusion would reduce the probability of their interaction. Indeed, the ‘mobile receptor’ hypothesis of the 1970s proposed that receptor–effector interactions at the plasma membrane are controlled by lateral mobility of interacting components. Recently, destabilisation of the actin cytoskeleton was reported to increase the diffusion of serotonin1A receptor and this increased diffusion was strongly correlated with the efficiency of ligand-mediated signalling [55], providing further support for such a model. Current Opinion in Immunology 2010, 22:299–307
Models of tonic BCR signalling. Several potential mechanistic models have been proposed for tonic BCR signalling in the absence of ligand. In each model, signalling BCRs are depicted in red and resting (nonsignalling) BCRs in green. Steady-state actin dynamism may play a role in tonic signalling by controlling BCR diffusion. The homotypic BCR aggregate model proposes that the BCR exists as an oligomeric complex on the surface of B cells and generates a tonic signal. The lipid raft compartmentalisation model proposes that the selective association of the pre-BCR with lipid raft domains results in constitutive signalling. The equilibrium model suggests that tonic signals are generated by the stochastic kinase-mediated phosphorylation of ITAM residues and their rapid dephosphorylation by tyrosine phosphatases. www.sciencedirect.com
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Moreover, we know that BCR stimulation induces rapid dephosphorylation of ERM proteins [56]. Thus, immunoreceptor signalling may fine tune diffusion dynamics during activation by modifying the interaction between ERM proteins and the actin cytoskeleton [56–58]. Interestingly, we detected a transient increase in BCR diffusion upon stimulation (Treanor and Batista, unpublished). We propose that BCR signalling triggers a localized dephosphorylation of ERM proteins and detachment of the membrane-skeleton permitting increased diffusion of unengaged BCR in close proximity, which may then gain accessibility to ligand or BCR microclusters. It may also be the case that ligand-bound BCR continues to diffuse and only becomes immobilized in large aggregates. Indeed, very recently, Lidke and colleagues [59] report that ligand-bound FceRI is signalling competent and mobile at low antigen doses, whereas receptor immobilisation was markedly increased at high antigen doses and increased cluster size, suggesting that perhaps receptor immobilisation is associated with signal attenuation and internalisation.
Conclusions It is now widely accepted that the plasma membrane is compartmentalised. However, how such compartmentalisation is defined and how it affects the organisation and dynamics of cell surface proteins continues to unfold. Recent evidence has shown that the actin cytoskeleton plays a key role in defining plasma membrane compartments and in the regulation of the lateral mobility of cell surface proteins. Emerging evidence suggests that protein islands, lipid microdomains, and the galectin lattice also contribute to the distribution and dynamics of a wide variety of cell surface proteins. Given that immunoreceptor signalling is critically linked to alterations in the distribution and dynamics of antigen receptors, unravelling how these varied mechanisms contribute to and interact with each other to regulate the steady-state characteristics of antigen receptors has important implications for both tonic and ligand-induced signalling.
Acknowledgement We thank the members of the Lymphocyte Interaction Lab for their useful discussion.
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40. Nishimura SY, Vrljic M, Klein LO, McConnell HM, Moerner WE: Cholesterol depletion induces solid-like regions in the plasma membrane. Biophys J 2006, 90:927-938. 41. Vrljic M, Nishimura SY, Moerner WE, McConnell HM: Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane. Biophys J 2005, 88:334-347. 42. Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schonle A et al.: Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 2009, 457:1159-1162. This study uses the enhanced lateral resolution of stimulated emission depletion (STED) microscopy to show that sphingolipids and GPIanchored proteins are transiently trapped in cholesterol-mediated domains of <20 nm diameter. 43. Brewer CF, Miceli MC, Baum LG: Clusters, bundles, arrays and lattices: novel mechanisms for lectin-saccharidemediated cellular interactions. Curr Opin Struct Biol 2002, 12:616-623. 44. Grigorian A, Torossian S, Demetriou M: T-cell growth, cell surface organization, and the galectin-glycoprotein lattice. Immunol Rev 2009, 230:232-246. 45. Lajoie P, Goetz JG, Dennis JW, Nabi IR: Lattices, rafts, and scaffolds: domain regulation of receptor signaling at the plasma membrane. J Cell Biol 2009, 185:381-385. 46. Chen IJ, Chen HL, Demetriou M: Lateral compartmentalization of T cell receptor versus CD45 by galectin-N-glycan binding and microfilaments coordinate basal and activation signaling. J Biol Chem 2007, 282:35361-35372. 47. Lajoie P, Partridge EA, Guay G, Goetz JG, Pawling J, Lagana A, Joshi B, Dennis JW, Nabi IR: Plasma membrane domain organization regulates EGFR signaling in tumor cells. J Cell Biol 2007, 179:341-356. 48. Hao S, August A: Actin depolymerization transduces the strength of B-cell receptor stimulation. Mol Biol Cell 2005, 16:2275-2284. 49. Weber M, Treanor B, Depoil D, Shinohara H, Harwood NE, Hikida M, Kurosaki T, Batista FD: Phospholipase C-gamma2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen. J Exp Med 2008, 205:853-868. 50. Schamel WW, Reth M: Stability of the B cell antigen receptor complex. Mol Immunol 2000, 37:253-259. 51. Reth M, Wienands J, Schamel WW: An unsolved problem of the clonal selection theory and the model of an oligomeric B-cell antigen receptor. Immunol Rev 2000, 176:10-18. 52. Guo B, Kato RM, Garcia-Lloret M, Wahl MI, Rawlings DJ: Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex. Immunity 2000, 13:243-253. 53. Monroe JG: ITAM-mediated tonic signalling through pre-BCR and BCR complexes. Nat Rev Immunol 2006, 6:283-294. 54. Lillemeier BF, Mortelmaier MA, Forstner MB, Huppa JB, Groves JT, Davis MM: TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat Immunol 2010, 11:90-96.
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This study uses high-speed photoactivated localisation microscopy, dual-colour fluorescence cross-correlation microscopy and transmission electron microscopy to show that in quiescent T cells, TCR and Lat exist on separate protein islands and that these domains concatenate after T cell activation. 55. Ganguly S, Pucadyil TJ, Chattopadhyay A: Actin cytoskeleton dependent dynamics of the human serotonin1A receptor correlates with receptor signaling. Biophys J 2008, 95:451-463. This study shows that destabilisation of the actin cytoskeleton causes an increase in the mobile fraction of serotonin1A receptor and a corresponding increase in the signalling efficiency of the receptor. 56. Gupta N, Wollscheid B, Watts JD, Scheer B, Aebersold R, DeFranco AL: Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics. Nat Immunol 2006, 7:625-633.
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57. Delon J, Kaibuchi K, Germain RN: Exclusion of CD43 from the immunological synapse is mediated by phosphorylationregulated relocation of the cytoskeletal adaptor moesin. Immunity 2001, 15:691-701. 58. Faure S, Salazar-Fontana LI, Semichon M, Tybulewicz VL, Bismuth G, Trautmann A, Germain RN, Delon J: ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation. Nat Immunol 2004, 5:272-279. 59. Andrews NL, Pfeiffer JR, Martinez AM, Haaland DM, Davis RW, Kawakami T, Oliver JM, Wilson BS, Lidke DS: Small, mobile FceRI receptor aggregates are signaling competent. Immunity 2009, 31:469-479. This study used single particle tracking to show that at low concentrations of antigen, which are still sufficient to induce activation, IgE-FceRI aggregates remained highly mobile whereas FceRI immobilisation correlated with increased cluster size and rates of receptor internalisation.
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Organisation and dynamics of antigen receptors: implications for lymphocyte signalling Bebhinn Treanor and Facundo D Batista In contrast to the fluid mosaic model proposed by Singer and Nicolson over thirty years ago, the emergent paradigm is one of plasma membrane compartmentalisation mediated by protein– protein interactions, differences in lipid composition, and interactions with the underlying actin cytoskeleton. Extensive research has focused on examining the distribution and organisation of cell surface receptors upon ligand binding; however, relatively little is known about the steady-state organisation and dynamics of cell surface proteins. Here, we discuss recent findings on the regulation of steady-state organisation and diffusion dynamics of immunoreceptors, and how this might impact on our understanding of basal and ligand-dependent signalling. Address Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK Corresponding author: Treanor, Bebhinn ([email protected]) and Batista, Facundo D ([email protected])
Current Opinion in Immunology 2010, 22:299–307 This review comes from a themed issue on Lymphocyte activation and effector functions Edited by Gabrielle Belz and John Wherry Available online 29th April 2010 0952-7915/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2010.03.009
Introduction Lymphocyte activation is triggered by immunoreceptor recognition of antigen and is associated with dramatic morphological changes and the spatial reorganisation of antigen receptors and signalling molecules. However, it appears that some, and perhaps all, immunoreceptors transmit a low level constitutive (tonic) signal independently of ligand [1]. This tonic signal is below the threshold of signalling to induce full-blown activation, but is essential for lymphocyte development and survival [2,3] (Figure 1A). Currently, the mechanism for this tonic signal is not clear, but given that ligand-induced activation is critically linked to alterations in the distribution and dynamics of antigen receptors, it is likely that the steady-state characteristics of antigen receptors has important implications for both tonic and ligand-induced signalling. Despite this, relatively little is known about www.sciencedirect.com
the steady-state organisation and dynamics of antigen receptors. Here, we consider potential mechanisms regulating steady-state diffusion of antigen receptors and discuss how this might impact on our understanding of tonic and ligand-dependent signalling.
Plasma membrane compartmentalisation The Singer–Nicolson fluid mosaic model of the cell membrane in the early 1970s proposed that the cell membrane was organised such that transmembrane proteins are interspersed in the phospholipid bilayer [4]. The model predicted a random distribution of molecular components in the membrane and the unrestricted lateral freedom of proteins and lipids. While this model no doubt had significant impact, several lines of experimental evidence were to emerge which could not be explained by this model. The first studies to question this model revealed that the diffusion of both proteins and lipids within the plasma membrane was several orders of magnitude slower than the diffusion of proteins within reconstituted membranes or liposomes [5–9]. These early studies employed fluorescence recovery after photobleaching (FRAP), a technique that enables the measurement of diffusion of fluorescently labelled membrane components from a non-bleached area into bleached areas. Subsequent FRAP and single particle tracking (SPT) studies clearly identified lateral restrictions in the mobility of both proteins and lipids within the plasma membrane [10– 13]. These measurements were made on the micrometer scale over milliseconds, providing what is referred to as macroscopic diffusion coefficients. More recently, SPT at a frame rate of 40,000 frames per second has revealed that proteins and lipids undergo ‘hop’ diffusion within the plasma membrane, that is, protein and lipid molecules appeared to undergo short-term confined diffusion within a small region (50–300 nm compartment) and long-term hop movement between compartments [14] (Figure 1B). The improved temporal resolution allowed researchers to see that diffusion within a compartment was consistent with diffusion within liposomes and reconstituted membranes, but the temporary confinement within these compartments and long-range hop diffusion resulted in macroscopic diffusion coefficients which were consistent with previous video-rate SPT and FRAP measurements. As a result of these studies it was proposed that the entire plasma membrane is partitioned into nanoscale-sized compartments with regard to translational diffusion of membrane proteins. But what defines these boundaries Current Opinion in Immunology 2010, 22:299–307
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Figure 1
that restrict the lateral diffusion of proteins and lipids within the membrane?
The regulation of protein diffusion on the plasma membrane Actin corrals
The earliest studies to suggest a role for the actin cytoskeleton in the lateral restriction of membrane proteins used experimental models in which the interaction between the plasma membrane and the underlying actin cortex was weakened; for example, by altering the spectrin-actin based membrane skeleton in erythrocytes [8,11,15]. Subsequent studies in which diffusion of membrane proteins with truncated cytoplasmic domains was markedly increased further suggested that the underlying actin cytoskeleton might modulate membrane protein diffusion [10,16]. Indeed, the diffusion of both proteins and lipids has been shown to increase upon treatment of cells with pharmacological agents that disrupt the actin cytoskeleton [13,14,17–19]. These and other studies led to the proposal of the membrane-skeleton fence, or membrane skeleton corralling model [20]. According to this model, transmembrane proteins protrude into the cytoplasm and collide with the membrane-skeleton causing temporary confinement or ‘corralling’ of the proteins within the membrane-skeleton mesh. Molecules transition between actin defined ‘corrals’ by ‘hop’ diffusion. This model has been supported by reconstruction of the underlying actin cytoskeleton by electron tomography, which revealed actin-defined compartments consistent in size (50–200 nm) with that of the compartments determined from high-speed SPT [21].
General concepts. (A) Thresholds of signalling. Schematic diagram to indicate that signalling is not an all or none event but rather there is a continuum of signalling, and the outcome of that signalling is determined by the magnitude or intensity of signalling. Lymphocyte survival is dependent on a low level constitutive (tonic) signal, which may be independent of ligand. Ligand-dependent immunoreceptor signalling exceeds this threshold and leads to cellular activation. (B) Hop diffusion. Schematic diagram to depict hop diffusion within the plasma membrane. Protein and lipid molecules undergo short-term confined diffusion within a small region and long-term hop movement between compartments. Red arrows indicate ‘hop’ transition between compartments. Scale bar represents approximately 50 nm. Current Opinion in Immunology 2010, 22:299–307
However, to directly visualise actin-defined corrals while simultaneously tracking single molecules necessitated the development of high-speed Dual-View total internal reflection fluorescence microscopy (TIRF) (Figure 2A). In contrast to conventional two-colour TIRF microscopy, which sequentially illuminates at different wavelengths and uses a motorized filter wheel to switch between emission filters, Dual-View TIRF simultaneously illuminates at two wavelengths and uses a splitter containing a dichroic and appropriate emission filters to simultaneously collect fluorescent emission. This means that the two fluorophore-labelled proteins of interest are actually visualised at the exact same moment, rather than milliseconds apart while the instrument switches laser illumination and emission filters. This is of critical importance for visualising dynamic processes. Using this technique, Lidke and colleagues provided the first direct demonstration of actin-defined corrals restricting steady-state diffusion of FceRI [22]. While it is not possible to visualise nanometer-sized actin structures because of limitations in optical resolution, this study revealed micron-scale actin features that limit receptor diffusion. Diffusion of single molecules of FceRI was www.sciencedirect.com
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Figure 2
An ezrin-defined and actin-defined membrane-skeleton regulates BCR diffusion. (A) Set up of Dual-View TIRF microscopy in which two fluorophores are simultaneously illuminated at the appropriate wavelengths and a splitter containing a dichroic and appropriate emission filters is used to simultaneously collect fluorescent emission from the sample. (B) Single frame of Dual-View TIRF microscopy showing single particles of BCR (red) and actin (green) (top row) and overlayed imaged (bottom row). Scale bar 2 mm. (C) Schematic model to show that an ezrin (blue) and actin (green) network regulates BCR (red) diffusion dynamics by creating barriers, which confine (red trajectories) BCR diffusion in actin-rich and ezrin-rich areas and define the boundaries where BCR can more freely diffuse (yellow trajectories).
limited to actin-poor regions and the average size of these actin-defined regions was approximately 1 mm, in contrast to the nanoscale confinement zones described by Kusumi and colleagues. Very recently, we have simultaneously visualised the actin cytoskeleton and single molecules of the B cell receptor (BCR) [23]. We observed single molecules of BCR with limited mobility trapped within actin-rich regions, which may consist of membrane compartments on the nanometer scale as suggested by the membrane-skeleton fence model [20]. In contrast, BCR diffusion was increased within actin-poor areas; however, the boundaries for diffusion tracks within these regions are still defined by the actin network, which appears to act as a diffusion barrier (Figure 2B,C). Moreover, we observed that mobile single particles of BCR become confined once they enter an actin-rich region. Taken together, these studies firmly identify the importance of the actin cytoskeleton in regulating the steady-state diffusion of a wide variety of membrane proteins. However, according to the membrane-skeleton fence model, long-range diffusion of transmembrane proteins occurs by proteins ‘hopping’ between adjacent compartments. What allows such a ‘hop’ to occur? Membrane-cytoskeleton linker proteins
Presumably, this ‘hop’ occurs when a space is formed between the plasma membrane and the underlying actin cytoskeleton that allows the passage of the cytoplasmic domain of the transmembrane protein. This is likely to occur when actin filaments temporarily dissociate from www.sciencedirect.com
the plasma membrane and could be regulated through proteins such as the ezrin-radixin-moesin (ERM) family of proteins. These proteins are a family of highly conserved and widely distributed membrane-associated proteins that provide a regulated linkage between plasma membrane proteins and the actin cytoskeleton. Phosphorylation of ERM proteins on a threonine within the C-terminal domain induces a conformational opening of the protein to expose a FERM domain in the N-terminus, which binds to integral membrane proteins, and an actinbinding domain in the C-terminus [24]. We recently employed high-speed Dual-View TIRF to simultaneously visualise GFP-tagged ezrin and single particles of BCR [23]. We observed that the BCR was largely slow moving, or confined, within ezrin-rich regions and more mobile in regions of reduced ezrin intensity (Figure 2C). Strikingly, ezrin-GFP was rapidly reorganized and appeared to create ‘gates’ [15] allowing the BCR to transition between compartments. Consistent with these observations, diffusion of cystic fibrosis transmembrane conductance regulator (CFTR) [25] and some G-protein coupled receptors [26] is increased in cells expressing chimeric molecules of ERM-binding protein 50 (EBP50) which lack the ERM-binding motif. Thus, ERM proteins may influence the diffusion of a wide variety of proteins. Indeed, at the frame rate used for single particle tracking, the ezrin network was highly dynamic and as such, we propose that ERM proteins provide a mechanism to very quickly modify membrane Current Opinion in Immunology 2010, 22:299–307
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protein diffusion by altering the association of the plasma membrane and the underlying actin cytoskeleton. Protein islands
In addition to the actin cytoskeleton and associated membrane linker proteins, emerging evidence suggests that the plasma membrane is compartmentalised into ‘protein islands’. The first evidence to suggest such an organisation utilised a combination of fluorescence resonance energy transfer (FRET) (measures molecular proximity) and electron and scanning force microscopy [27]. This study provided evidence of a higher hierarchical level of receptor clustering in lymphoid cells in which nanometer-scale islets of MHC class I were organised into micrometer-sized ‘island groups’ of 400–800 nm in size. Further support for such hierarchical protein clustering was observed more recently using transmission electron microscopy (TEM) of ‘plasma membrane sheets’, where the cytoplasmic surface of the plasma membrane is exposed by adhering cells to coated EM grids and then ‘ripping’ the adherent membrane away from the rest of the cell [28,29,30]. Using this method, Lillemeier and colleagues very elegantly showed that membrane-associated proteins were found clustered in cholesterolenriched domains that were segregated by ‘protein free’ and cholesterol-low membrane domains [28]. Actin staining showed that these structures are connected to the cytoskeleton suggesting that it may be important for their formation and/or maintenance. How might such an organisation affect protein diffusion within the membrane? Protein islands might induce an ‘oligomerisation-induced trapping’ [20] effect leading to reduced diffusion and enhanced confinement. It should be noted that given the low level of protein labelling in most SPT studies, it is not clear whether tracked particles are in fact monomers or contained within such ‘protein islands’. Moreover, it has yet to be established if molecules within these ‘protein islands’ diffuse independently or whether the ‘island’ diffuses as a whole. However, using a mixture of IgE coupled to two different fluorophores, Andrews and colleagues found that despite co-confinement of IgE molecules, the trajectories of individual molecules did not appear to be correlated [22]. This might suggest that individual molecules within an island can diffuse independently, however correlated trajectories might exist on longer time scales if diffusion of the ‘protein island’ is much slower. Lipid microdomains
Perhaps the most widely discussed revision to the Singer– Nicolson fluid mosaic model is the postulation that the cell membrane is composed of specialised lipid microdomains with distinct composition and function [31]. These cholesterol-rich microdomains, or ‘membrane rafts’, which selectively associate with proteins through non-covalent interactions, were proposed to function as Current Opinion in Immunology 2010, 22:299–307
rafts for the transport of selected membrane proteins or as relay stations in intracellular signalling. According to this idea, proteins in the membrane are localised to either lipid rafts, or the intervening fluid regions of glycerolipids. The idea of these lipid domains was based on the observation of membrane fragment insolubility in nonionic detergents (referred to as detergent-resistant membranes; DRMs). In lymphocytes, several studies have suggested the constitutive localisation of Src family kinases, such as Lck and Lyn [32,33] and the transient localisation of immunoreceptors upon activation to lipid microdomains [reviewed in 34]. But how would such lipid microdomains affect the lateral mobility of cell surface proteins? If the diffusion characteristics in the raft domain are similar to those in the liquid-ordered domain in artificial membranes, then it would be expected that diffusion would be reduced within these domains [35]. Moreover, electrostatic interaction between the cytoplasmic domain of proteins and phospholipids within the membrane may alter the conformation of membrane proteins and thus the lateral mobility [36]. However, several GPI-anchored and putative ‘raft’-associated proteins display high lateral mobility within the plasma membrane [37–39], suggesting that lipid domains may have a limited influence on diffusion within the plasma membrane. Moreover, disruption of lipid rafts using cholesterol-depleting agents results in a loss of membrane fluidity and decreased diffusion of membrane proteins [40,41], further complicating the examination of the effect of membrane rafts on the lateral mobility of membrane proteins. Still, it may be that the small size of lipid rafts precludes accurate measurement of diffusion within these domains. Indeed, Eggeling and colleagues have shown that sphingolipids and glycosylphosphatidylinositiol (GPI)-anchored proteins are transiently confined within cholesterol-dependent domains on the scale of <20 nm [42]. Observation of such an effect was made using stimulated emission depletion (STED), a microscopy technique that provides enhanced lateral resolution, of the order predicted for lipid raft size. Whether this temporary confinement is purely because of raft characteristics, or is indicative of raft association with the actin cytoskeleton, as suggested by Kusumi and colleagues [20], remains to be determined. Galectin lattice
While much attention has focused on the influence of membrane and cytoskeletal features in defining physical barriers to membrane proteins, the potential influence of extracellular aspects has been largely overlooked. However, glycan-based domains generated by galectin binding to cell surface glycoproteins have been proposed [43]. Galectins are a family of b-galactoside-binding proteins that can cross-link glycoproteins to form an extracellular molecular lattice. This galectin lattice may influence glycoprotein localisation and lateral mobility at the cell www.sciencedirect.com
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surface [reviewed in 44,45]. Indeed, recent evidence suggests a role for the galectin lattice in the compartmentalisation of the T cell receptor and CD45 [46]. Moreover, a possible role for the galectin lattice in the regulation of membrane protein diffusion has recently emerged [47]. This study showed that the galectin lattice reduced the lateral mobility of EGF receptor. However, the organisation and composition as well as the dynamics of galectin lattices in different cell types have yet to be determined. How this additional layer of membrane complexity integrates together with protein scaffolds and the actin cytoskeleton represents an exciting new area. Indeed, the emergence of several lines of evidence implicating the galectin lattice in the regulation of basal and activationinduced signalling and autoimmunity [reviewed in 44] suggests that it may play an important role in the regulation of immunoreceptor signalling.
How might this observation provide insight into tonic BCR signalling that is absolutely necessary for B cell development and survival [2,3]? In contrast to ligandinduced signalling, where the activation of cells is simultaneously induced by triggering many receptors, tonic signalling is much more difficult to measure. How do you synchronize a constitutive signal that is emitted by only a few receptors at any one moment? Given this difficulty, the mechanism for tonic signalling is not clear, but appears to be ligand independent [1]. We propose that the steady-state dynamism of the actin cytoskeleton may play an important role in regulating tonic BCR signalling by regulating the diffusion dynamics of the BCR. Our observations suggest that the signalling induced by alteration of the actin cytoskeleton is strongly correlated with changes in BCR diffusion, and in particular, with a reduction in the proportion of very slow (or immobile) BCR.
Implications for signalling It is clear that numerous cell surface proteins including immunoreceptors exhibit restricted steady-state diffusion. This raises the question of what is the functional significance of restricted steady-state diffusion? Surprisingly, we recently found that simple alteration of the actin cytoskeleton was sufficient to induce robust intracellular signalling in B cells in the absence of BCR stimulation [23]. This signalling was comparable to that observed upon BCR cross-linking and resulted in not only early signalling events, such as robust calcium flux, but also activation of downstream signalling pathways including ERK and Akt, and even transcriptional activation. We found that the signal induced by alteration of the actin cytoskeleton was abrogated in B cells lacking key BCR signalling molecules such as PLCg2 and Vav, strongly suggesting that this signal is mediated by the BCR.
How might changes in BCR diffusion facilitate signalling? One possible explanation might be that the membraneskeleton restricts BCR mobility and thus limits the interaction between the BCR and activated kinases or coreceptors. Disruption of the diffusion barrier defined by the actin cytoskeleton increases BCR diffusion and thus may increase the probability that the BCR will encounter an activated kinase or coreceptor. It may also be that the actin cytoskeleton defines the distribution of proteins at the cell surface, which may limit BCR signalling, for example, by immobilising BCR and phosphatases together (Figure 3). Moreover, since ligand-induced BCR signalling leads to alterations in the actin cytoskeleton [48], it is conceivable that tonic BCR signalling may to some degree regulate steady-state actin organisation or dynamics. In line with this we observed that B cells deficient in key BCR signalling molecules such as Lyn
Figure 3
A role for actin-restricted BCR diffusion in tonic signalling? We suggest that the steady-state dynamism of the actin cytoskeleton may play a role in regulating tonic BCR signalling by regulating the diffusion dynamics of the BCR. For example, the membrane-skeleton restricts BCR mobility and thus may limit the interaction between the BCR and activated kinases or co-receptors, or immobilise BCR and phosphatases together. Stochastic alterations in the distribution or density of the actin cytoskeleton (indicated in light brown) may release BCRs and lead to increased BCR diffusion and thus increase the probability that the BCR will encounter an activated kinase or coreceptor, which produces a low level or transient signal (left panel). This transient signal is rapidly switched off but continual actin dynamism permits previously resting BCRs to signal (right panel). www.sciencedirect.com
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and Syk have dramatically altered organisation of the actin cytoskeleton [23,49]. Moreover, we found that diffusion of the BCR was reduced in B cells lacking PLCg2 and Vav, suggesting that tonic signalling through the BCR may indeed influence steady-state actin dynamics or organisation. Such interplay between tonic BCR signalling and actin dynamics may provide a mechanism for regulating this low level constitutive signalling.
Figure 4
Of course, several additional models for tonic signalling have been proposed (Figure 4), which are not necessarily antithetical to our model. For example, homotypic BCR aggregation [50,51] proposes that the BCR exists as an oligomeric complex on the surface of B cells and generates a tonic signal. Alternatively, it has been proposed that the selective association of the preBCR with lipid raft domains results in constitutive signalling [52], or that tonic signals are generated by the homeostatic equilibrium established by the stochastic activation of kinases and their rapid termination by tyrosine phosphatases [53]. What is implicit in each of these models is that the organisation and mobility of the BCR is likely to be an important aspect controlling tonic signalling. How might the organisation and mobility of the immunoreceptors impact on ligand-induced signalling? Interestingly, Hao and colleagues recently reported that BCR stimulation is accompanied by the rapid depolymerisation of the actin cytoskeleton. Importantly, they found that pre-treatment of B cells with actin depolymerising agents enhanced ligand-induced BCR signalling. These observations suggest that disruption of the actin cytoskeleton is a critical aspect of ligand-induced signalling. We suggest that this is likely important because it alters the organisation and mobility of the BCR. As previously discussed, this may permit the association of the BCR with coreceptors or activated kinases. Interestingly, in the context of ‘protein islands’, Lillemeier and colleagues recently reported that TCR and Lat were preclustered into separate domains in unstimulated T cells and these domains transiently concatenate after antigen recognition [54]. Notably, treatment with actin depolymerising agents led to the formation of large clusters suggesting that the actin cytoskeleton prevents the aggregation of protein islands. Since functional association between these interacting components is necessary for cellular activation, restricted diffusion would reduce the probability of their interaction. Indeed, the ‘mobile receptor’ hypothesis of the 1970s proposed that receptor–effector interactions at the plasma membrane are controlled by lateral mobility of interacting components. Recently, destabilisation of the actin cytoskeleton was reported to increase the diffusion of serotonin1A receptor and this increased diffusion was strongly correlated with the efficiency of ligand-mediated signalling [55], providing further support for such a model. Current Opinion in Immunology 2010, 22:299–307
Models of tonic BCR signalling. Several potential mechanistic models have been proposed for tonic BCR signalling in the absence of ligand. In each model, signalling BCRs are depicted in red and resting (nonsignalling) BCRs in green. Steady-state actin dynamism may play a role in tonic signalling by controlling BCR diffusion. The homotypic BCR aggregate model proposes that the BCR exists as an oligomeric complex on the surface of B cells and generates a tonic signal. The lipid raft compartmentalisation model proposes that the selective association of the pre-BCR with lipid raft domains results in constitutive signalling. The equilibrium model suggests that tonic signals are generated by the stochastic kinase-mediated phosphorylation of ITAM residues and their rapid dephosphorylation by tyrosine phosphatases. www.sciencedirect.com
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Moreover, we know that BCR stimulation induces rapid dephosphorylation of ERM proteins [56]. Thus, immunoreceptor signalling may fine tune diffusion dynamics during activation by modifying the interaction between ERM proteins and the actin cytoskeleton [56–58]. Interestingly, we detected a transient increase in BCR diffusion upon stimulation (Treanor and Batista, unpublished). We propose that BCR signalling triggers a localized dephosphorylation of ERM proteins and detachment of the membrane-skeleton permitting increased diffusion of unengaged BCR in close proximity, which may then gain accessibility to ligand or BCR microclusters. It may also be the case that ligand-bound BCR continues to diffuse and only becomes immobilized in large aggregates. Indeed, very recently, Lidke and colleagues [59] report that ligand-bound FceRI is signalling competent and mobile at low antigen doses, whereas receptor immobilisation was markedly increased at high antigen doses and increased cluster size, suggesting that perhaps receptor immobilisation is associated with signal attenuation and internalisation.
Conclusions It is now widely accepted that the plasma membrane is compartmentalised. However, how such compartmentalisation is defined and how it affects the organisation and dynamics of cell surface proteins continues to unfold. Recent evidence has shown that the actin cytoskeleton plays a key role in defining plasma membrane compartments and in the regulation of the lateral mobility of cell surface proteins. Emerging evidence suggests that protein islands, lipid microdomains, and the galectin lattice also contribute to the distribution and dynamics of a wide variety of cell surface proteins. Given that immunoreceptor signalling is critically linked to alterations in the distribution and dynamics of antigen receptors, unravelling how these varied mechanisms contribute to and interact with each other to regulate the steady-state characteristics of antigen receptors has important implications for both tonic and ligand-induced signalling.
Acknowledgement We thank the members of the Lymphocyte Interaction Lab for their useful discussion.
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This study uses high-speed photoactivated localisation microscopy, dual-colour fluorescence cross-correlation microscopy and transmission electron microscopy to show that in quiescent T cells, TCR and Lat exist on separate protein islands and that these domains concatenate after T cell activation. 55. Ganguly S, Pucadyil TJ, Chattopadhyay A: Actin cytoskeleton dependent dynamics of the human serotonin1A receptor correlates with receptor signaling. Biophys J 2008, 95:451-463. This study shows that destabilisation of the actin cytoskeleton causes an increase in the mobile fraction of serotonin1A receptor and a corresponding increase in the signalling efficiency of the receptor. 56. Gupta N, Wollscheid B, Watts JD, Scheer B, Aebersold R, DeFranco AL: Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics. Nat Immunol 2006, 7:625-633.
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